An antioxidant peptide derived from the mucus of the pearl oyster *Pinctada salina* var. *hepuensis*, its preparation and application.

Abstract

This invention discloses an antioxidant active peptide from the mucus of the Hepu pearl oyster, its preparation, and its application. The amino acid sequence of the antioxidant active peptide from the Hepu pearl oyster mucus is Arg-Trp-Ala-Ser-Trp. The preparation method of the active peptide includes collecting pearl oysters, collecting mucus, collecting supernatant, identifying the supernatant, and screening and identifying the active peptide. The antioxidant active peptide obtained from the mucus of the Hepu pearl oyster exhibits high activity, effectively scavenging free radicals and reducing H2O2 damage to skin fibroblasts; it can also promote skin wound healing. The application of this antioxidant active peptide in drugs or devices for reducing cellular oxidative stress shows good promise.

Description

Technical Field

[0001] This invention belongs to the field of biopeptide technology, specifically relating to an antioxidant active peptide from the mucus of the Hepu pearl oyster and its preparation and application. Background Technology

[0002] Antioxidant peptides have advantages such as wide availability, high bioactivity, and few side effects. They can be extracted from various organisms, including plants, animals, and microorganisms, and their digestion and absorption in the body are relatively simple, making them easily utilized by the human body in the form of food or medicine. In particular, antioxidant peptides extracted from marine organisms, belonging to the polypeptide family, often exhibit excellent antioxidant capabilities due to their unique amino acid sequences and structures. Developing new marine-derived polypeptide antioxidants can not only broaden the sources of antioxidants but also help improve their efficacy and application scope.

[0003] Chinese Patent CN114031669A (publication date: February 11, 2022) discloses an antioxidant active peptide from thick-shelled mussels, its preparation, and its application, relating to the field of peptide technology. The patent discloses two thick-shelled mussel antioxidant peptides, Gln-Glu-Thr-Tyr and Tyr-Glu-Leu-His-Asp, which can reduce the production of intracellular reactive oxygen species (ROS) and exert a protective effect. By activating the Keap1 / Nrf2 signaling pathway, they exert antioxidant effects and protect vascular endothelial cells, potentially providing a candidate drug for the prevention and treatment of cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, and heart failure.

[0004] Chinese Patent CN114317654A (publication date: April 12, 2022) discloses a method for preparing marine biological antioxidant active peptides, relating to the field of antioxidant active peptide technology. The method includes: S1) extracting collagen from fragmented squid tissue; S2) mixing the extracted collagen, trypsin, papain, and flavor protease in water for enzymatic hydrolysis to obtain an enzymatic hydrolysate; S3) subjecting the enzymatic hydrolysate to salting-out precipitation and dialysis to obtain marine biological antioxidant active peptides. Compared with existing technologies, using squid as raw material to extract antioxidant active peptides and employing different enzymatic hydrolysis methods can effectively and thoroughly catalyze proteins, thereby obtaining high-quality, rationally composed, stable, and reliable active polypeptides.

[0005] Chinese Patent CN116904542A (publication date: October 20, 2023) discloses an antioxidant active peptide based on oyster shells and its preparation method, relating to the field of comprehensive utilization technology of oyster shells. This patent addresses the lack of research on antioxidant active peptides based on oyster shells, which has resulted in less than ideal comprehensive utilization of oyster shells, limiting the development and utilization of oyster shell resources. The preparation method first involves thoroughly refining the oyster shells to facilitate protein extraction. The proteins are then dissolved for excellent extraction. Multiple proteases are used to screen for suitable proteases for the proteins in oyster shells, enabling complete enzymatic hydrolysis to form active peptides. The oyster shell hydrolysate is then subjected to stepwise ultrafiltration to concentrate the antioxidant components, improving the antioxidant properties of the active peptides and ensuring full utilization of oyster shell resources. This method is of great significance for the development and utilization of oyster shell resources.

[0006] The search revealed that although some patents currently exist regarding the preparation of antioxidant peptides using shellfish as raw materials, their research focuses primarily on how to utilize shellfish shells to prepare antioxidant peptides, and the preparation process is complex.

[0007] The Hepu pearl oyster is a common shellfish used for pearl production. Its surface mucus is an underutilized biological resource. Currently, there is limited research on antioxidant active peptides derived from the surface mucus of the Hepu pearl oyster. The amino acid sequences of the peptides are unclear, and preparation methods and applications need further development. Summary of the Invention

[0008] To address the above technical problems, this invention provides an antioxidant active peptide from the mucus of the Hepu pearl oyster, its preparation, and its application.

[0009] An antioxidant peptide derived from the mucus of the Hepu pearl oyster, wherein the amino acid sequence of the antioxidant peptide is Arg-Trp-Ala-Ser-Trp.

[0010] The antioxidant active peptide can bind to antioxidant proteases;

[0011] The antioxidant protease is any one of SOD, CAT, and keap1.

[0012] A method for preparing an antioxidant peptide from the mucus of the Hepu pearl oyster, comprising the following steps:

[0013] S1: Collect pearl oysters and remove impurities from their shells;

[0014] S2: Open the clams and collect the mucus. After opening the clams, place the clam meat and mucus together in a plastic basket with holes to separate them. Collect the filtered mucus.

[0015] S3: Initial separation of mucus, the mucus is centrifuged and the supernatant is collected;

[0016] S4: Screening peptides, using molecular docking to predict and screen antioxidant active peptides from the supernatant;

[0017] S5: Synthesize peptides using solid-phase synthesis and verify their antioxidant properties.

[0018] Furthermore, the centrifugation speed in step S3 is 10000-12000 rpm, and the centrifugation time is 10-15 min.

[0019] Furthermore, in step S6, the antioxidant activity is verified using the free radical scavenging rate as an evaluation index; the free radicals include DPPH, ABTS, and hydroxyl groups.

[0020] Furthermore, in step S6, AutoDock Vina is used to simulate the binding ability of active peptides with antioxidant enzymes SOD, CAT, and Keap1, and the ability of peptides to scavenge free radicals is detected.

[0021] Application of an antioxidant peptide from the surface mucus of the pearl oyster of Hepu in the preparation of drugs or devices to reduce cellular oxidative stress.

[0022] Compared with the prior art, the advantages and effects of the present invention are as follows:

[0023] 1. This invention provides an antioxidant active peptide Arg-Trp-Ala-Ser-Trp from the surface mucus of the pearl oyster of Hepu, which can promote the proliferation of human fibroblasts and can be used to promote skin wound healing. It can also be used to promote and discover new drugs that promote wound healing.

[0024] 2. This invention provides a method for preparing antioxidant active peptides from the mucus on the surface of the pearl oyster. The preliminary separation of the pearl oyster's surface mucus helps to quickly identify proteins and peptides. The screening of antioxidant peptides through molecular docking, followed by solid-phase synthesis, helps to improve the efficiency of developing marine-derived active peptides.

[0025] 3. This invention provides an application of an antioxidant active peptide in the mucus of the Hepu pearl oyster. The antioxidant active peptide has good free radical scavenging ability and can reduce H2O2 damage to cells. The application of the antioxidant active peptide in drugs or devices that reduce cellular oxidative stress has good prospects.

[0026] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the preferred embodiments of this application are described in detail below with reference to the accompanying drawings.

[0027] The above and other objects, advantages and features of this application will become more apparent to those skilled in the art from the following detailed description of specific embodiments in conjunction with the accompanying drawings. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0029] in:

[0030] Figure 1 LC-MS spectrum of the peptide prepared in this invention

[0031] Figure 2 This is a schematic diagram of the 2D simulated structure of the polypeptide prepared in this invention.

[0032] Figure 3 This is a schematic diagram illustrating the simulated binding of the polypeptide prepared in this invention with antioxidant enzymes.

[0033] Figure 3 (a) is a schematic diagram illustrating the simulated binding of the polypeptide prepared in this invention with the antioxidant enzyme SOD.

[0034] Figure 3 (b) is a schematic diagram illustrating the simulated binding of the polypeptide prepared in this invention with the antioxidant enzyme CAT.

[0035] Figure 3 (c) is a schematic diagram illustrating the simulated binding of the polypeptide prepared in this invention with the antioxidant enzyme Keap1.

[0036] Figure 4 This is a comparison chart of the free radical scavenging results of the peptides prepared in this invention.

[0037] Figure 4 (a) Statistical chart of DPPH free radical scavenging results of the peptides prepared in this invention.

[0038] Figure 4 (b) is a statistical chart showing the ABTS free radical scavenging results of the peptides prepared in this invention.

[0039] Figure 4 (c) is a statistical chart showing the hydroxyl radical scavenging results of the peptides prepared in this invention.

[0040] Figure 5This is a comparison chart showing the results of the peptides prepared in this invention promoting fibroblast migration.

[0041] Figure 5 (a) shows the results of fibroblast migration in the control group.

[0042] Figure 5 (b) is a diagram showing the results of polypeptide-composed fibroblasts before migration.

[0043] Figure 5 (c) Results of fibroblast migration in the control group

[0044] Figure 5 (d) shows the results of fibroblast migration after polypeptide composition.

[0045] Figure 6 This is a comparison chart showing the protection of cells from H2O2 damage by the peptides prepared in this invention.

[0046] Figure 6 (a) is a graph showing the results of protecting cells in the control group from H2O2 damage.

[0047] Figure 6 (b) is a graph showing the results of H2O2 damage to the protected cells in the control group.

[0048] Figure 6 (c) is a graph showing the results of the peptide group protecting cells from H2O2 damage.

[0049] Figure 7 Comparison of the results of the polypeptide prepared in this invention promoting wound healing in diabetic rats.

[0050] Figure 7 (a) Image showing the wound healing results in the control group of diabetic rats.

[0051] Figure 7 (b) shows the wound healing results in diabetic rats using the polypeptide group. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. In the following description, specific details such as specific configurations and components are provided merely to help fully understand the embodiments of this application. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. In addition, for clarity and brevity, descriptions of known functions and structures are omitted in the embodiments.

[0053] It should be understood that the phrase "an embodiment" or "this embodiment" throughout the specification means that a specific feature, structure, or characteristic related to the embodiment is included in at least one embodiment of this application. Therefore, "an embodiment" or "this embodiment" appearing throughout the specification does not necessarily refer to the same embodiment. Furthermore, these specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0054] Furthermore, reference numerals and / or letters may be repeated in different examples within this application. Such repetition is for the purpose of simplification and clarity and does not in itself indicate a relationship between the various embodiments and / or settings discussed.

[0055] In this article, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. The term " / and" in this article describes another type of relationship between related objects, indicating that two relationships can exist. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the related objects before and after it are in an "or" relationship.

[0056] In this article, the term "at least one" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, "at least one of A and B" can mean: A exists alone, A and B exist simultaneously, or B exists alone.

[0057] It should also be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion.

[0058] Example 1

[0059] This embodiment describes a method for preparing antioxidant active peptides from the mucus of the Hepu pearl oyster.

[0060] Based on specific experimental results, the method for preparing antioxidant peptides includes the following steps:

[0061] S1: Collect pearl oysters and remove the shells, mud, sand, and seaweed;

[0062] S2: Open the scallops. Place the scallop meat and mucus in a mesh with a pore size of 0.5-1cm, let it stand for 60 minutes, separate the scallop meat and mucus, and collect the filtered mucus.

[0063] S3: The mucus is centrifuged at 10,000 rpm, and the supernatant is collected;

[0064] S4: Sequence identification of the initially separated mucus peptides was performed, and peptides were obtained by screening using molecular docking simulation method;

[0065] S5: Polypeptide synthesis using solid-phase synthesis.

[0066] The amino acid sequence of the polypeptide is Arg-Trp-Ala-Ser-Trp.

[0067] The technical effect of this embodiment is that preparing antioxidant peptides through a simple method helps to improve the development efficiency of marine-derived bioactive peptides.

[0068] Example 2

[0069] Based on Example 1, this example describes a method for identifying synthetic polypeptides.

[0070] like Figure 1 As shown, Figure 1 The LC-MS spectrum of the polypeptide prepared in this invention shows that the synthesized polypeptide exhibits obvious characteristic peaks, indicating that the synthesized polypeptide has high purity.

[0071] The technical effect of this embodiment: The antioxidant active peptides prepared by the present invention from the mucus on the surface of the pearl oyster have high purity.

[0072] Example 3

[0073] Based on Example 2, this example establishes a molecular model of the peptide Arg-Trp-Ala-Ser-Trp.

[0074] Modeling of highly active peptide molecules was performed using Discovery Studio software. Please refer to [link / reference]. Figure 2 , Figure 2 This is a schematic diagram of the 2D simulated structure of the peptide prepared in this invention. The predicted structure of the peptide Arg-Trp-Ala-Ser-Trp exhibits a compact ring conformation, in which hydrophobic residues W and A are located on one side of the ring, forming a hydrophobic pocket. This structure is beneficial for interaction with the hydrophobic regions of the target protein.

[0075] The technical effect of this embodiment: The present invention uses Discovery Studio software to model the predicted highly active peptide molecules, and can determine whether such a structure is conducive to interaction with the hydrophobic region of the target protein.

[0076] Example 4

[0077] Based on Examples 1-3, this example is a verification of the oxidative activity of the peptide Arg-Trp-Ala-Ser-Trp.

[0078] Molecular docking simulations were performed using the open-source molecular docking tool AutoDockVina software to simulate the interaction between the peptide Arg-Trp-Ala-Ser-Trp and the antioxidant proteins SOD, CAT, and Keap1.

[0079] Prior to docking simulation, the structures of the peptide and target protein are preprocessed, including adding hydrogen atoms, calculating charge, and adjusting partition flexibility. Then, a docking algorithm is selected for simulation. By evaluating binding energy and analyzing interaction modes, the potential biological activity and mechanism of action of the docked peptide are revealed.

[0080] like Figure 3 As shown, Figure 3 This is a schematic diagram illustrating the simulated binding of the polypeptide prepared in this invention with an antioxidant enzyme. Wherein, Figure 3 (a) is a schematic diagram of the simulated binding of the polypeptide prepared in this invention with the antioxidant enzyme SOD. The results show that the hydrophobic residues W and A of the polypeptide Arg-Trp-Ala-Ser-Trp may interact with the hydrophobic pocket of SOD, thereby stabilizing the structure of the enzyme and possibly enhancing its catalytic activity. This binding may help improve the scavenging efficiency of SOD against superoxide radicals, thereby enhancing the antioxidant defense of cells.

[0081] Figure 3 (b) is a schematic diagram of the peptide prepared in this invention and the antioxidant enzyme CAT. The results show that the compact cyclic conformation and hydrophobic residues of the peptide may bind to the corresponding regions of CAT through hydrophobic interactions, thereby enhancing the structural stability and catalytic ability of the enzyme. Figure 3 (c) is a schematic diagram of the simulated binding of the polypeptide prepared in this invention with the antioxidant enzyme Keap1. The results show that the compact cyclic conformation and hydrophobic residues may enable it to form a tight interaction with the binding pocket of Keap1, thereby interfering with the normal binding of Nrf2 and activating the Nrf2-mediated antioxidant response.

[0082] The technical effect of this embodiment is that by analyzing the simulation results through molecular docking simulation, it can be found that the peptide has potential antioxidant activity.

[0083] Example 5

[0084] Based on Example 4, this example is an evaluation of the ability of the peptide Arg-Trp-Ala-Ser-Trp to scavenge DPPH, ABTS and hydroxyl radicals.

[0085] like Figure 4 As shown, Figure 4 This is a comparison chart of the free radical scavenging results of the peptides prepared in this invention (P<0.01). Among them, Figure 4(a) is a statistical graph showing the DPPH radical scavenging results of the peptides prepared in this invention. The results show that the DPPH radical scavenging rate increases with the increase of peptide solution concentration. When the Arg-Trp-Ala-Ser-Trp concentration is 0.5 mg / mL, the DPPH radical scavenging rate is 29.8%; when the Arg-Trp-Ala-Ser-Trp concentration is 1.5 mg / mL, the DPPH radical scavenging rate is 52.5%.

[0086] Figure 4 (b) is a statistical graph showing the ABTS radical scavenging results of the peptides prepared in this invention. The results show that the ABTS radical scavenging rate increases with the increase of peptide solution concentration. When the Arg-Trp-Ala-Ser-Trp concentration is 0.5 mg / mL, the ABTS radical scavenging rate is 27.1%; when the Arg-Trp-Ala-Ser-Trp concentration is 1.5 mg / mL, the ABTS radical scavenging rate is 41.3%.

[0087] Figure 4 (c) is a statistical graph showing the hydroxyl radical scavenging results of the peptides prepared in this invention. The results show that when the concentration of Arg-Trp-Ala-Ser-Trp is 0.8 mg / mL, the hydroxyl radical scavenging rate is 8.9%; when the concentration of Arg-Trp-Ala-Ser-Trp is 1.0 mg / mL, the hydroxyl radical scavenging rate is 8.9%; and when the concentration of Arg-Trp-Ala-Ser-Trp is 1.8 mg / mL, the hydroxyl radical scavenging rate is 26.5%.

[0088] The technical effects of this embodiment are as follows: By comparing the DPPH, ABTS and hydroxyl radical scavenging rates of the peptide Arg-Trp-Ala-Ser-Trp at different concentrations, it can be found that the peptide has a significant ability to scavenge DPPH, ABTS and hydroxyl radicals (P<0.01), and the antioxidant capacity of the peptide solution is positively correlated with its concentration.

[0089] Example 6

[0090] Based on Example 5, this example verifies the promoting effect of the peptide Arg-Trp-Ala-Ser-Trp on fibroblast migration.

[0091] like Figure 5 As shown, Figure 5 This is a comparison chart showing the results of the polypeptides prepared in this invention promoting fibroblast migration. Among them, Figure 5 (a) shows the results of fibroblasts in the control group (without peptides) before migration. Figure 5 (b) is a diagram showing the results of fibroblasts before migration of polypeptide-based fibroblasts. As can be seen from the diagram, the distance between fibroblasts in both the control group and the polypeptide group before migration was 250 μm. Figure 5 (c) is a diagram showing the migration results of fibroblasts in the control group. As can be seen from the diagram, after 12 hours, the distance between fibroblasts in the control group was about 200 μm, indicating that the migration of fibroblasts was not obvious. Figure 5 (d) shows the results of fibroblast migration after peptide-based fibroblasts migrate. As can be seen from the figure, after 12 hours, the distance between peptide-based fibroblasts was about 50 μm, indicating that the peptide Arg-Trp-Ala-Ser-Trp significantly promotes fibroblast migration.

[0092] The technical effect of this embodiment is that it verifies that the peptide Arg-Trp-Ala-Ser-Trp promotes fibroblast migration.

[0093] Example 7

[0094] Based on Example 6, this example verifies the effect of the peptide Arg-Trp-Ala-Ser-Trp in protecting cells from H2O2 damage.

[0095] like Figure 6 As shown, Figure 6 This is a comparison chart showing the protection of cells from H2O2 damage by the polypeptide prepared in this invention. Figure 6 (a) shows the results of H2O2 damage detection for the control group (without added peptides). The results indicate that in the absence of H2O2, the cells exhibited lower fluorescence intensity and lower ROS production.

[0096] Figure 6 (b) is a graph showing the results of H2O2 damage to the control group protected cells. The results indicate that H2O2 treatment increased fluorescence intensity and increased ROS production in the cells.

[0097] Figure 6 (c) shows the results of the peptide group protecting cells from H2O2 damage. The results indicate that the fluorescence intensity of the cells decreased after incubation with the peptide, which means that the peptide can reduce the production of ROS in the cells.

[0098] The technical effect of this embodiment is that it verifies that the peptide Arg-Trp-Ala-Ser-Trp can protect cells from damage caused by H2O2.

[0099] Example 8

[0100] Based on Example 7, this example verifies the effect of the peptide Arg-Trp-Ala-Ser-Trp on promoting wound healing in diabetic rats.

[0101] like Figure 7 As shown, Figure 7 The image shows a comparison of the results of the polypeptides prepared in this invention promoting wound healing in diabetic rats; wherein, Figure 7(a) shows the wound healing results in the control group (without peptides) of diabetic rats. Figure 7 (b) shows the wound healing results of diabetic rats in the polypeptide group;

[0102] The results showed that after 18 days, the wounds of diabetic rats in the control group still had obvious wounds, while the wounds of diabetic rats in the polypeptide group were basically healed after polypeptide treatment, and obvious new skin could be observed.

[0103] The technical effect of this embodiment: It verifies that the peptide Arg-Trp-Ala-Ser-Trp can effectively promote the healing of diabetic wounds.

[0104] In summary, this invention provides an antioxidant active peptide from the mucus of the Hepu pearl oyster, its preparation, and its application. The amino acid sequence of the antioxidant active peptide from the mucus of the Hepu pearl oyster is Arg-Trp-Ala-Ser-Trp, which has high activity, can effectively scavenge free radicals, protect cells from H2O2 damage, and promote skin wound healing. The application of this antioxidant active peptide in drugs or devices that alleviate cellular oxidative stress shows good promise, and this invention is innovative.

[0105] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antioxidant active peptide from the body surface mucus of Pinctada fucata, characterized in that, The amino acid sequence of the antioxidant active peptide is Arg-Trp-Ala-Ser-Trp.

2. The Pinctada fucata body surface mucus antioxidant active peptide according to claim 1, characterized in that, The antioxidant active peptide can be combined with an antioxidant protease.

3. The Pinctada fucata somatic mucus antioxidant active peptide according to claim 2, characterized in that, The antioxidant protease is any one of SOD, CAT and keap1.

4. The method for preparing the body surface mucus antioxidant active peptide of Pinctada fucata according to any one of claims 1-3, characterized in that, The method comprises the following steps: S1: collecting pearl mussels and removing shell impurities; S2: opening the mussels and collecting mucus, placing the mussel meat together with the mucus in a plastic basket with a pore size after opening the mussels, separating the mussel meat and the mucus, and collecting the filtered mucus; S3: primary separation of mucus, collecting the supernatant after centrifugal treatment of the mucus; S4: screening polypeptides, predicting and screening antioxidant active peptides from the supernatant by molecular docking; S5: synthesizing polypeptides, synthesizing polypeptides by solid-phase synthesis method, and verifying the antioxidant activity.

5. The method for preparing the Pinctada fucata body surface mucus antioxidant active peptide according to claim 4, characterized in that, The centrifugal speed of the centrifugal treatment in step S3 is 10,000-12,000 rpm, and the centrifugal time is 10-15 min.

6. The method for preparing the Pinctada fucata body surface mucus antioxidant active peptide according to claim 4 or 5, characterized in that, The antioxidant activity in step S5 is verified by taking the free radical scavenging rate as the evaluation index; the free radicals include DPPH, ABTS and hydroxyl.

7. Use of a Pinctada martensii body surface mucus antioxidant active peptide in the preparation of a drug for promoting the healing of diabetic wounds.

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