Composition for promoting healing of diabetic wound as well as preparation method and application of composition

Abstract

The invention belongs to the technical field of biology, and particularly relates to a composition for promoting healing of diabetic wounds and a preparation method and application thereof, and the composition utilizes the synergistic effect of bovine placenta extract and various components to remarkably promote healing of the diabetic wounds. All the components are biocompatible materials, so that no irritation or anaphylactic reaction is caused, and the safety is high. Through local smearing administration, noninvasive administration is realized, and systemic side effects of traditional drugs are avoided. In the preparation process, through the steps of enzymolysis, ultrafiltration, filtration sterilization and the like, the purity and activity are ensured, and the preparation is simple.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a composition for promoting diabetic wound healing, a preparation method thereof, and an application thereof. Background Art

[0002] The wound healing ability of diabetic patients is significantly reduced, often leading to chronic ulcers and infections, and even amputation in severe cases. Traditional treatment methods such as antibiotics, surgical debridement, and growth factor therapy have problems such as limited efficacy and large side effects. Bovine placenta extract is rich in various bioactive substances, such as growth factors, extracellular matrix components, and stem cells, and has the potential to promote tissue regeneration and repair. However, the extraction and preparation processes are complex and the cost is relatively high, which limits its wide application. Summary of the Invention

[0003] To solve the problems raised in the background art, the present invention provides a composition for promoting diabetic wound healing, a preparation method thereof, and an application thereof.

[0004] The technical solution of the present invention is as follows: The present invention provides a composition for promoting diabetic wound healing, and the composition includes: Bovine placenta extract, antimicrobial peptide, antioxidant, penetration enhancer; The mass ratios of bovine placenta extract to antimicrobial peptide, antioxidant, and penetration enhancer are 1:0.2 - 0.3, 1:0.2 - 0.3, and 1:0.1 - 0.2 respectively.

[0005] Further, the mass ratios of bovine placenta extract to antimicrobial peptide, antioxidant, and penetration enhancer are 1:0.25, 1:0.25, and 1:0.15 respectively.

[0006] The antioxidant is coenzyme Q10, and the penetration enhancer is azone.

[0007] The composition further includes a pH regulator.

[0008] The present invention also provides a preparation method of the composition for promoting diabetic wound healing, including: The bovine placenta tissue is enzymolyzed, centrifuged, and concentrated in sequence to obtain bovine placenta extract; Antimicrobial peptide and antioxidant are added to the bovine placenta extract in sequence, mixed, after adding the pH regulator, the penetration enhancer is added, and after mixing, it is sterilized.

[0009] The obtained bovine placenta extract is obtained by cutting the neonatal bovine placenta tissue into pieces, adding collagenase and hyaluronidase for enzymolysis; the enzymolyzed mixture is centrifuged, the supernatant is collected, and the supernatant is concentrated to obtain bovine placenta extract.

[0010] The pH regulator is to adjust the pH to 6.5 - 7.0 by using sodium citrate solution.

[0011] Furthermore, during the preparation process, the antioxidant is coenzyme Q10 and the penetration enhancer is azone.

[0012] The present invention also provides an application of the composition for promoting diabetic wound healing in the preparation of a product for improving the diabetic wound healing rate.

[0013] Beneficial effects: The composition for promoting diabetic wound healing of the present invention utilizes the synergistic effect of bovine placental extract and various components to significantly promote the healing of diabetic wounds. And all components are biocompatible materials, without irritation or allergic reactions, and have high safety. By administering through local application, non-invasive administration is achieved, avoiding the systemic side effects of traditional drugs. During the preparation process, through steps such as enzymatic hydrolysis, ultrafiltration and filtration sterilization, the purity and activity are ensured, and the preparation is simple. Description of the Drawings

[0014] Figure 1 It is a comparison of wound healing photos of different groups of diabetic mouse models in Experiment 1 of animal experiments.

[0015] Figure 2 It is a comparison of wound healing rates of different groups of diabetic mouse models in Experiment 1 of animal experiments.

[0016] Figure 3 It is a comparison of wound healing photos of different groups of diabetic rat models in Experiment 2 of animal experiments.

[0017] Figure 4 It is a comparison of wound healing rates of different groups of diabetic rat models in Experiment 2 of animal experiments. Detailed Embodiments

[0018] The following examples are intended to illustrate the present invention, rather than further limiting the present invention.

[0019] The present invention provides a composition for promoting diabetic wound healing, and the composition includes: bovine placental extract, antimicrobial peptide, antioxidant, penetration enhancer; The mass ratios of bovine placental extract to antimicrobial peptide, antioxidant, and penetration enhancer are 1:0.2 - 0.3, 1:0.2 - 0.3, and 1:0.1 - 0.2 respectively.

[0020] Preferably, the mass ratios of bovine placental extract to antimicrobial peptide, antioxidant, and penetration enhancer are 1:0.25, 1:0.25, and 1:0.15 respectively.

[0021] Among them, the antioxidant is coenzyme Q10 and the penetration enhancer is azone.

[0022] In addition, the composition further comprises a pH regulator.

[0023] The present invention also provides a method for preparing the composition for promoting diabetic wound healing, comprising: The bovine placenta tissue is successively subjected to enzymatic hydrolysis, centrifugation, and concentration to obtain a bovine placenta extract; In deionized water, the bovine placenta extract, antimicrobial peptide, and antioxidant are successively added and mixed. After adding the pH regulator, a penetration enhancer is added, and the mixture is filtered and sterilized after mixing.

[0024] Specifically, the obtaining of the bovine placenta extract is to cut the neonatal bovine placenta tissue into pieces, add collagenase and hyaluronidase, and carry out enzymatic hydrolysis; the hydrolyzed mixture is centrifuged, the supernatant is collected, and the supernatant is concentrated to obtain the bovine placenta extract.

[0025] Among them, the pH regulator is to adjust the pH to 6.5 - 7.0 using a sodium citrate solution.

[0026] During the preparation process, the antioxidant is coenzyme Q10, and the penetration enhancer is azone.

[0027] The present invention also provides an application of the composition for promoting diabetic wound healing in the preparation of a product for improving the diabetic wound healing rate.

[0028] The composition for promoting diabetic wound healing of the present application utilizes the synergistic effect of the bovine placenta extract and various components to significantly promote the healing of diabetic wounds. And all components are biocompatible materials, without irritation or allergic reactions, and have high safety. By administering through local application, non-invasive drug delivery is achieved, avoiding the systemic side effects of traditional drugs. During the preparation process, through steps such as enzymatic hydrolysis, ultrafiltration, and filtration sterilization, the purity and activity are ensured, and the preparation is simple.

[0029] Example 1 (1) Preparation of bovine placenta extract (11) Collect bovine placenta tissue from healthy neonatal bovine placentas, remove blood vessels and amniotic membranes, and rinse thoroughly with physiological saline.

[0030] (12) Enzymatic hydrolysis treatment: Cut the bovine placenta tissue into pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37°C for 2 hours.

[0031] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0032] (14) Ultrafiltration concentration: Use a 10 kDa ultrafiltration membrane to concentrate the supernatant to obtain a bovine placenta extract.

[0033] (2) Preparation of the composition (21) Add bovine placental extract, antimicrobial peptide, and antioxidant coenzyme Q10 to deionized water in sequence, and stir evenly until completely dissolved.

[0034] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0035] (23) Add azone and stir evenly.

[0036] (24) Filter the mixture through a 0.22 μm filter membrane to sterilize it, obtain the composition, and dispense it into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antimicrobial peptide, antioxidant coenzyme Q10, and permeation enhancer azone are 1:0.25, 1:0.25, and 1:0.15 respectively.

[0037] Example 2 Compared with Example 1, the difference lies in the change in the mass ratio of the antimicrobial peptide in the composition, which is as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse it thoroughly with physiological saline.

[0038] (12) Enzymatic hydrolysis treatment: Cut the bovine placental tissue into pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0039] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0040] (14) Ultrafiltration concentration: Concentrate the supernatant with a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0041] (2) Preparation of the composition (21) Add bovine placental extract, antimicrobial peptide, and antioxidant coenzyme Q10 to deionized water in sequence, and stir evenly until completely dissolved.

[0042] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0043] (23) Add azone and stir evenly.

[0044] (24) Filter the mixture through a 0.22 μm filter membrane to sterilize it, obtain the composition, and dispense it into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antimicrobial peptide, antioxidant coenzyme Q10, and permeation enhancer azone are 1:0.2, 1:0.25, and 1:0.15 respectively.

[0045] Example 3 Compared with Example 1, the difference lies in the change in the mass ratio of the antimicrobial peptide in the composition, specifically as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0046] (12) Enzymatic digestion treatment: Cut the bovine placental tissue into small pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37°C for 2 hours.

[0047] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0048] (14) Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0049] (2) Preparation of the composition (21) Add bovine placental extract, antimicrobial peptide and antioxidant coenzyme Q10 to deionized water in sequence, and stir evenly until completely dissolved.

[0050] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0051] (23) Add azone and stir evenly.

[0052] (24) Filter the mixture through a 0.22 μm filter membrane to sterilize, obtain the composition and dispense it into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antimicrobial peptide, antioxidant coenzyme Q10, and penetration enhancer azone are 1:0.3, 1:0.25, and 1:0.15 respectively.

[0053] Example 4 Compared with Example 1, the difference lies in the change in the mass ratio of the antioxidant coenzyme Q10 in the composition, specifically as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0054] (12) Enzymatic digestion treatment: Cut the bovine placental tissue into small pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37°C for 2 hours.

[0055] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0056] (14) Ultrafiltration concentration: The supernatant was concentrated using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0057] (2) Preparation of the composition (21) In deionized water, bovine placental extract, antimicrobial peptide, and antioxidant coenzyme Q10 were added in sequence and stirred evenly until completely dissolved.

[0058] (22) The pH of the solution was adjusted to 6.5 - 7.0 with sodium citrate.

[0059] (23) Azone was added and stirred evenly.

[0060] (24) The mixture was filtered through a 0.22 μm filter membrane to sterilize, and the obtained composition was dispensed into a sterile container. The concentration of bovine placental extract in the composition was 10 mg / ml. Among them, the mass ratios of bovine placental extract to antimicrobial peptide, antioxidant coenzyme Q10, and penetration enhancer azone were 1:0.25, 1:0.2, and 1:0.15 respectively.

[0061] Example 5 Compared with Example 1, the difference lies in the change in the mass ratio of antioxidant coenzyme Q10 in the composition, specifically as follows: (1) Preparation of bovine placental extract (11) Bovine placental tissue was collected from healthy newborn bovine placentas, blood vessels and amniotic membranes were removed, and it was rinsed clean with physiological saline.

[0062] (12) Enzymatic hydrolysis treatment: The bovine placental tissue was minced, and 0.1% collagenase and 0.05% hyaluronidase were added by mass percentage, and digested at 37°C for 2 hours.

[0063] (13) Centrifugal separation: The digested mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected.

[0064] (14) Ultrafiltration concentration: The supernatant was concentrated using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0065] (2) Preparation of the composition (21) In deionized water, bovine placental extract, antimicrobial peptide, and antioxidant coenzyme Q10 were added in sequence and stirred evenly until completely dissolved.

[0066] (22) The pH of the solution was adjusted to 6.5 - 7.0 with sodium citrate.

[0067] (23) Azone was added and stirred evenly.

[0068] (24) Filter and sterilize the mixture through a 0.22 μm filter membrane, and obtain the composition which is then aliquoted into sterile containers. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antibacterial peptide, antioxidant coenzyme Q10, and penetration enhancer azone are 1:0.25, 1:0.3, and 1:0.15, respectively.

[0069] Example 6 Compared with Example 1, the difference lies in the change in the mass ratio of the penetration enhancer azone in the composition, which is as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0070] (12) Enzymatic digestion: Cut the bovine placental tissue into pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0071] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0072] (14) Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0073] (2) Preparation of the composition (21) Add bovine placental extract, antibacterial peptide, and antioxidant coenzyme Q10 to deionized water in sequence, and stir evenly until completely dissolved.

[0074] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0075] (23) Add azone and stir evenly.

[0076] (24) Filter and sterilize the mixture through a 0.22 μm filter membrane, and obtain the composition which is then aliquoted into sterile containers. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antibacterial peptide, antioxidant coenzyme Q10, and penetration enhancer azone are 1:0.25, 1:0.25, and 1:0.1, respectively.

[0077] Example 7 Compared with Example 1, the difference lies in the change in the mass ratio of the penetration enhancer azone in the composition, which is as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0078] (12)Enzymatic hydrolysis treatment: The bovine placenta tissue was minced and, by mass percentage, 0.1% collagenase and 0.05% hyaluronidase were added, followed by digestion at 37°C for 2 hours.

[0079] (13)Centrifugal separation: The digested mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected.

[0080] (14)Ultrafiltration concentration: The supernatant was concentrated using a 10 kDa ultrafiltration membrane to obtain bovine placenta extract.

[0081] (2)Preparation of the composition (21)In deionized water, bovine placenta extract, antimicrobial peptide, and antioxidant coenzyme Q10 were added sequentially and stirred evenly until completely dissolved.

[0082] (22)The pH of the solution was adjusted to 6.5 - 7.0 with sodium citrate.

[0083] (23)Azone was added and stirred evenly.

[0084] (24)The mixture was filtered through a 0.22 μm filter membrane to sterilize, and the obtained composition was dispensed into a sterile container. The concentration of bovine placenta extract in the composition was 10 mg / ml. Among them, the mass ratios of bovine placenta extract to antimicrobial peptide, antioxidant coenzyme Q10, and penetration enhancer azone were 1:0.25, 1:0.25, and 1:0.2, respectively.

[0085] Comparative Example 1 Compared with Example 1, the difference was that the antimicrobial peptide was not added, and the remaining operations and component addition amounts were the same as in Example 1. Specifically as follows: (1)Preparation of bovine placenta extract (11)Bovine placenta tissue was collected from healthy newborn bovine placentas, blood vessels and amnion were removed, and it was rinsed clean with physiological saline.

[0086] (12)Enzymatic hydrolysis treatment: The bovine placenta tissue was minced and, by mass percentage, 0.1% collagenase and 0.05% hyaluronidase were added, followed by digestion at 37°C for 2 hours.

[0087] (13)Centrifugal separation: The digested mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected.

[0088] (14)Ultrafiltration concentration: The supernatant was concentrated using a 10 kDa ultrafiltration membrane to obtain bovine placenta extract.

[0089] (2)Preparation of the composition (21)In deionized water, bovine placenta extract and antioxidant coenzyme Q10 were added sequentially and stirred evenly until completely dissolved.

[0090] (22)Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0091] (23)Add azone and stir evenly.

[0092] (24)Filter and sterilize the mixture through a 0.22 μm filter membrane, obtain the composition and dispense it into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antioxidant coenzyme Q10 and penetration enhancer azone are 1:0.25 and 1:0.15 respectively.

[0093] Comparative Example 2 Compared with Example 1, the difference is that antioxidant coenzyme Q10 is not added, and the rest of the operations and component addition amounts are the same as those in Example 1. Specifically as follows: (1)Preparation of bovine placental extract (11)Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse it clean with physiological saline.

[0094] (12)Enzymatic hydrolysis treatment: Cut the bovine placental tissue into pieces, add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0095] (13)Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes and collect the supernatant.

[0096] (14)Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0097] (2)Preparation of the composition (21)Sequentially add bovine placental extract and antimicrobial peptide to deionized water, and stir evenly until completely dissolved.

[0098] (22)Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0099] (23)Add azone and stir evenly.

[0100] (24)Filter and sterilize the mixture through a 0.22 μm filter membrane, obtain the composition and dispense it into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antimicrobial peptide and penetration enhancer azone are 1:0.25 and 1:0.15 respectively.

[0101] Comparative Example 3 Compared with Example 1, the difference is that penetration enhancer azone is not added, and the rest of the operations and component addition amounts are the same as those in Example 1. Specifically as follows: (1)Preparation of bovine placental extract (11)Collect bovine placenta tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0102] (12)Enzymatic digestion treatment: Cut the bovine placenta tissue into small pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0103] (13)Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0104] (14)Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placenta extract.

[0105] (2)Preparation of the composition (21)In deionized water, sequentially add bovine placenta extract, antimicrobial peptide, and antioxidant coenzyme Q10, and stir evenly until completely dissolved.

[0106] (22)Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0107] (23)Filter and sterilize the mixture through a 0.22 μm filter membrane, and dispense the obtained composition into a sterile container. The concentration of bovine placenta extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placenta extract to antimicrobial peptide and antioxidant coenzyme Q10 are 1:0.25 and 1:0.25 respectively.

[0108] Comparative Example 4 Compared with Example 1, the difference is that antimicrobial peptide and antioxidant coenzyme Q10 are not added, and the remaining operations and ingredient addition amounts are the same as those in Example 1. Specifically as follows: (1)Preparation of bovine placenta extract (11)Collect bovine placenta tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0109] (12)Enzymatic digestion treatment: Cut the bovine placenta tissue into small pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0110] (13)Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0111] (14)Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placenta extract.

[0112] (2)Preparation of the composition (21)In deionized water, add bovine placenta extract, and stir evenly until completely dissolved.

[0113] (22)Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0114] (23)Add azone and stir evenly.

[0115] (24)Filter and sterilize the mixture through a 0.22 μm filter membrane, and obtain the composition which is dispensed into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratio of bovine placental extract to the penetration enhancer azone is 1:0.15.

[0116] Comparative Example 5 Compared with Example 1, the difference is that the antibacterial peptide and the penetration enhancer azone are not added, and the remaining operations and component addition amounts are the same as those in Example 1. Specifically as follows: (1)Preparation of bovine placental extract (11)Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0117] (12)Enzymatic hydrolysis treatment: Cut the bovine placental tissue into pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0118] (13)Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0119] (14)Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0120] (2)Preparation of the composition (21)In deionized water, sequentially add bovine placental extract and the antioxidant coenzyme Q10, and stir evenly until completely dissolved.

[0121] (22)Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0122] (23)Filter and sterilize the mixture through a 0.22 μm filter membrane, and obtain the composition which is dispensed into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratio of bovine placental extract to the antioxidant coenzyme Q10 is 1:0.25.

[0123] Comparative Example 6 Compared with Example 1, the difference is that the antioxidant coenzyme Q10 and the penetration enhancer azone are not added, and the remaining operations and component addition amounts are the same as those in Example 1. Specifically as follows: (1)Preparation of bovine placental extract (11)Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0124] (12)Enzymatic hydrolysis treatment: The bovine placenta tissue was minced and, by mass percentage, 0.1% collagenase and 0.05% hyaluronidase were added, and digestion was carried out at 37 °C for 2 hours.

[0125] (13)Centrifugal separation: The digested mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected.

[0126] (14)Ultrafiltration concentration: The supernatant was concentrated using a 10 kDa ultrafiltration membrane to obtain bovine placenta extract.

[0127] (2)Preparation of the composition (21)In deionized water, bovine placenta extract and antimicrobial peptide were successively added and stirred evenly until completely dissolved.

[0128] (22)The pH of the solution was adjusted to 6.5 - 7.0 with sodium citrate.

[0129] (23)The mixed solution was filtered through a 0.22 μm filter membrane to sterilize, and the obtained composition was dispensed into a sterile container. The concentration of bovine placenta extract in the composition was 10 mg / ml. Among them, the mass ratio of bovine placenta extract to antimicrobial peptide was 1:0.25.

[0130] Comparative Example 7 Compared with Example 1, the difference was that antimicrobial peptide, antioxidant coenzyme Q10, and penetration enhancer azone were not added, and the remaining operations and component addition amounts were the same as those in Example 1. Specifically as follows: (1)Preparation of bovine placenta extract (11)Bovine placenta tissue was collected from healthy newborn bovine placentas, blood vessels and amnion were removed, and it was rinsed clean with physiological saline.

[0131] (12)Enzymatic hydrolysis treatment: The bovine placenta tissue was minced and, by mass percentage, 0.1% collagenase and 0.05% hyaluronidase were added, and digestion was carried out at 37 °C for 2 hours.

[0132] (13)Centrifugal separation: The digested mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected.

[0133] (14)Ultrafiltration concentration: The supernatant was concentrated using a 10 kDa ultrafiltration membrane to obtain bovine placenta extract.

[0134] (2)Preparation of the composition (21)In deionized water, bovine placenta extract was added and stirred evenly until completely dissolved.

[0135] (22)The pH of the solution was adjusted to 6.5 - 7.0 with sodium citrate.

[0136] (23) Filter and sterilize the mixed solution through a 0.22 μm filter membrane, obtain the composition and dispense it into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml.

[0137] Comparative Example 8 Compared with Example 1, the difference is that the proportion of the antimicrobial peptide in the composition is down-regulated, specifically as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse it thoroughly with physiological saline.

[0138] (12) Enzymatic digestion treatment: Cut the bovine placental tissue into pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0139] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0140] (14) Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0141] (2) Preparation of the composition (21) Add bovine placental extract, antimicrobial peptide and antioxidant coenzyme Q10 to deionized water in sequence, and stir evenly until completely dissolved.

[0142] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0143] (23) Add azone and stir evenly.

[0144] (24) Filter and sterilize the mixed solution through a 0.22 μm filter membrane, obtain the composition and dispense it into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antimicrobial peptide, antioxidant coenzyme Q10, and penetration enhancer azone are 1:0.1, 1:0.25, and 1:0.15 respectively.

[0145] Comparative Example 9 Compared with Example 1, the difference is that the proportion of the antimicrobial peptide in the composition is up-regulated, specifically as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse it thoroughly with physiological saline.

[0146] (12) Enzymatic digestion treatment: Cut the bovine placental tissue into pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0147]

[0147] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes and collect the supernatant.

[0148]

[0148] (14) Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0149]

[0149] (2) Preparation of the composition (21) Add bovine placental extract, antimicrobial peptide, and antioxidant coenzyme Q10 to deionized water in sequence, and stir evenly until completely dissolved.

[0150]

[0150] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0151]

[0151] (23) Add azone and stir evenly.

[0152]

[0152] (24) Filter the mixture through a 0.22 μm filter membrane to sterilize, and dispense the obtained composition into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antimicrobial peptide, antioxidant coenzyme Q10, and penetration enhancer azone are 1:0.4, 1:0.25, and 1:0.15 respectively.

[0153] Comparative Example 10 Compared with Example 1, the difference is that the proportion of antioxidant coenzyme Q10 in the composition is decreased, specifically as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amnion, and rinse thoroughly with physiological saline.

[0154]

[0154] (12) Enzymatic digestion treatment: Cut the bovine placental tissue into pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0155]

[0155] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes and collect the supernatant.

[0156]

[0156] (14) Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0157]

[0157] (2) Preparation of the composition (21) Add bovine placental extract, antimicrobial peptide, and antioxidant coenzyme Q10 to deionized water in sequence, and stir evenly until completely dissolved.

[0158]

[0158] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0159]

[0159] (23) Add azone and stir evenly.

[0160] (24) Filter the mixture through a 0.22 μm filter membrane to sterilize it, and obtain the composition which is dispensed into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antibacterial peptide, antioxidant coenzyme Q10, and penetration enhancer azone are 1:0.25, 1:0.1, and 1:0.15 respectively.

[0161] Comparative Example 11 Compared with Example 1, the difference is that the proportion of antioxidant coenzyme Q10 in the composition is increased, specifically as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amniotic membranes, and rinse thoroughly with physiological saline.

[0162] (12) Enzymatic digestion: Cut the bovine placental tissue into pieces, and add 0.1% collagenase and 0.05% hyaluronidase by mass percentage, and digest at 37 °C for 2 hours.

[0163] (13) Centrifugal separation: Centrifuge the digested mixture at 3000 rpm for 10 minutes, and collect the supernatant.

[0164] (14) Ultrafiltration concentration: Concentrate the supernatant using a 10 kDa ultrafiltration membrane to obtain bovine placental extract.

[0165] (2) Preparation of the composition (21) Add bovine placental extract, antibacterial peptide, and antioxidant coenzyme Q10 to deionized water in sequence, and stir evenly until completely dissolved.

[0166] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0167] (23) Add azone and stir evenly.

[0168] (24) Filter the mixture through a 0.22 μm filter membrane to sterilize it, and obtain the composition which is dispensed into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antibacterial peptide, antioxidant coenzyme Q10, and penetration enhancer azone are 1:0.25, 1:0.4, and 1:0.15 respectively.

[0169] Comparative Example 12 Compared with Example 1, the difference is that the proportion of penetration enhancer azone in the composition is decreased, specifically as follows: (1) Preparation of bovine placental extract (11) Collect bovine placental tissue from healthy newborn bovine placentas, remove blood vessels and amniotic membranes, and rinse thoroughly with physiological saline.

[0170] (12)Enzymatic hydrolysis treatment: The bovine placenta tissue was minced and, by mass percentage, 0.1% collagenase and 0.05% hyaluronidase were added, and digestion was carried out at 37 °C for 2 hours.

[0171] (13)Centrifugal separation: The digested mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected.

[0172] (14)Ultrafiltration concentration: The supernatant was concentrated using a 10 kDa ultrafiltration membrane to obtain bovine placenta extract.

[0173] (2)Preparation of the composition (21)In deionized water, bovine placenta extract, antimicrobial peptide, and antioxidant coenzyme Q10 were sequentially added and stirred evenly until completely dissolved.

[0174] (22)The pH of the solution was adjusted to 6.5 - 7.0 with sodium citrate.

[0175] (23)Azone was added and stirred evenly.

[0176] (24)The mixture was filtered through a 0.22 μm filter membrane to sterilize, and the obtained composition was dispensed into a sterile container. The concentration of bovine placenta extract in the composition was 10 mg / ml. Among them, the mass ratios of bovine placenta extract to antimicrobial peptide, antioxidant coenzyme Q10, and permeation enhancer azone were 1:0.25, 1:0.25, and 1:0.05, respectively.

[0177] Comparative Example 13 Compared with Example 1, the difference was that the proportion of permeation enhancer azone in the composition was increased, specifically as follows: (1)Preparation of bovine placenta extract (11)Bovine placenta tissue was collected from healthy newborn bovine placentas, blood vessels and amniotic membranes were removed, and it was rinsed clean with physiological saline.

[0178] (12)Enzymatic hydrolysis treatment: The bovine placenta tissue was minced and, by mass percentage, 0.1% collagenase and 0.05% hyaluronidase were added, and digestion was carried out at 37 °C for 2 hours.

[0179] (13)Centrifugal separation: The digested mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected.

[0180] (14)Ultrafiltration concentration: The supernatant was concentrated using a 10 kDa ultrafiltration membrane to obtain bovine placenta extract.

[0181] (2)Preparation of the composition (21)In deionized water, bovine placenta extract, antimicrobial peptide, and antioxidant coenzyme Q10 were sequentially added and stirred evenly until completely dissolved.

[0182] (22) Adjust the pH of the solution to 6.5 - 7.0 with sodium citrate.

[0183] (23) Add azone and stir evenly.

[0184] (24) Filter the mixture through a 0.22 μm filter membrane to sterilize it, and obtain the composition which is dispensed into a sterile container. The concentration of bovine placental extract in the composition is 10 mg / ml. Among them, the mass ratios of bovine placental extract to antimicrobial peptide, antioxidant coenzyme Q10, and penetration enhancer azone are 1:0.25, 1:0.25, and 1:0.3 respectively.

[0185] Experimental effect verification I. Cell experiment Based on HUVEC (human umbilical vein endothelial cells), a diabetic wound cell model is constructed to comprehensively simulate the pathological characteristics of diabetic microangiopathy, as follows: 1. Cells and reagents Cell line: HUVEC (human umbilical vein endothelial cells, passages within 4 generations are used to maintain functional stability).

[0186] Culture medium: Special medium for endothelial cells (EGM - 2, containing 2% FBS and growth factors).

[0187] Inducers: Glucose, advanced glycation end products (AGEs).

[0188] 2. Construction of the diabetic wound model, combined treatment with high glucose and AGEs, cell seeding. Seed HUVEC in a 6 - well plate (density 5×10 4 cells / well), and culture at 37°C and 5% CO 2 until 70% confluence.

[0189] Use high glucose + AGEs to treat and induce a diabetic cell injury model. For the model group, change the medium to the one containing 30 mM glucose + 200 μg / mL AGEs. For the experimental treatment group, change the medium to the one containing 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition. The treatment time is 48 hours for both groups. Among them, 200 μg / ml of the composition is obtained by diluting the compositions of Examples 1 - 7 and Comparative Examples 1 - 13 (the concentration of bovine placental extract in the composition is 10 mg / ml) with cell culture medium to a working concentration of 200 μg / ml of bovine placental extract in the composition, that is, in the cell culture system, take the compositions of Examples 1 - 7 and Comparative Examples 1 - 13 and add them to the medium respectively to make their working concentration 200 μg / ml.

[0190] The specific grouping of the model group and the experimental treatment group is as follows: Model group: Medium with 30 mM glucose + 200 μg / mL AGEs.

[0191] Experimental treatment group: Example 1: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Example 1.

[0192] Example 2: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Example 2.

[0193] Example 3: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Example 3.

[0194] Example 4: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Example 4.

[0195] Example 5: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Example 5.

[0196] Example 6: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Example 6.

[0197] Example 7: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Example 7.

[0198] Comparative Example 1: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Comparative Example 1.

[0199] Comparative Example 2: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Comparative Example 2.

[0200] Comparative Example 3: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Comparative Example 3.

[0201] Comparative Example 4: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Comparative Example 4.

[0202] Comparative Example 5: Medium with 30 mM glucose + 200 μg / mL AGEs + 200 μg / ml of the composition of Comparative Example 5.

[0203] Comparative Example 6: Medium of 30 mM glucose + 200 μg / mL AGEs + 200 μg / mL of the composition of Comparative Example 6.

[0204] Comparative Example 7: Medium of 30 mM glucose + 200 μg / mL AGEs + 200 μg / mL of the composition of Comparative Example 7.

[0205] Comparative Example 8: Medium of 30 mM glucose + 200 μg / mL AGEs + 200 μg / mL of the composition of Comparative Example 8.

[0206] Comparative Example 9: Medium of 30 mM glucose + 200 μg / mL AGEs + 200 μg / mL of the composition of Comparative Example 9.

[0207] Comparative Example 10: Medium of 30 mM glucose + 200 μg / mL AGEs + 200 μg / mL of the composition of Comparative Example 10.

[0208] Comparative Example 11: Medium of 30 mM glucose + 200 μg / mL AGEs + 200 μg / mL of the composition of Comparative Example 11.

[0209] Comparative Example 12: Medium of 30 mM glucose + 200 μg / mL AGEs + 200 μg / mL of the composition of Comparative Example 12.

[0210] Comparative Example 13: Medium of 30 mM glucose + 200 μg / mL AGEs + 200 μg / mL of the composition of Comparative Example 13.

[0211] 3. Cell viability detection by CCK-8 assay. After the treatment, CCK-8 reagent was added and incubated for 2 hours, and the OD value was measured at 450 nm to compare the effects of different compositions on cell viability. See Table 1.

[0212] Table 1 Absorbance values of different groups

[0213] As can be seen from Table 1, compared with the model group and Comparative Example 1-13 groups, the absorbance (OD value) at 450 nm of the compositions of Examples 1-7 of the present application was significantly increased, indicating that the compositions of the present invention can significantly increase the cell survival level and cell viability (P < 0.01).

[0214] 4. The supernatant of each group was collected to detect the concentration of IL-6, and ELISA for the secretion of inflammatory factors was performed to evaluate the intensity of the inflammatory response. See Table 2 for details.

[0215] Table 2 IL-6 expression levels of different groups

[0216] As can be seen from Table 2, compared with the model group and Comparative Example Groups 1-13, the compositions of Examples 1-7 of the present application can significantly reduce the level of the cellular inflammatory factor IL-6 in the diabetic cell model and significantly improve the inflammatory response of diabetic wounds (P<0.01).

[0217] II. Animal experiments The healing effect of the composition of the present application on diabetic wounds was verified through animal experiments on diabetic mice and diabetic rats, as follows: Experiment 1 In the diabetic mouse model, the wound healing rate of the wounds treated with the composition of the present application was significantly faster than that of the control group. The details of the two animal experiments are as follows.

[0218] 1. Experimental materials Animal strain: Ten healthy male C57BL / 6 mice, 6-8 weeks old, weighing 18-22 g. They were adaptively fed for one week with free access to food and water.

[0219] Main reagents: High-fat feed (with fat content above 45%), streptozotocin (STZ), sodium citrate buffer (0.1 mol / L, pH 4.5), blood glucose meter and test strips.

[0220] Main instruments: Electronic balance, centrifuge, blood glucose meter, syringe.

[0221] 2. Model construction Inducing insulin resistance with a high-fat diet: The mice were fed with a high-fat diet for 8-12 weeks to induce insulin resistance.

[0222] Establishing a diabetes model: After the high-fat diet feeding ended, the mice were fasted for 6 hours and then intraperitoneally injected with STZ solution at a dose of 50-100 mg / kg / day for 1-3 consecutive days. Pay attention to the operation specifications when injecting STZ to avoid damaging the internal organs of the mice. The STZ solution was prepared by dissolving STZ with pre-cooled sodium citrate buffer to form a 1% STZ solution, which was prepared and used immediately to avoid repeated freezing and thawing. After injection, the high-fat diet feeding continued.

[0223] Model verification: 72 hours after the last injection of STZ, blood was collected from the tail vein, and the fasting blood glucose (FBG) was measured using a blood glucose meter. Mice with FBG≥11.1 mmol / L were considered to have successfully established a diabetes model.

[0224] 3. Preparation of a diabetic skin wound model Two weeks after successful establishment of the diabetes model, blood glucose was measured weekly to monitor blood glucose levels, and a local full-thickness skin excision wound model was made on the back of the mice. The mice with diabetes were fasted for 12 h before model establishment. During model establishment, they were anesthetized by intraperitoneal injection of sodium pentobarbital solution (1%, 50 mg / kg, I.P.). First, the hair was shaved on the lumbosacral region with a razor, and the skin was disinfected with 75% alcohol cotton balls. A full-thickness skin excision wound with a diameter of 6 mm was made in the surgical area down to the subcutaneous tissue to create a full-thickness skin excision wound model. After the operation, the wound surface stopped bleeding naturally and was not bandaged.

[0225] 4. Experiment on promoting wound healing of diabetic mice by the composition: The mice were randomly divided into two groups: Control group and composition group, with 5 mice in each group. Among them, the wound surface of the Control group was smeared with sterile normal saline; in the composition group, under sterile conditions, the composition of Example 1 was dripped onto the wound surface at a dose of 200 μl / mouse, and the concentration of bovine placental extract in the composition was 1 mg / ml (the composition of Example 1 was diluted with sterile normal saline to a concentration of bovine placental extract in the composition of 1 mg / ml) and smeared evenly. All the mice were placed in a clean cage, numbered and fed individually, given clean drinking water and allowed to eat freely, and the wounds were kept clean to prevent wound infection. The next day, under sterile conditions, the composition of Example 1 was aspirated with a pipette and dripped onto the wound surface and smeared evenly. The composition of the present application was evenly smeared on the surface of the diabetic wound once a day. On the experimental day, i.e., day 0, 3, 6, 9, 12, and 15, the wounds of the mice were photographed ( Figure 1 ). ImageJ was used to calculate the wound area of the mice. The wound healing rate = (initial area - measured area on the observation day) ÷ initial area × 100%, and statistical analysis was performed on the wound healing rate. The average value of the wound healing rates of 5 mice in each group was taken as the wound healing rate value of the group ( Figure 2 ). After the wound healed, the wound scar was photographed to explore the effect of the composition of the present application on wound repair. The results showed that compared with the Control group, the composition of the present application had an obvious promoting effect on the healing of diabetic wounds and could increase the wound healing speed of diabetic mice, indicating that the composition of the present application is a very promising biological preparation for promoting diabetic wound healing.

[0226] All of the above experimental operations need to comply with the ethical norms of animal experiments.

[0227] Experiment Two In the diabetic rat model, the wound healing speed of the wounds treated with the composition of the present application was significantly faster than that of the control group. The specific details of the two groups of animal experiments are as follows.

[0228] 1. Experimental materials Experimental animals: Ten healthy male SD rats (full name: Sprague-Dawley rats), 6-8 weeks old, weighing 220-300 g. They were adaptively fed for one week with free access to food and water.

[0229] Main reagents: Streptozotocin (STZ), sodium citrate buffer (0.1 mol / L, pH 4.5), blood glucose meter and test strips Main instruments: Electronic balance, centrifuge, blood glucose meter, syringe 2. Model construction Model establishment: After the rats were fasted for 6 hours, they were intraperitoneally injected with STZ solution at a dose of 50 mg / kg / day for 5 consecutive days. The STZ solution was prepared by dissolving STZ in pre-cooled sodium citrate buffer to form a 1% STZ solution, which was used immediately after preparation. After injection, they had free access to food and water.

[0230] Model verification: 72 hours after the last injection of STZ, blood was collected from the tail vein, and fasting blood glucose (FBG) was measured using a blood glucose meter. Rats with FBG ≥ 11.1 mmol / L were considered to have successfully established a diabetes model.

[0231] 3. Preparation of diabetic skin wound model Two weeks after the successful establishment of the diabetes model, blood glucose was measured weekly to monitor the blood glucose level. A local full-thickness skin excision wound model was made on the back of the rats. The rats were fasted 12 hours before diabetes modeling. During modeling, they were anesthetized by intraperitoneal injection of pentobarbital sodium solution (1%, 50 mg / kg, I.P). First, the hair on the lumbosacral region was shaved with a razor, and the skin was disinfected with 75% alcohol cotton balls. A full-thickness skin excision wound with a diameter of 6 mm was established in the surgical area, reaching deep to the subcutaneous tissue to create a full-thickness skin excision wound model. After the operation, the wound surface stopped bleeding naturally and was not bandaged.

[0232] 4. Experiment on the promotion of wound healing in diabetic rats by the composition The rats that had completed the preparation of the diabetic skin wound model were randomly divided into two groups: the Control group and the composition group, with 5 rats in each group. Among them, the wound surface of the Control group was smeared with sterile normal saline; in the composition group, the composition of Example 1 was dropped on the wound surface at a dose of 200 μl / rat under sterile conditions, and the concentration of bovine placental extract in the composition was 1 mg / ml (the composition of Example 1 was diluted with sterile normal saline to a concentration of bovine placental extract in the composition of 1 mg / ml) and smeared evenly. All rats were placed in a clean cage, numbered and fed individually, given clean drinking water and free access to food, and the wounds were kept clean to prevent wound infection. The next day, under sterile conditions, the composition of Example 1 was aspirated with a pipette and dropped on the wound surface and smeared evenly. The composition of the present invention was evenly smeared on the surface of the diabetic wound once a day. The wounds of the rats were photographed on the day of the experiment (day 0), day 3, day 6, day 9, day 12, and day 15 (Figure 3 ). The wound area of rats was calculated using ImageJ. The wound healing rate = (initial area - measured area on the observation day) ÷ initial area × 100%, and statistical analysis was performed on the wound healing rate. The average value of the wound healing rates of 5 rats in each group was taken as the wound healing rate value of that group ( Figure 4 ). After wound healing, the wound scars were photographed to explore the effect of the composition of the present application on wound repair. The results showed that compared with the Control group, the composition of the present application had a significant promoting effect on the healing of diabetic wounds, indicating that the composition of the present application is a promising biological agent for promoting wound healing.

[0233] All experimental operations must comply with the ethical norms of animal experiments.

Claims

1. A composition for promoting diabetic wound healing, characterized in that: The composition comprises: Bovine placenta extract, antimicrobial peptides, antioxidants, penetration enhancers; The mass ratios of bovine placenta extract to antimicrobial peptides, antioxidants, and penetration enhancers were 1:0.2~0.3, 1:0.2~0.3, and 1:0.1~0.2, respectively.

2. The composition for promoting diabetic wound healing according to claim 1, characterized in that: The mass ratios of bovine placenta extract to antimicrobial peptides, antioxidants, and permeation enhancers were 1:0.25, 1:0.25, and 1:0.15, respectively.

3. The composition for promoting diabetic wound healing according to claim 1, characterized in that: The antioxidant is coenzyme Q10, and the penetration enhancer is azone.

4. The composition for promoting diabetic wound healing according to claim 1, characterized in that: The composition also includes a pH adjuster.

5. A method for preparing the composition for promoting diabetic wound healing as claimed in claim 4, characterized in that: include: The bovine placenta tissue is successively subjected to enzymatic hydrolysis, centrifugation and concentration to obtain the bovine placenta extract; Antimicrobial peptides and antioxidants are sequentially added to the bovine placenta extract, mixed, a pH adjuster is added, and then a penetration enhancer is added, and the mixture is sterilized after mixing.

6. The method for preparing the composition for promoting diabetic wound healing according to claim 5, characterized in that: The bovine placenta extract is obtained by cutting up the newborn bovine placenta tissue, adding collagenase and hyaluronidase for enzymolysis; centrifuging the mixture after enzymolysis, collecting the supernatant, and concentrating the supernatant to obtain the bovine placenta extract.

7. The method for preparing the composition for promoting diabetic wound healing according to claim 5, characterized in that: The pH regulator is sodium citrate, which is used to adjust the pH of the solution to 6.5-7.

0.

8. The method for preparing the composition for promoting diabetic wound healing according to claim 5, characterized in that: The antioxidant is coenzyme Q10, and the penetration enhancer is azone.

9. Use of the composition for promoting diabetic wound healing as claimed in claim 1 in preparing a product for improving the healing rate of diabetic wounds.

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