Oxidized konjac glucomannan hydrogel for promoting wound healing of diabetic patient and preparation method of oxidized konjac glucomannan hydrogel

Abstract

The invention provides oxidized konjac glucomannan hydrogel for promoting wound healing of diabetic patients and a preparation method of the oxidized konjac glucomannan hydrogel, and belongs to the technical field of biomedical engineering. According to the invention, resveratrol is loaded in a dynamic cross-linked hydrogel matrix formed by carboxymethyl chitosan and oxidized konjac glucomannan, so that a multi-mechanism synergistic composite hydrogel is formed; the prepared resveratrol-loaded oxidized konjac glucomannan hydrogel is safe and efficient, has good oxidation resistance and a function of promoting wound healing, and has good development and application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to an oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients and its preparation method. Background Technology

[0002] Diabetic patients often experience metabolic disorders due to prolonged hyperglycemia, leading to multi-system dysfunction and significantly hindering wound healing, making them prone to developing chronic, intractable ulcers, especially diabetic foot ulcers (DFUs). As one of the most serious complications of diabetes, DFU is the end-stage manifestation of lower limb disease in diabetic patients. It is defined as a chronic wound below the ankle that penetrates the full thickness of the skin, often accompanied by infection, ischemia, or nerve damage, and is characterized by high morbidity, high recurrence rate, high amputation rate, and high mortality. According to the International Diabetes Federation, the global annual incidence of diabetic foot ulcers is 3%-13%, with approximately 15% of diabetic patients developing DFU in their lifetime, of which about 20% ultimately require amputation. In China, the proportion of diabetic patients with DFU reaches 12%-25%, with those having diabetes for more than 10 years and being over 70 years of age considered high-risk groups. The five-year recurrence rate of DFU is as high as 65%, and the lifetime morbidity is 19%-34%. Even more alarming is the fact that the 5-year mortality rate for patients with diabetic ulceration (DFU) is 2.5 times higher than that of patients without ulcers, and the 5-year mortality rate after amputation exceeds 70%, far surpassing the prognosis of some malignant tumors. Furthermore, chronic diabetic wounds not only severely reduce patients' quality of life but also exacerbate the financial burden on families and healthcare systems, resulting in a heavy economic burden. The cost of treating a single case of DFU in the United States ranges from $8,000 to $63,000, depending on the severity of the infection and the type of amputation. It is estimated that 10% of total global adult healthcare expenditure is spent on diabetes and its complications, with DFU-related costs accounting for a significant proportion.

[0003] Slow wound healing in diabetic chronic diseases is a complex result of the interaction of multiple pathological factors. The core mechanisms can be summarized into five categories: hyperglycemia-driven microenvironmental imbalance, oxidative stress damage, chronic inflammation, impaired angiogenesis, and microbial infection. These factors, through a self-reinforcing vicious cycle, collectively hinder the normal transition of the wound from the inflammatory phase to the proliferative and remodeling phases. Traditional dry dressings easily lead to wound dehydration and crusting, hindering cell migration and growth factor release. Hydrogels, by mimicking the "moist healing" theory, provide a moderately moist environment for the wound, thereby increasing cell migration efficiency and promoting collagen synthesis.

[0004] Currently, researchers have developed various functionalized hydrogels for diabetic chronic wounds. For example, one characteristic of diabetic chronic wounds is macrophage polarization imbalance, leading to the continuous release of inflammatory factors. Gelatin hydrogels loaded with IL-4 have been developed to induce macrophage transformation to the M2 type, reduce ROS generation, and promote tissue repair. However, macrophage imbalance in diabetic wounds involves a complex cytokine network, and single-factor regulation is insufficient to reverse the chronic inflammatory microenvironment. For instance, impaired angiogenesis in diabetic wounds is closely related to the inhibition of pro-angiogenic factor signaling. Hydrogels loaded with pro-angiogenic factors (such as VEGF) or with ECM-mimicking topologies have been developed to guide the directional migration of endothelial cells through these topologies. Alternatively, collagen-heparin hydrogels achieve controlled release of VEGF through the high affinity of heparin for growth factors, increasing vascular density by 2.3 times in diabetic mouse models. Furthermore, conductive hydrogels (such as polypyrrole / gelatin composites) can stimulate endothelial cell proliferation through electrical signals, accelerating capillary network formation. However, the above hydrogels still have problems such as the inability to continuously inhibit oxidative damage, low efficiency in promoting angiogenesis, or the possibility of endothelial cell apoptosis caused by electrical stimulation, which limits their ability to promote wound healing. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides an oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients and its preparation method.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0007] This invention provides a method for preparing oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients, comprising the following steps:

[0008] Resveratrol was mixed evenly with carboxymethyl chitosan solution and oxidized konjac glucomannan solution to obtain oxidized konjac glucomannan hydrogel loaded with resveratrol, which is oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients.

[0009] This invention uses oxidized konjac glucomannan instead of traditional cross-linking agents. Oxidized konjac glucomannan introduces aldehyde groups through oxidation with sodium periodate, which can react with the amino groups of carboxymethyl chitosan via a Schiff base reaction to form an injectable, dynamically cross-linked hydrogel suitable for complex wound surfaces. However, single hydrogels are insufficient in terms of antioxidant and immunomodulatory functions, making it difficult to cope with the excessive reactive oxygen species and persistent inflammatory environment in diabetic wounds. Therefore, this invention loads resveratrol into the dynamically cross-linked hydrogel system, utilizing its potent antioxidant, anti-inflammatory, and angiogenesis-promoting functions to form a multi-mechanism synergistic composite hydrogel. The resulting resveratrol-loaded oxidized konjac glucomannan hydrogel is safe and efficient, showing promising development and application prospects.

[0010] Furthermore, the mass ratio of oxidized konjac glucomannan to carboxymethyl chitosan is 5:1 to 1:9. For example, the mass ratio of oxidized konjac glucomannan to carboxymethyl chitosan is 5:1, 4:1, 3:1, 2:1, 1:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:9.

[0011] Furthermore, in the oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients, the resveratrol loading is 50-200 μg / mL, for example, the resveratrol loading is 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL or 200 μg / mL.

[0012] Furthermore, the oxidized konjac glucomannan is prepared by oxidizing konjac glucomannan with sodium periodate.

[0013] Furthermore, the preparation method of the oxidized konjac glucomannan is as follows:

[0014] (1) Dissolve konjac glucomannan in water, add sodium periodate, and react at 30-50 °C for 10-15 h under dark conditions;

[0015] (2) Add an appropriate amount of ethylene glycol to terminate the reaction, centrifuge to remove the precipitate, and collect the supernatant;

[0016] (3) The supernatant was dialyzed and dried to obtain oxidized konjac glucomannan.

[0017] Furthermore, the mass ratio of konjac glucomannan to sodium periodate is 100:40-100:150, for example, the mass ratio of konjac glucomannan to sodium periodate is 100:40, 100:60, 100:80, 100:100, 100:120 or 100:150.

[0018] Furthermore, the molecular weight cutoff in the dialysis treatment is 8000-14000 Da, for example, the molecular weight cutoff in the dialysis treatment is 8000 Da, 8500 Da, 9000 Da, 9500 Da, 10000 Da, 11000 Da, 12000 Da, 13000 Da or 14000 Da.

[0019] The present invention also provides an oxidized konjac glucomannan hydrogel prepared by the method described above.

[0020] The present invention also provides the application of the oxidized konjac glucomannan hydrogel in the preparation of products that promote wound healing in diabetic patients.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] This invention loads resveratrol onto a dynamically cross-linked hydrogel matrix formed by carboxymethyl chitosan and oxykonjac glucomannan, creating a multi-mechanism synergistic composite hydrogel. The resulting resveratrol-loaded oxykonjac glucomannan hydrogel is safe and efficient, exhibiting good antioxidant properties and wound-healing functions, and has promising development and application prospects. Attached Figure Description

[0023] Figure 1 Fourier transform infrared spectra of materials OKGM, KGM and hydrogel matrix OC (1:1);

[0024] Figure 2 Scanning electron microscope images of hydrogel matrices OC(5:1), OC(1:1), OC(1:5), and OC(1:9);

[0025] Figure 3 The rheological properties of hydrogel matrices OC(1:9) (A), OC(1:5) (B), OC(1:1) (C), and OC(5:1) (D) are shown in the test results.

[0026] Figure 4 The swelling ratios of the hydrogel matrices OC(5:1), OC(1:1), OC(1:5), and OC(1:9) are given.

[0027] Figure 5 The resveratrol release rate of the hydrogel;

[0028] Figure 6 Alternating strain diagram (A) and injectable photograph (B) of the hydrogel;

[0029] Figure 7 The free radical scavenging rate of the hydrogel;

[0030] Figure 8 The hemolysis status (A) and hemolysis rate (B) of the hydrogel;

[0031] Figure 9 The results are from the cytotoxicity assay of the hydrogel.

[0032] Figure 10 The results are from a cell proliferation experiment using hydrogels.

[0033] Figure 11 The effect of hydrogel on skin wound healing in diabetic mice (A) and wound healing rate (B);

[0034] Figure 12 H&E stained sections of mice after hydrogel treatment;

[0035] Figure 13 Masson-stained sections of mice after hydrogel treatment;

[0036] Figure 14 Immunohistochemical staining (A) and expression level (B) of CD31 in mice after hydrogel treatment. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] This invention provides a method for preparing oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients, comprising the following steps:

[0039] Resveratrol was mixed evenly with carboxymethyl chitosan solution and oxidized konjac glucomannan solution to obtain oxidized konjac glucomannan hydrogel loaded with resveratrol, which is oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients.

[0040] In some examples, the mass ratio of oxidized konjac glucomannan to carboxymethyl chitosan is 5:1 to 1:9. For example, the mass ratio of oxidized konjac glucomannan to carboxymethyl chitosan can be 5:1, 4:1, 3:1, 2:1, 1:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or 1:9.

[0041] In some examples, the resveratrol loading is 50-200 μg / mL. For example, the resveratrol loading can be 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, 130 μg / mL, 140 μg / mL, 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, or 200 μg / mL.

[0042] In some examples, the oxidized konjac glucomannan is prepared by oxidizing konjac glucomannan with sodium periodate, as follows:

[0043] (1) Dissolve konjac glucomannan in water, add sodium periodate, and react at 30-50 °C for 10-15 h under dark conditions;

[0044] (2) Add an appropriate amount of ethylene glycol to terminate the reaction, centrifuge to remove the precipitate, and collect the supernatant;

[0045] (3) The supernatant was dialyzed and dried to obtain oxidized konjac glucomannan.

[0046] In some examples, the mass ratio of konjac glucomannan to sodium periodate is 100:40-100:150, for example, the mass ratio of konjac glucomannan to sodium periodate is 100:40, 100:60, 100:80, 100:100, 100:120 or 100:150.

[0047] In some examples, the molecular weight cutoff in the dialysis treatment is 8000-14000 Da. For example, the molecular weight cutoff in the dialysis treatment can be 8000 Da, 8500 Da, 9000 Da, 9500 Da, 10000 Da, 11000 Da, 12000 Da, 13000 Da, or 14000 Da.

[0048] Example 1

[0049] Oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients

[0050] 1. Preparation of oxidized konjac glucomannan

[0051] (1) Weigh 2.5 g of konjac glucomannan (KGM) powder, add it to 250 mL of deionized water, and stir magnetically for 5 h until completely dissolved;

[0052] (2) Add 1.6 g of sodium periodate and react at 40 °C for 12 h under light-protected conditions;

[0053] (3) Add 5 mL of ethylene glycol to terminate the reaction, centrifuge (3000 rpm, 5 min) to remove the precipitate, and collect the supernatant;

[0054] (4) Put the supernatant into a dialysis bag (molecular weight cutoff of 11000 Da) and dialyze in deionized water for 3 days, changing the water 2-3 times a day;

[0055] (5) After the dialysis solution was frozen at -80℃ overnight, it was freeze-dried for 72 h to obtain white oxidized konjac glucomannan (OKGM) powder.

[0056] 2. Preparation of resveratrol-loaded konjac glucomannan hydrogel

[0057] (1) Prepare a 5 wt% carboxymethyl chitosan (CMCS) solution: Weigh CMCS and dissolve it in deionized water, and stir magnetically until transparent to obtain a 5 wt% CMCS solution.

[0058] (2) Prepare a 5 wt% OKGM solution: Weigh OKGM powder and dissolve it in deionized water. Heat and stir until completely dissolved to obtain a 5 wt% OKGM solution.

[0059] (3) Mix 5 wt% CMCS solution and 5 wt% OKGM solution according to the mass ratio of OKGM to CMCS of 5:1, 1:1, 1:5 and 1:9, vortex to mix, let stand to form hydrogel matrix, and label them as OC(5:1), OC(1:1), OC(1:5) and OC(1:9) respectively.

[0060] (4) Loading resveratrol: Resveratrol was dissolved in dimethyl sulfoxide (DMSO) and then added to 5 wt% CMCS solution. Subsequently, it was mixed evenly with 5 wt% OKGM solution so that the mass ratio of OKGM to CMCS was 1:1. Oxidized konjac glucomannan hydrogels loaded with resveratrol with loading amounts of 50, 100 and 200 μg / mL were prepared and labeled as OC-R50, OC-R100 and OC-R200, respectively.

[0061] 3. Detection of injectable hydrogels

[0062] (1) Chemical structure analysis: Fourier transform infrared spectroscopy (FTIR) was used for analysis. A certain mass of sample was mixed with potassium bromide, pressed into a pellet, and then measured using a Fourier transform infrared spectrometer with a resolution of 2 cm⁻¹. -1 The scanning wavelength range is 4000-400 cm. -1 A total of 64 scans were performed. The results are as follows: Figure 1 As shown in the figure, OKGM is at 1730 cm. -1 The characteristic peak of aldehyde groups appears at 1620 cm⁻¹, and the hydrogel OC(1:1) (labeled OKGM / CMCS in the figure) shows a peak at 1620 cm⁻¹. - The characteristic peak of C=N bond is shown at position ¹, confirming the occurrence of Schiff base crosslinking reaction.

[0063] (2) Microscopic morphology observation: Field emission scanning electron microscopy was used to observe the microstructure of hydrogels OC(5:1), OC(1:1), OC(1:5), and OC(1:9). The hydrogel samples were swollen for 12 h and then freeze-dried. After the freeze-dried samples were taken out and subjected to liquid nitrogen brittle fracture, the samples were placed on a sample stage with conductive adhesive and the cross-section of the samples was sputtered with gold (30 mA, 60 s). The micromorphology of the hydrogels was then observed.

[0064] The results are as follows Figure 2 As shown in the figure, scanning electron microscopy revealed continuous, irregular, and porous three-dimensional network structures in hydrogels of different proportions, indicating that the hydrogels can provide an environment conducive to wound healing and enhance the diffusion and penetration of active substances and gases. Among them, the OC (1:1) hydrogel exhibited a more stable and uniform pore structure, a higher degree of cross-linking, and greater structural stability.

[0065] (3) Rheological property testing: Using a DHR-1 rotational rheometer, a suitable amount of hydrogel sample was placed in the test area. The height of the clamp was adjusted, and excess hydrogel was removed before rheological testing. Frequency scanning experiment was conducted with the following parameters set: temperature: 25 ℃, constant strain: 1%, frequency range: 1-100 Hz. The storage modulus (G') and loss modulus (G'') were recorded.

[0066] The results are as follows Figure 3 As shown, Figure 3In the figure, A, B, C, and D represent the rheological property test results of OC(1:9), OC(1:5), OC(1:1), and OC(5:1), respectively; E and F represent the comparison of the storage modulus and loss modulus results of OC(1:9), OC(1:5), OC(1:1), and OC(5:1), respectively. As shown in the figure, the G' of each group of hydrogels is greater than G'', indicating the formation of stable gels. Comparing the G' of each group of hydrogel matrices, it can be found that OC(1:1) has the largest G' modulus value of 291 Pa, which remains stable within the test frequency range, while OC(1:9) has the smallest G'. This is because high crosslinking density increases the mechanical strength and structural stability of the hydrogel, indicating that hydrogel OC(1:1) has better mechanical properties.

[0067] (4) Swelling performance test: Weigh the hydrogel sample with an initial mass M0 and immerse it in PBS (pH 7.4). Weigh the mass M of the hydrogel sample after swelling at regular intervals. t And calculate the swelling ratio SR=(M t −M0) / M0×100%.

[0068] The results are as follows Figure 4 As shown in the figure, all hydrogels exhibit a certain degree of swelling performance. With the increase of the OKGM ratio, the swelling ratio of the hydrogels first decreases and then increases. The OC (1:9) and OC (5:1) groups of hydrogels show degradation and deformation, making it difficult to maintain their shape and resulting in mass loss. In contrast, the OC (1:1) hydrogel can not only absorb exudate from the wound but also avoid excessive swelling. It maintains its shape during use, which is more in line with the application requirements of wound dressings.

[0069] (5) Release of resveratrol: Weigh 1 g of the resveratrol-loaded konjac glucomannan hydrogel sample and place it in a centrifuge tube. Then add 10 mL of pure water, extract 1 mL of the supernatant at a predetermined time, and replenish the corresponding volume of pure water. Measure the absorbance at the corresponding time points using a UV spectrophotometer, and calculate the resveratrol concentration in the supernatant according to the standard curve.

[0070] The results are as follows Figure 5 As shown in the figure, the release of resveratrol increases with time. The resveratrol in the oxidized konjac glucomannan hydrogel loaded with resveratrol in each group is released rapidly in the first 8 hours, and the cumulative release in 8 hours exceeds 50%. The release rate begins to slow down after 12 hours from the start of the experiment and reaches the release peak at 24 hours.

[0071] (6) Self-healing and injectability testing: Alternating strain testing was performed on the hydrogel OC(1:1) using a rheometer. During the test, step strains ranging from γ = 1% to γ ​​= 300% were applied alternately, with each strain interval being 100 s and the angular frequency fixed at 10 rad / s. The hydrogel was extruded into a specific shape using a 1 mL syringe, and photographs were taken to evaluate the injectability of the hydrogel. The results of the alternating strain test are as follows: Figure 6 As shown in (A), when a 300% strain is applied to the hydrogel, the G′ value immediately drops from 98 Pa to 38 Pa, and is lower than G′′, indicating that the hydrogel network is broken and the sample exhibits fluid behavior. When the strain is reduced to 1%, G′ and G′′ immediately recover, indicating that the hydrogel recovers its elasticity, begins to self-heal, and transforms into a solid hydrogel. After multiple high-strain damages, the G′ value of the hydrogel is close to the initial G′ value, indicating that the hydrogel suffers very little damage after recovery and has a strong self-healing ability. Repeated step-strain experiments show that the hydrogel has a rapid and efficient self-healing ability. Injectable images are shown below. Figure 6 As shown in (B), the hydrogel can be easily ejected from the syringe with a needle and remains solid.

[0072] (7) Antioxidant performance test of hydrogels: 200 mg of each group of hydrogel samples were weighed, ground, and placed in centrifuge tubes. 1 mL of anhydrous ethanol and 1 mL of DPPH· solution were added, and the mixture was reacted at room temperature for 24 h in the dark. 200 μL of the supernatant was pipetted into a 96-well plate, and 1 mL of anhydrous ethanol was added to 1 mL of DPPH· solution as a blank control. The absorbance of the solution at a wavelength of 517 nm was measured using a microplate reader. Finally, the scavenging rate of DPPH· free radicals was calculated based on the measured absorbance.

[0073] The results of the free radical scavenging ability test of each group of hydrogels are as follows: Figure 7 As shown in the figure, hydrogel OC (1:1) has a weak ability to scavenge DPPH·, while the DPPH· scavenging ability of hydrogel loaded with resveratrol increases with the increase of resveratrol loading in the hydrogel, showing a dose-dependent effect. Among them, hydrogel OC-R200 has a free radical scavenging rate as high as 85%.

[0074] Example 2

[0075] Functional testing of resveratrol-loaded oxykonjac glucomannan hydrogel (OC-R100)

[0076] To evaluate the safety and wound-healing effect of resveratrol-loaded oxidized konjac glucomannan hydrogel (OC-R100) on diabetic wounds, C57BL / 6 mice were used as an animal model in this invention.

[0077] 1. Safety evaluation

[0078] (1) Blood compatibility test: Appropriate amounts of hydrogel samples of OC (5:1), OC (1:1), OC (1:5), and OC (1:9) were soaked in physiological saline at a ratio of 0.1 g / mL for 24 h. The samples were then filtered through a 0.22 μm bacterial filter to obtain hydrogel extracts. 500 μL of hydrogel extract was placed in a 1.5 mL centrifuge tube, 500 μL of distilled water was placed in a 1.5 mL centrifuge tube as a positive control, and 500 μL of physiological saline was placed in a 1.5 mL centrifuge tube as a negative control. 500 μL of 2% erythrocyte suspension was added to each centrifuge tube, and the tubes were incubated at 37 ℃ for 1 h. After incubation, the tubes were centrifuged at 3000 rpm for 15 min. The color of each group was observed and photographed. The absorbance of the supernatant at 545 nm was measured using an ELISA reader, and the hemolysis rate was calculated.

[0079] The results are as follows Figure 8 As shown, where, Figure 8 (A) are photographs of the hydrogel extract, positive control group, and negative control group after contact with blood for 1 hour and centrifugation for 15 minutes. As shown in the figure, hydrogels OC(5:1), OC(1:1), OC(1:5), and OC(1:9) can maintain the integrity of blood cell structure and do not cause hemolysis. Figure 8 (B) shows the hemolysis rate of each group of experiments. As can be seen from the figure, the hemolysis rates of hydrogels OC(5:1), OC(1:1), OC(1:5), and OC(1:9) are much lower than the 5% specified in the international standard. The above results indicate that hydrogels OC(5:1), OC(1:1), OC(1:5), and OC(1:9) do not cause severe hemolysis.

[0080] (2) Cytotoxicity test: Take L929 cells in good growth condition, adjust the cell density, and add 100 μL of cell suspension per well (1 × 10⁻⁶ cells per well). 4 Cells were seeded into 96-well plates, with blank wells containing only the same volume of culture medium and no cells. The plates were incubated for 24 h. The old culture medium was removed, and 100 μL of complete culture medium containing hydrogel extracts (OC (5:1), OC (1:1), OC (1:5), or OC (1:9)) was added to each well. Four replicates were prepared for each group, and the plates were incubated for 24 h. The old culture medium was removed, and the plates were washed twice with PBS buffer. 100 μL of prepared CCK-8 reagent (CCK-8:complete culture medium = 1:9) was added to each well, and the plates were incubated at 37 °C in the dark for 1.5 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0081] Figure 9The figure shows the relative survival rates of mouse fibroblast L929 cells cultured in each group of hydrogel extracts. As can be seen from the figure, there was no significant difference in the relative survival rates of L929 cells cultured in OC(5:1), OC(1:1), OC(1:5), and OC(1:9) hydrogel extracts, indicating that each hydrogel has good cell compatibility.

[0082] (3) Cell proliferation capacity assay: Take L929 cells in good growth condition, adjust the cell density, and add 100 μL of cell suspension per well (1 × 10⁻⁶ cells per well). 4 Cells were seeded into 96-well plates, with blank wells containing only the same volume of culture medium and no cells. The plates were incubated for 24 h. The old culture medium was removed, and 100 μL of complete culture medium containing different concentrations of free resveratrol and hydrogel extract (prepared by soaking in physiological saline at a ratio of 0.1 g / mL for 24 h, then filtering through a 0.22 μm filter) was added to each well. The free resveratrol concentration gradients were 5, 10, 15, 20, and 25 μg / mL. Four replicates were set up for each group, and the cells were incubated for 24 h and 48 h. After incubation, the old culture medium was removed, and the cells were washed twice with PBS buffer. 100 μL of prepared CCK-8 reagent (CCK-8:complete culture medium = 1:9) was added to each well, and the plates were incubated at 37 ℃ in the dark for 1.5 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0083] Figure 10 (A) Relative survival rates of mouse fibroblasts cultured with different concentrations of free resveratrol. As shown in the figure, the viability of L929 cells first increased and then decreased with the increase of free resveratrol concentration. The highest viability was observed at a concentration of 10 μg / mL, which also promoted the viability of mouse fibroblasts. When the concentration was greater than 20 μg / mL, cytotoxicity was observed. Figure 10 (B) shows the CCK-8 assay results of L929 cells co-cultured with OC(1:1), OC-R50, OC-R100, and OC-R200 hydrogel extracts for 24 h and 48 h. As shown in the figure, cell viability initially increased and then decreased with increasing resveratrol loading. The OC-R100 group exhibited the highest relative cell viability, reaching 159%, which was higher than that of free resveratrol at a concentration of 10 μg / mL. The OC-R200 group, however, showed cytotoxicity, resulting in a decrease in L929 cell viability. These results indicate that the resveratrol-loaded hydrogel extract has a better promoting effect on cell viability than resveratrol at the same concentration.

[0084] 2. Functional Evaluation

[0085] (1) Observation of wound healing in diabetic mice: Healthy female BALB / c mice aged 6-8 weeks were used. The mice were first fasted for 12 hours but allowed to drink water. Then, 1% streptozotocin was injected intraperitoneally at a dose of 55 mg / kg for 5 consecutive days. After the injection, the mice were fasted for another 2 hours but allowed to drink water. Then, food was added to the mice. Blood glucose was monitored for 2 weeks. The mice were considered diabetic when the blood glucose level was stable above 16.7 mmol / L. 24 diabetic mice were randomly divided into a control group, an OC (1:1) group and an OC-R100 group, with 8 mice in each group. An 8 mm full-thickness skin defect was created on the back using a punch. The control group was wiped with physiological saline. The OC (1:1) group and the OC-R100 group were covered with OC (1:1) and OC-R100 hydrogel, respectively. After the corresponding treatment, each group was fixed with sterile dressings. All treatment groups were dressed every two days. The wound healing status was recorded by taking pictures on days 0, 3, 7 and 14.

[0086] Figure 11 (A) and (B) show the healing photos and wound healing rates of skin wounds in different groups of diabetic mice, respectively. The experimental results indicate that OC-R100 hydrogel can provide a moist and sealed environment for wound recovery, promote cell migration, reduce inflammatory response, accelerate tissue regeneration, and promote wound healing.

[0087] (2) Histological analysis of wounds in diabetic mice: Three mice from each group were randomly selected on days 7 and 14. After the mice were euthanized by isoflurane, skin tissue was obtained from the wounds. Histological observation and analysis of the mouse skin tissue were performed by H&E staining, Masson staining and CD31 immunohistochemical staining.

[0088] The H&E staining results of the control group, OC (1:1) group, and OC-R100 group are as follows: Figure 12 As shown, the results indicate that OC-R100 hydrogel can reduce inflammatory cell infiltration. Masson staining results are as follows. Figure 13 As shown, the results indicate that OC-R100 hydrogel can promote collagen deposition. CD31 immunohistochemical staining and expression levels are shown below. Figure 14 As shown, by detecting the percentage of CD31-positive areas in the wound tissue of diabetic mice, it was demonstrated that OC-R100 hydrogel can promote angiogenesis and wound healing in diabetic mice.

[0089] In summary, this invention forms a multi-mechanism synergistic composite hydrogel by loading resveratrol into a dynamically cross-linked hydrogel matrix formed by carboxymethyl chitosan and oxykonjac glucomannan. The prepared resveratrol-loaded oxykonjac glucomannan hydrogel is safe and efficient, with good antioxidant properties and wound healing promotion function, and has good development and application prospects in the preparation of antioxidant and wound healing promotion products.

[0090] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients, characterized in that, Includes the following steps: Resveratrol was mixed evenly with carboxymethyl chitosan solution and oxidized konjac glucomannan solution to obtain oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients.

2. The method for preparing oxidized konjac glucomannan hydrogel for promoting wound healing in diabetic patients according to claim 1, characterized in that, The mass ratio of oxidized konjac glucomannan to carboxymethyl chitosan is 5:1-1:

9.

3. The method for preparing oxidized konjac glucomannan hydrogel for promoting wound healing in diabetic patients according to claim 2, characterized in that, The mass ratio of oxidized konjac glucomannan to carboxymethyl chitosan is 1:

1.

4. The method for preparing oxidized konjac glucomannan hydrogel for promoting wound healing in diabetic patients according to claim 1, characterized in that, In the oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients, the resveratrol loading is 50-200 μg / mL.

5. The method for preparing oxidized konjac glucomannan hydrogel for promoting wound healing in diabetic patients according to claim 4, characterized in that, The resveratrol loading in the oxidized konjac glucomannan hydrogel that promotes wound healing in diabetic patients is 100 μg / mL.

6. The method for preparing oxidized konjac glucomannan hydrogel for promoting wound healing in diabetic patients according to claim 1, characterized in that, The oxidized konjac glucomannan is prepared by oxidizing konjac glucomannan with sodium periodate.

7. The method for preparing oxidized konjac glucomannan hydrogel for promoting wound healing in diabetic patients according to claim 6, characterized in that, The preparation method of the oxidized konjac glucomannan is as follows: (1) Dissolve konjac glucomannan in water, add sodium periodate, and react at 30-50 ℃ for 10-15 h under light-protected conditions; (2) Add an appropriate amount of ethylene glycol to terminate the reaction, centrifuge to remove the precipitate, and collect the supernatant; (3) The supernatant was dialyzed and dried to obtain oxidized konjac glucomannan.

8. The method for preparing oxidized konjac glucomannan hydrogel for promoting wound healing in diabetic patients according to claim 7, characterized in that... The mass ratio of konjac glucomannan to sodium periodate is 100:40-100:

150.

9. Oxidized konjac glucomannan hydrogel prepared by the method according to any one of claims 1-8.

10. The use of the oxidized konjac glucomannan hydrogel of claim 9 in the preparation of products that promote wound healing in diabetic patients.

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