Collagen modified superoxide dismutase as well as preparation method and application thereof

Abstract

The invention provides collagen modified superoxide dismutase and a preparation method and application thereof, the preparation method comprises the following steps: dissolving collagen in water, sequentially adding superoxide dismutase, EDC and NHC into the water, and stirring in a water bath heat preservation state to obtain a flowable gel sample; diluting the obtained gel sample with water, centrifuging, and collecting a lower-layer viscous liquid; and adding the centrifuged viscous liquid into a PBS (phosphate buffer solution), and dialyzing to obtain the collagen modified superoxide dismutase. Trehalose and mannitol are added into the collagen modified superoxide dismutase, the mixture is stirred and mixed evenly, the mixture is poured into a mold to be freeze-dried and extruded to be flat, and the repairing patch is obtained. The crosslinking modified superoxide dismutase obtained in the invention has good stability, and the obtained repair patch is convenient to use, can promote wound healing, and has wide application prospects and practical significance.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular, relates to a collagen-modified superoxide dismutase and a preparation method and application thereof. Background Art

[0002] Oral ulcers mostly occur locally in the oral mucosa and are self-limiting, cyclical, and recurrent. They are characterized by the appearance of superficial ulcers that are approximately round or oval in shape, the size of rice grains, on the mucosa of the gums, tongue, cheeks, palate, and lips. The number and size of ulcers vary, the edges are neat, and the distribution is uneven, with the clinical characteristics of "yellow, red, concave, and painful". Although it has a certain degree of self-limitation, the pain is obvious when it occurs and is accompanied by systemic symptoms such as low fever, swollen lymph nodes, and headache. It also seriously affects daily behaviors such as eating, speaking, and sleeping, causing great physical and mental pain to patients. At present, the cause and pathogenesis of oral ulcers are still unclear and there is no ideal cure, but active and effective intervention is conducive to accelerating ulcer healing, reducing pain and recurrence. Treatment also often uses a combination of local anti-inflammatory and systemic treatment, but the treatment effect is not obvious. Currently, most drugs used in clinical treatment of oral ulcers are pills, capsules, external powders or pastes. The effective concentration of this dosage form in mucosal tissue is low or the concentration is difficult to maintain, and it is easily affected by saliva and cannot be enriched in the lesion site.

[0003] Superoxide dismutase (SOD) is a type of protease substance that is widely present in organisms in nature and has the function of scavenging free radicals and maintaining the body's oxidative balance. It is the main antioxidant enzyme for the human body to resist free radical damage. SOD can convert harmful superoxide free radicals into hydrogen peroxide through reactions, and then decompose them into water using catalase (CAT) and peroxidase (POD). SOD is also regarded as the most magical enzyme and the garbage scavenger in the human body. In addition, SOD can effectively remove excess free radicals on the surface of ulcers, thereby achieving the purpose of alleviating and curing ulcers. However, due to the high cost of SOD extraction, limited sources, large molecular weight, easy inactivation, and difficulty in absorption, the production and application of SOD is still relatively small. With the development of science and technology, the use of genetic engineering technology to artificially synthesize SOD has become the main production method. Compared with natural SOD, artificially synthesized SOD has lower cost, smaller molecular weight, more stable state and higher activity, but the enzyme activity of liquid SOD will gradually decrease 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 diseases. Summary of the invention

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

[0006] To solve the above problems, the technical solution adopted by the present invention is: Technical topic 1: A collagen-modified superoxide dismutase, the preparation method of which is as follows: S1: Dissolve collagen in water to prepare a collagen solution, add superoxide dismutase, EDC and NHC in sequence, stir in a water bath to make it cross-linked, and obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with water and centrifuge it to collect the viscous liquid at the bottom layer; S3: adding PBS buffer to the viscous liquid obtained after centrifugation in S2 to prepare a mixed solution, placing the mixed solution in a dialysis bag for dialysis, collecting the treated cross-linked liquid in the bag, and obtaining 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.

[0007] As a further embodiment of the present invention, the concentration of the collagen solution in S1 is 50-100 mg / mL.

[0008] As a further embodiment of the present invention, the final enzyme activity content of the superoxide dismutase described in S1 is 400~1000U / mL.

[0009] As a further embodiment of the present invention, the molar ratio of EDC to NHS in S1 is 1-3:1; and the total content of EDC and NHS is 60-100 mmol / L.

[0010] As a further embodiment of the present invention, the water bath temperature in S1 is 30-40°C; the stirring speed is 700-1500 r / min; and the stirring time is 10-30 min.

[0011] 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; and the centrifugal speed is 8000 to 12000 r / min.

[0012] 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.

[0013] Technical theme 2: A method for preparing collagen-modified superoxide dismutase as described in technical subject one.

[0014] Technical Theme 3: A use of collagen-modified superoxide dismutase as described in Technical Topic 1 in the preparation of oral ulcer repair drugs.

[0015] 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, the mixture is stirred and poured into a mold for freeze-drying to obtain a sample, and the freeze-dried sample is squeezed flat to obtain a repair patch; the added mass of the trehalose is 5% to 10% of the mass of the collagen-modified superoxide dismutase; the added mass of the mannitol is 5% to 10% of the mass of the collagen-modified superoxide dismutase.

[0016] The beneficial effects of adopting the above technical solution are: 1. The addition of collagen effectively increases the enzyme activity time of SOD. After cross-linking, collagen binds to SOD, which can stabilize the molecular conformation of SOD and prolong the enzyme activity time of SOD.

[0017] 2. The cooperation between collagen and SOD can not only better play the role of exogenous SOD itself, but also promote the body to produce more SOD.

[0018] 3. The oral repair patch provided by the present invention can prevent saliva from dissolving drugs and ensure unidirectional drug release. In addition, the patch can not only protect the ulcer surface, but also enable the drug to act directly on the ulcer surface, prolong the retention time, increase the effective concentration of local acting substances, and promote wound healing. More importantly, it is easy to use, requires less use, and is more adaptable to patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the recombinant collagen-SOD composite sample obtained in Example 16 of the present invention.

[0020] Figure 2 This is the recombinant collagen-SOD composite sample obtained in Example 17 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is clearly and completely described below in conjunction with specific embodiments.

[0022] EDC was purchased from Bio-Tech, product number ST1299-5g, molecular weight 191.7; NHS was purchased from Thermo Fisher Scientific, catalog number 24500, molecular weight 115.09; The superoxide dismutase (SOD) activity is 10000U / mg.

[0023] Example 1 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0024] Example 2 S1: Dissolve 5 g of recombinant type II humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0025] Example 3 S1: Dissolve 5 g of recombinant type III humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0026] Example 4 S1: Dissolve 5 g of recombinant type IV humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0027] Example 5 S1: Dissolve 5 g of recombinant type XVII humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0028] Example 6 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 enzyme activity of SOD 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, keep warm in a 37℃ water bath, and stir at a speed of 1100r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0029] Example 7 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 4 mg of superoxide dismutase to make the final enzyme activity of SOD 400 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0030] Example 8 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 10 mg of superoxide dismutase to make the final enzyme activity of SOD 1000 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0031] Example 9 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 0.865 g of EDC and 0.173 g of NHC to make the final concentration of EDC 45 mmol / L and the final concentration of NHC 15 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0032] Example 10 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.435 g of EDC and 0.287 g of NHC to make the final concentration of EDC 75 mmol / L and the final concentration of NHC 25 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0033] Embodiment 11 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 0.765 g of EDC and 0.460 g of NHC to make the final concentration of EDC 40 mmol / L and the final concentration of NHC 40 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0034] Example 12 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 30°C water bath, and stir at a speed of 1500 r / min for 10 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 8000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0035] Example 13 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 40°C water bath, and stir at 700 r / min for 30 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 12000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0036] Embodiment 14 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 8 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 8 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0037] Embodiment 15 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 12 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 12 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0038] Comparative Example 1 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to it so that the final enzyme activity of SOD is 500 U / mL, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes to obtain a water-like sample with high fluidity; S2: dilute the sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, and repeat the dilution and centrifugation 3 times; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, and collect the treated liquid in the bag.

[0039] Comparative Example 2 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 it, so that the final enzyme activity of SOD is 500U / mL, then add 1.15g of EDC and 0.23g of NHC, so that the final concentration of EDC is 60mmol / L, and the final concentration of NHC is 20mmol / L, keep warm in a 37℃ water bath, and stir at a speed of 1100r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0040] Comparative Example 3 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 it, so that the final enzyme activity of SOD is 500U / mL, then add 1.15g of EDC and 0.23g of NHC, so that the final concentration of EDC is 60mmol / L, and the final concentration of NHC is 20mmol / L, keep warm in a 37℃ water bath, and stir at a speed of 1100r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0041] Comparative Example 4 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 enzyme activity of SOD 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, keep warm in a 37℃ water bath, and stir at a speed of 1100r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0042] Comparative Example 5 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 16 mg of superoxide dismutase to make the final enzyme activity of SOD 1600 U / mL, then add 1.15 g of EDC and 0.23 g of NHC to make the final concentration of EDC 60 mmol / L and the final concentration of NHC 20 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0043] Comparative Example 6 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 0.575 g of EDC and 0.115 g of NHC to make the final concentration of EDC 30 mmol / L and the final concentration of NHC 10 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0044] Comparative Example 7 S1: Dissolve 5 g of recombinant type I humanized collagen in 100 mL of water to prepare a concentrated solution with a concentration of 50 mg / mL, add 5 mg of superoxide dismutase to make the final enzyme activity of SOD 500 U / mL, then add 1.725 g of EDC and 0.345 g of NHC to make the final concentration of EDC 90 mmol / L and the final concentration of NHC 30 mmol / L, keep warm in a 37°C water bath, and stir at a speed of 1100 r / min for 20 minutes for cross-linking to obtain a block sample with poor fluidity; S2: dilute the gel sample obtained in S1 with 10 times the volume of water, centrifuge at 10,000 rpm, repeat the dilution and centrifugation 3 times to remove the cross-linking agent as much as possible; S3: Add 10 times PBS buffer to the viscous liquid obtained after centrifugation in S2 to make a mixed solution, put it into a dialysis bag, dialyze for 60 hours, replace the dialysate every 12 hours, collect the treated cross-linked liquid in the bag, and obtain SOD-recombinant collagen complex, that is, collagen-modified superoxide dismutase.

[0045] Example 16 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 1, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0046] Embodiment 17 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 2, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0047] Embodiment 18 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 3, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0048] Embodiment 19 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 4, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0049] Embodiment 20 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 5, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0050] Embodiment 21 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked superoxide dismutase obtained in Example 6, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0051] Embodiment 22 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 7, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0052] Embodiment 23 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 8, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at -10°C for 25 h and maintained at a vacuum of 10 Pa, and kept at 30°C for 30 h and maintained at a vacuum of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0053] Embodiment 24 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 9, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0054] Embodiment 25 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 10, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0055] Embodiment 26 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 11, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0056] Embodiment 27 Add 0.5 g of trehalose and 0.5 g of mannitol to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 12, stir and mix, pour into a mold, put into a freeze dryer, freeze-dry at -30°C for 7 h, keep a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and keep a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid is placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0057] Embodiment 28 Add 0.5 g of trehalose and 0.5 g of mannitol to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 13, stir and mix, pour into a mold, put into a freeze dryer, freeze-dry at -30°C for 7 h, keep a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and keep a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. Place the freeze-dried solid under a pressing plate and squeeze it flat to obtain a repair patch for oral ulcers.

[0058] Embodiment 29 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 14, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0059] Embodiment 30 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Example 15, the mixture was stirred and evenly mixed, poured into a mold, and placed in a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0060] Embodiment 31 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Comparative Example 1, the mixture was stirred and evenly mixed, poured into a mold, and put into a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0061] Embodiment 32 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Comparative Example 2, the mixture was stirred and evenly mixed, poured into a mold, and put into a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0062] Embodiment 33 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Comparative Example 3, the mixture was stirred and evenly mixed, poured into a mold, and put into a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0063] Embodiment 34 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Comparative Example 4, the mixture was stirred and evenly mixed, poured into a mold, and put into a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0064] Embodiment 35 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Comparative Example 5, the mixture was stirred and evenly mixed, poured into a mold, and put into a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0065] Embodiment 36 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Comparative Example 6, the mixture was stirred and evenly mixed, poured into a mold, and put into a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0066] Embodiment 37 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked modified superoxide dismutase obtained in Comparative Example 7, the mixture was stirred and evenly mixed, poured into a mold, and put into a freeze dryer. The mixture was freeze-dried at -30°C for 7 h, kept at a constant temperature of -10°C for 25 h while maintaining a vacuum degree of 10 Pa, and kept at a constant temperature of 30°C for 30 h while maintaining a vacuum degree of 10 Pa to obtain a porous sponge-like recombinant collagen-SOD composite sample. The freeze-dried solid was placed under a pressing plate and squeezed flat to obtain a repair patch for oral ulcers.

[0067] Comparative Example 8 0.5 g of trehalose and 0.5 g of mannitol were added to 5 g of the collagen cross-linked superoxide dismutase obtained in Example 1, and the mixture was stirred to obtain a collagen-modified superoxide dismutase gel.

[0068] Comparative Example 9 Add 0.5 g of trehalose and 0.5 g of mannitol to 5 g of superoxide dismutase, stir to mix, pour into a mold, put into a freeze dryer, freeze-dry at -30 ° C for 7 hours, keep a constant temperature of -10 ° C for 25 hours and maintain a vacuum degree of 10 Pa, keep a constant temperature of 30 ° C for 30 hours and maintain a vacuum degree of 10 Pa, to obtain a porous sponge-like recombinant collagen-SOD composite sample, put the freeze-dried solid under a pressing plate and squeeze it flat to obtain a repair patch for oral ulcers.

[0069] Test Example 1: Cytotoxicity Test L929 cells (P6) in the logarithmic growth phase were plated at a cell density of 1×10 4 Each well was added with 100 μL, and 5 replicate wells were set for each group, and the cells were cultured for 24 h. After the sample solution and cells were co-cultured for 24 h, the culture medium was discarded, and 50 μL MTT (thiazolyl blue) solution (1 mg / mL) was added to each test well, and the plate was incubated in a 37 °C incubator for 2 h. The MTT solution was discarded, and 100 μL isopropanol solution was added to each well, and the plate was mixed for 10 min. The plate was placed on a well titer plate photometer equipped with a 570 nm filter to measure the absorbance (reference wavelength 650 nm), and the cell viability was calculated. The experimental results are shown in Table 1. Among them, the sample solution of the blank control group was MEM complete medium without any sample added; The sample solution of the experimental group was the SOD-recombinant collagen complex obtained in Example 1 (200 U / mL); The sample solution of the positive control group is a DMSO (dimethyl sulfoxide) solution with a mass fraction of 99.90%.

[0070] Table 1 Cytotoxicity test results The results show that the collagen-modified superoxide dismutase obtained by the present invention has no toxic side effects on human cells and can promote cell growth.

[0071] Test Example 2: Cell migration test After HaCaT (human immortalized keratinocytes) cells were subcultured for 2-3 generations, the cell suspension was centrifuged at 1000 rpm for 5 min. The cell suspension was resuspended in complete medium and the density was adjusted to (5-15) × 10 5 cells / well, 2 mL / well; after culturing the cells for 24 h, use a 200 μL pipette tip to gently push down the longitudinal (transverse) scratches to form scratches, rinse the cells 3 times with PBS to remove the scratched cells; add the sample solution to the scratch culture medium, culture in a 37 ℃, 5% CO2 incubator, and take pictures under a 40x microscope at 0 h, 24 h, and 48 h with the intersection of the transverse and longitudinal scratches as the core; use software to quantify and count the area of ​​the "scratch" area in the photo, calculate the cell migration rate based on the cell area, and evaluate the effect of the sample on cell migration. The experimental results are shown in Table 2; Among them, the blank group sample solution was serum-free basic DMEM medium without any sample added; The sample solution of the experimental group was 200 U / mL of the SOD-recombinant collagen complex obtained in Example 1.

[0072] Table 2 Cell migration test results The results show that the collagen-modified superoxide dismutase obtained in the present invention has a significant promoting effect on the migration of human immortalized keratinocytes, can promote the healing of human epidermal affected areas, and has a positive effect on the healing of oral ulcers.

[0073] Test Example 3: Enzyme activity detection test S1: Refer to GB / T41906 superoxide dismutase activity detection method. At about 25°C, add 2.35mL of 0.01mol / L tris(hydroxymethyl)aminomethane hydrochloric acid buffer solution, 2.00mL of distilled water, and 0.15mL of 4.5mmol / L pyrogallol hydrochloric acid solution in a 10mL colorimetric tube in sequence. Immediately after adding 4.5mmol / L pyrogallol hydrochloric acid solution, mix and pour into a colorimetric dish. Determine the absorbance at 0s and 1min under a wavelength of 325nm. The difference between the two is the pyrogallol self-oxidation rate △A 325 (min-1); This test determines △A 325 (min-1) is 0.060; S2: Determine the rate at which the sample inhibits the self-oxidation of pyrogallol: (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; (2) After adding the pyrogallol hydrochloric acid solution, mix immediately and pour into a cuvette; measure the absorbance at 0 s and 1 min at a wavelength of 325 nm; (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. Immediately after adding the pyrogallol hydrochloric acid solution, mix the mixture and pour it into a cuvette; measure the absorbance at 0 s and 1 min at a wavelength of 325 nm, respectively; (4) Repeat step (3) to obtain test solution n, until the difference in absorbance between 0s and 1min of test solution n at 325nm is △A' 325 (min-1) is 1 / 2△A 325 (min-1), that is, △A' 325 (min-1) was 0.030, and the enzyme activity was calculated. The results are shown in Table 3; Among them, the blank group sample solution was 2.00 mL of distilled water; The sample solution of control group 1 was 1.98 mL of distilled water and 20.0 μL of the mixture of SOD and recombinant collagen obtained in comparative example 1; The sample solution of control group 2 was 1.98 mL of distilled water and 20.0 μL of the mixture of SOD and recombinant collagen obtained in comparative example 1 after being stored at room temperature for 30 days; The sample solution of experimental group 1 was 1.98 mL of distilled water and 20.0 μL of the SOD-recombinant collagen complex obtained in Example 1; The sample solution of experimental group 2 was 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; The sample solution of experimental group 3 was 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; (3) Calculation of results: Where: U / mL—SOD enzyme activity unit; △A 325 -pyrogallol autoxidation rate; △A' 325 —The test solution inhibits the rate of pyrogallol auto-oxidation; V—volume of enzyme solution obtained from the comparative example or example added, in milliliters (mL); D—the dilution factor of the enzyme solution obtained in the comparative example or the example; 4.5—Total volume of reaction solution, in milliliters (mL); C—SOD enzyme concentration of test solution.

[0074] Table 3 Enzyme activity detection test results The results show that: when the complex of uncrosslinked SOD and recombinant type I humanized collagen is placed at room temperature for 1 month in a moist state, the enzyme activity decreases rapidly and is basically inactivated after 30 days; however, after crosslinking, the enzyme activity stability time of the collagen-modified superoxide dismutase obtained by the present invention is significantly prolonged. When placed in a moist state for 1 month, the enzyme activity decreases by about 5%, and when placed for 3 months, the enzyme activity decreases by about 20%, indicating that it has good stability and can maintain activity in the drug for a long time to exert its therapeutic effect.

[0075] Effect Example 1 (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 surface of the rabbit's oral mucosa. 0.2 mL of 30% glacial acetic acid was injected into the tube. After 30 seconds, a cotton swab was used to dip out the glacial acetic acid. An ulcer was formed the next day with a diameter of about 0.2 to 0.3 cm.

[0076] (2) Comparison of ulcer healing: After the ulcer was formed, the repair patch obtained in Examples 16 to 42, bFGF growth factor (control group), and no treatment measures (blank group) were used on the ulcer surface, and the ulcer healing area was observed. The patch was replaced and recorded every six hours, and the absence of mucosal ulcers and congestion was used as the healing standard.

[0077] Table 4 Ulcer healing conditions of the repair patches obtained in Examples 16 to 37 and Comparative Examples 8 to 9, bFGF growth factor, and no treatment measures Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; 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 in a water bath to make it cross-linked, and obtain a flowable gel sample; S2: dilute the gel sample obtained in S1 with water and centrifuge it to collect the viscous liquid at the bottom layer; S3: adding PBS buffer to the viscous liquid obtained after centrifugation in S2 to prepare a mixed solution, placing the mixed solution in a dialysis bag for dialysis, collecting the treated cross-linked liquid in the bag, and obtaining 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.

2. A collagen-modified superoxide dismutase according to claim 1, characterized in that: The concentration of the collagen solution in S1 is 50-100 mg / mL.

3. The collagen-modified superoxide dismutase according to claim 1, characterized in that: The final enzyme activity content of superoxide dismutase described in S1 is 400~1000U / mL.

4. The collagen-modified superoxide dismutase according to claim 1, characterized in that: S1: The molar ratio of EDC to NHS is 1-3:1; the total content of EDC and NHS is 60-100 mmol / L.

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

6. 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 centrifugal speed is 8000 to 12000 r / min.

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

8. A method for preparing the collagen-modified superoxide dismutase according to any one of claims 1 to 7.

9. Use of the collagen-modified superoxide dismutase according to any one of claims 1 to 7 in the preparation of an oral ulcer repair drug.

10. The use according to claim 9, characterized in that: The application method is as follows: trehalose and mannitol are added to the obtained collagen-modified superoxide dismutase, the mixture is stirred and poured into a mold for freeze-drying to obtain a sample, and the freeze-dried sample is squeezed flat to obtain a repair patch; the added mass of the trehalose is 5% to 10% of the mass of the collagen-modified superoxide dismutase; the added mass of the mannitol is 5% to 10% of the mass of the collagen-modified superoxide dismutase.

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