Preparation method of a pH-responsive antioxidant hydrogel for promoting wound healing

Abstract

The present invention discloses a preparation method of a pH-responsive antioxidant hydrogel capable of promoting wound healing, comprising the following steps: extraction and purification of purple sweet potato anthocyanins, preparation of a purple sweet potato anthocyanin solution, preparation of a κ-carrageenan solution, preparation of a hydrogel to obtain a hard gel, and cross-linking reaction. In the present invention, anthocyanins with pH-responsive color change and antioxidant and anti-inflammatory effects on the wound site are extracted from purple sweet potatoes and added into the κ-carrageenan hydrogel, thereby endowing the anthocyanin / κ-carrageenan hydrogel with multiple functions of antioxidant, anti-inflammatory and color change indication, and capable of accelerating wound healing.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer material preparation, and particularly relates to a preparation method of a pH-responsive antioxidant hydrogel capable of promoting wound healing. Background Art

[0002] As the largest organ of the human body, the skin is vulnerable to various pathogenic or harmful factors, resulting in skin damage. Wound healing is a complex process, which can be divided into four overlapping stages: hemostasis, inflammation, proliferation, and tissue remodeling. For wound healing, the excessive production of reactive oxygen species (ROS) at the wound site will disrupt the balance between oxidants and antioxidants, leading to slow tissue regeneration and wound healing, severe disability, and even death. Therefore, wound dressing biomaterials with antioxidant properties are needed to accelerate wound healing. Hydrogels based on natural polysaccharides have become promising matrices due to their manufacturing flexibility, ability to mimic the physical properties of the extracellular matrix (ECM), ability to retain therapeutic biomolecules in their network, and biocompatibility, and can effectively promote wound healing. Among natural polysaccharides, carrageenan is widely used in the field of wound dressings due to its high hygroscopicity, gelation, biocompatibility, and biodegradability. However, it also has functional defects (such as antioxidant, anti-inflammatory, etc.).

[0003] Affected by internal and external factors, the pH value of the wound is different in different states such as healing, inflammation, and ulcer. Research shows that the pH value of healthy skin is slightly acidic (pH = 4-6), and when the skin barrier is damaged, the pH value becomes alkaline (pH = 9). During the wound healing process, the environmental state should develop from an alkaline state to a neutral state and finally to an acidic state. If the pH value increases and tends to be alkaline, it indicates that an infection may have occurred. During the entire healing process, the pH value of the skin shows dynamic changes. The pH should change from alkaline to neutral and then back to weakly acidic. Therefore, by monitoring the pH value change of the wound, the wound healing status and whether an infection has occurred can be understood.

[0004] Anthocyanin is a plant polyphenol that gives color to many fruits and flowers. Anthocyanin is widely present in the human diet through crops, berries, fruits, vegetables, and red wine. Due to its abundant phenolic hydroxyl groups, it can form a series of physically synthesized hydrogels with various polymers. Research shows that anthocyanin has promoting effects on health, including antioxidant, anti-inflammatory, antidiabetic, angiogenic, neuroprotective, hepatoprotective, and cardioprotective properties. In addition, anthocyanin also has pH color response characteristics, and its response range is between 2-13, and the color change is more obvious between 5-9. For this reason, the applicant prepared a hydrogel loaded with anthocyanin using carrageenan as the matrix and used it for wound dressings, which has multiple functions of antioxidant, anti-inflammatory, and color change indication to a certain extent, and has very important practical significance. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method of a pH-responsive antioxidant hydrogel that can promote wound healing. Anthocyanins with pH-responsive color change and antioxidant and anti-inflammatory effects on the wound site are extracted from purple sweet potatoes and added into κ-carrageenan hydrogel, thereby endowing the anthocyanin / κ-carrageenan hydrogel with multiple functions of antioxidant, anti-inflammatory and color change indication, and accelerating wound healing.

[0006] To achieve the above object, the solution of the present invention is: a preparation method of a pH-responsive antioxidant hydrogel that can promote wound healing, comprising the following steps:

[0007] Step (1), extraction and purification of purple sweet potato anthocyanins:

[0008] S1, First, take 50 g of purple sweet potato powder and add it to 1000 mL of 0.1% HCl-ethanol solvent, add a certain amount of cellulase, and ultrasonicate at 50 °C for 30 min to obtain a crude extract of purple sweet potato anthocyanins;

[0009] S2, Then, load the treated AB-8 resin into a chromatography column, control the flow rate at 30 rpm / min, dilute the crude extract of purple sweet potato anthocyanins, and pass it through the chromatography column at a flow rate of 20 rpm per 500 mL under natural acidity conditions. After the crude extract of purple sweet potato anthocyanins is loaded into the column, wash it with distilled water to remove the adsorbed sugars and other impurities in the chromatography column;

[0010] S3, Finally, elute the purple sweet potato anthocyanins in the chromatography column with an ethanol solution, centrifuge and rotary evaporate to obtain a concentrated solution, and freeze-dry for standby;

[0011] Step (2), preparation of anthocyanin / κ-carrageenan hydrogel:

[0012] S1, First, disperse the purple sweet potato anthocyanins prepared in step (1) in distilled water to prepare three different concentrations of purple sweet potato anthocyanin solutions;

[0013] S2, Secondly, wet κ-carrageenan with glycerol and dissolve it in deionized water to obtain a κ-carrageenan solution, and stir and heat it to 80 °C for about 30 min in a magnetic stirrer;

[0014] S3, Thirdly, mix the purple sweet potato anthocyanin solution prepared in S1 and the κ-carrageenan solution prepared in S2 in a certain proportion, and continue to stir the mixture with a magnetic stirrer for 5 min, and ultrasonically degas to obtain a pre-gel solution;

[0015] S4, Next, pour the pre-gel solution into a 55 mm mold, keep it at room temperature for 2-5 minutes, and after forming a solid gel, crosslink it with a potassium chloride solution for further stabilization;

[0016] S5. Finally, wash the solid gel prepared in S4 twice with distilled water to remove the excess KCl, thereby obtaining the anthocyanin / κ-carrageenan hydrogel.

[0017] Furthermore, the addition amount of the cellulase described in step (1) S1 is 54 U / mL.

[0018] Furthermore, the crude purple sweet potato anthocyanin extract described in step (1) S2 is diluted to 1 mg / mL.

[0019] Furthermore, the ethanol solution described in step (1) S3 is an ethanol solution with a concentration of 60%.

[0020] Furthermore, the mass concentrations of the purple sweet potato anthocyanin solutions described in step (2) S1 are 1% w / v, 3% w / v, and 5% w / v, respectively.

[0021] Furthermore, after wetting 1 g of κ-carrageenan with 0.5 g of glycerol in step (2) S2, it is dissolved in 50 mL of deionized water to obtain a 2% κ-carrageenan solution.

[0022] Furthermore, the purple sweet potato anthocyanin solution and the κ-carrageenan solution in step (2) S3 are mixed in a ratio of 1:50 by mass concentration.

[0023] Furthermore, the mold described in step (2) S4 is a glass petri dish.

[0024] Furthermore, the potassium chloride solution described in step (2) S4 is a potassium chloride solution with a concentration of 0.75%.

[0025] After adopting the above scheme, the beneficial effects of the present invention are as follows:

[0026] The anthocyanin adopted in the present invention is extracted from purple sweet potatoes, which is harmless to the human body and has good pH responsiveness. Therefore, the anthocyanin / κ-carrageenan hydrogel prepared in the present invention is extremely sensitive and very responsive to environmental acids and bases; under different pH conditions, the color of the anthocyanin / κ-carrageenan hydrogel changes spontaneously. When the pH value is 5 - 7, it is purplish red; when the pH value is 7 - 8, it is blue; when the pH value is greater than 8, it is green, and the color change can reach stability within 15 - 30 s. Therefore, the pH value of the wound can be monitored by observing the color change, so as to understand the information on the wound healing status.

[0027] The anthocyanin / κ-carrageenan hydrogel prepared in the present invention has excellent mechanical properties and biocompatibility. The introduction of anthocyanin makes up for the functional defects of κ-carrageenan hydrogel in terms of antioxidant and anti-inflammatory properties, can effectively inhibit the ROS level in cells, has an obvious proliferative effect on the growth of cells, thereby promoting the closure of the wound gap and shortening the wound healing time.

[0028] The preparation method of the present invention is simple, the reaction conditions are mild, and it is easy to implement; moreover, the anthocyanin has a wide source and low cost, and has excellent properties such as antioxidant, anti-inflammatory, anti-diabetic, and angiogenesis, and is an ideal ligand. Description of the Drawings

[0029] Figure 1 It is the SEM images of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention under 500X;

[0030] Figure 2 It is the swelling rate of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention;

[0031] Figure 3 It is the pH-responsive characteristics of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention;

[0032] Figure 4 It is the antioxidant activity of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention;

[0033] Figure 5 It is the biocompatibility of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention;

[0034] Figure 6 It is the ability of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention to scavenge intracellular ROS;

[0035] Figure 7 It is the cell migration experiment of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention. Detailed Embodiments

[0036] To better understand the present invention, the technical solutions and technical effects of the present invention will be further described below in conjunction with the drawings, examples, and experimental examples. It should be noted that the following examples are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0037] The present invention provides a preparation method of a pH-responsive antioxidant hydrogel that can promote wound healing, including the following steps:

[0038] Step (1), extraction and purification of purple sweet potato anthocyanin (PSPE):

[0039] S1. First, take 50 g of purple sweet potato powder and add it to 1000 mL of 0.1% HCl-ethanol solvent. Add cellulase at a concentration of 54 U / mL and ultrasonicate for 30 min at 50 °C to obtain a crude extract of purple sweet potato anthocyanins.

[0040] S2. Then, pack the treated AB-8 resin into a chromatography column, control the flow rate at 30 rpm / min, dilute the crude extract of purple sweet potato anthocyanins to 1 mg / mL, and pass it through the chromatography column at a flow rate of 20 rpm per 500 mL under natural acidity conditions. After the crude extract of purple sweet potato anthocyanins is loaded into the column, wash it with distilled water to remove the adsorbed sugars and other impurities in the chromatography column.

[0041] S3. Finally, elute the purple sweet potato anthocyanins in the chromatography column with a 60% ethanol solution (pH = 3), centrifuge and rotary evaporate to obtain a concentrated solution, and freeze-dry it for standby.

[0042] Step (2). Preparation of anthocyanin / κ-carrageenan hydrogel:

[0043] S1. First, disperse the purple sweet potato anthocyanins prepared in step (1) in distilled water to prepare three different concentrations of purple sweet potato anthocyanin solutions with mass concentrations of 1% w / v, 3% w / v, and 5% w / v, respectively.

[0044] S2. Second, moisten 1 g of κ-carrageenan with 0.5 g of glycerol and dissolve it in 50 mL of deionized water to obtain a 2% κ-carrageenan solution. Stir and heat it to 80 °C for about 30 min on a magnetic stirrer.

[0045] S3. Third, mix the purple sweet potato anthocyanin solution prepared in S1 and the κ-carrageenan solution prepared in S2 at a ratio of 1:50 (v / v), and continue to stir evenly for 5 minutes using a magnetic stirrer, and then ultrasonically degas to obtain a pre-gel solution.

[0046] S4. Next, pour the pre-gel solution into a 55-mm mold or glass petri dish, keep it at room temperature for 2 - 5 minutes, and after forming a solid gel, crosslink it with a 0.75% potassium chloride solution for further stabilization.

[0047] S5. Finally, wash the solid gel prepared in S4 twice with distilled water to remove the excess KCl, thereby obtaining the anthocyanin / κ-carrageenan hydrogel (κC-PSPE).

[0048] Example 1

[0049] After wetting 1 g of κ-carrageenan with 0.5 g of glycerol, it was dissolved in 50 mL of deionized water to obtain a 2% κ-carrageenan solution, which was stirred and heated to 80 °C for about 30 min in a magnetic stirrer; then, the pre-gel solution was cast into a 55 mm mold or a glass Petri dish and kept at room temperature for 2 - 5 minutes. After forming a solid gel, it was further stabilized by cross-linking with 0.75% potassium chloride solution, and then washed twice with distilled water to remove excess KCl, obtaining κ-carrageenan hydrogel, named κC, as a blank control.

[0050] Example 2

[0051] Purification of purple sweet potato PSPE: 50 g of purple sweet potato powder was added to 1000 mL of 0.1% HCl-ethanol solvent, and 54 U / mL of cellulase was added. It was ultrasonicated at 50 °C for 30 min to obtain a crude extract of purple sweet potato PSPE; the treated AB-8 resin was loaded into a chromatography column, and the flow rate was controlled at 30 rpm / min. The crude extract of purple sweet potato PSPE was diluted to 1 mg / mL and passed through the chromatography column at a flow rate of 20 rpm per 500 mL under natural acidity conditions. After the crude extract of purple sweet potato PSPE was loaded into the column, it was perfused with distilled water to wash away the adsorbed sugars and other impurities in the chromatography column; the purple sweet potato PSPE in the chromatography column was eluted with 60% ethanol solution (pH = 3), and after centrifugation and rotary evaporation, a concentrated solution was obtained and stored for freeze-drying.

[0052] Preparation of κC-PSPE: The purple sweet potato PSPE prepared above was dispersed in distilled water to prepare a purple sweet potato PSPE solution with a mass concentration of 1% w / v; 1 g of κ-carrageenan was wetted with 0.5 g of glycerol and then dissolved in 50 mL of deionized water to obtain a 2% κ-carrageenan solution, which was stirred and heated to 80 °C for about 30 min in a magnetic stirrer; then, the purple sweet potato PSPE solution and the κ-carrageenan solution were mixed in a ratio of 1:50 (v / v), and the mixture was continuously stirred evenly for 5 minutes using a magnetic stirrer and degassed by ultrasonic treatment; the pre-gel solution was cast into a 55 mm mold or a glass Petri dish and kept at room temperature for 2 - 5 minutes. After forming a solid gel, it was further stabilized by cross-linking with 0.75% potassium chloride solution; finally, it was washed twice with distilled water to obtain 1% κC-PSPE.

[0053] Example 3

[0054] Purification of Purple Sweet Potato PSPE: Take 50 g of purple sweet potato powder and add it to 1000 mL of 0.1% HCl-ethanol solvent. Add cellulase at a concentration of 54 U / mL and ultrasonicate for 30 min at 50 °C to obtain a crude extract of purple sweet potato PSPE. Load the treated AB-8 resin into a chromatography column, control the flow rate at 30 rpm / min, appropriately dilute the crude extract of purple sweet potato PSPE, and pass it through the chromatography column at a flow rate of 20 rpm per 500 mL under natural acidity conditions. After the crude extract of purple sweet potato PSPE is loaded into the column, wash it with distilled water to remove the adsorbed sugars and other impurities. Finally, elute the purple sweet potato PSPE in the chromatography column with 60% ethanol solution (pH = 3), centrifuge and rotary evaporate to obtain a concentrated solution, and freeze-dry it for standby;

[0055] Preparation of κC-PSPE: Disperse the purple sweet potato PSPE prepared above in distilled water to prepare a purple sweet potato PSPE solution with a mass concentration of 3% w / v. Moisten 1 g of κ-carrageenan with 0.5 g of glycerol and dissolve it in 50 mL of deionized water to obtain a 2% κ-carrageenan solution. Stir and heat it to 80 °C for about 30 min on a magnetic stirrer. Then mix the purple sweet potato PSPE solution and the κ-carrageenan solution in a ratio of 1:50 (v / v), and continue to stir the mixture evenly for 5 minutes on a magnetic stirrer and ultrasonically degas. Pour the pre-gel solution into a 55 mm mold or a glass petri dish and keep it at room temperature for 2 - 5 minutes. After forming a solid gel, further stabilize it by cross-linking with 0.75% potassium chloride solution. Finally, wash it twice with distilled water to obtain 3% κC-PSPE.

[0056] Example 4

[0057] Purification of Purple Sweet Potato PSPE: Take 50 g of purple sweet potato powder and add it to 1000 mL of 0.1% HCl-ethanol solvent. Add cellulase at a concentration of 54 U / mL and ultrasonicate for 30 min at 50 °C to obtain a crude extract of purple sweet potato PSPE. Load the treated AB-8 resin into a chromatography column, control the flow rate at 30 rpm / min, appropriately dilute the crude extract of purple sweet potato PSPE, and pass it through the chromatography column at a flow rate of 20 rpm per 500 mL under natural acidity conditions. After the crude extract of purple sweet potato PSPE is loaded into the column, wash it with distilled water to remove the adsorbed sugars and other impurities. Finally, elute the purple sweet potato PSPE in the chromatography column with 60% ethanol solution (pH = 3), centrifuge and rotary evaporate to obtain a concentrated solution, and freeze-dry it for standby;

[0058] Preparation of κC-PSPE: The obtained purple sweet potato PSPE was dispersed in distilled water to prepare a purple sweet potato PSPE solution with a mass concentration of 5% w / v; 1 g of κ-carrageenan was wetted with 0.5 g of glycerol and dissolved in 50 mL of deionized water to obtain a 2% κ-carrageenan solution, which was stirred and heated to 80 °C for about 30 min on a magnetic stirrer; then the purple sweet potato PSPE solution and the κ-carrageenan solution prepared in S2 were mixed at a ratio of 1:50 (v / v), and the mixture was continuously stirred evenly for 5 minutes using a magnetic stirrer, degassed by ultrasonic treatment, the pre-gel solution was cast into a 55 mm mold or a glass petri dish, and kept at room temperature for 2 - 5 minutes. After forming a solid gel, it was further stabilized by cross-linking with 0.75% potassium chloride solution; finally, it was washed twice with distilled water to obtain 5% κC-PSPE.

[0059] Experimental Example 1

[0060] In this experimental example, the morphologies of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% obtained in Examples 1 - 4 were observed by scanning electron microscopy. Before the experiment, each freeze-dried hydrogel was placed on the sample stage using double-sided conductive tape and subjected to gold spraying treatment. A thin layer of gold or platinum was sprayed on the surface of the sample using an ion sputtering instrument, and the morphology of each hydrogel was observed under a scanning electron microscope after gold spraying. Figure 1 SEM images of the morphologies of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% freeze-dried hydrogels at a magnification of 500 times.

[0061] The hydrogel showed a 3D network structure similar to that of a typical hydrogel, presenting a porous honeycomb structure, as Figure 1 shown, where a is κC, b is κC-PSPE1%, c is κC-PSPE3%, and d is κC-PSPE5%. It can be clearly seen that the fracture surface of κC is quite flat and smooth, with a large number of holes on the surface; in contrast, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% formed an interconnected porous network structure, which can be reflected from the roughness of the cross-section. Adding anthocyanins does not cause a significant change in the pore size, but these porous structures not only provide a larger effective surface area for nutrient exchange and cell adhesion, but also facilitate cell penetration or tissue formation in the hydrogel structure.

[0062] Experimental Example 2

[0063] An important advantage of hydrogels as wound dressings is their ability to maintain a suitable moist environment at the wound site while absorbing excess wound exudate. In this experimental example, the swelling properties of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% obtained in Examples 1 - 4 were tested. Figure 2is the swelling ratio of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention.

[0064] Weigh 4.017 g of NaCl, 0.1775 g of NaHCO3, 0.1125 g of KCl, 0.1155 g of K2HPO4·3H2O, 0.1475 g of MgCl2, 0.146 g of CaCl2, 0.036 g of Na2SO4, and 3.059 g of (HOCH2)3CNH2 (tris(hydroxymethyl)aminomethane, THAM), and add them to distilled water at 37 °C. Make up the volume to 500 mL. During this process, adjust the pH value to 7.4 with HCl to prepare the simulated body fluid SBF. Take each hydrogel sample (1 cm × 1 cm × 1 cm), dry it in an oven at 60 °C until the mass remains unchanged, immerse it in 50 mL of SBF at 37 °C to simulate the actual body condition, and take out the hydrogel samples at different time intervals. After removing the surface liquid with a clean filter paper, weigh them. The swelling ratio of the hydrogel is calculated as follows:

[0065]

[0066] Note: WB and WA are the weights of the hydrogel before and after swelling, respectively.

[0067] As Figure 2 shown, all κC-PSPEs showed excellent SBF absorption ability in the SBF solution as the soaking time increased, that is, the swelling ratio increased exponentially with the increase of the soaking time. Among them, the high swelling ability of κC may be due to the high hydrophilicity of carrageenan crosslinked with KCl. K + forms a bridge between two adjacent helices of carrageenan containing sulfate groups to control the gelation degree, stability, and strength of the carrageenan-based hydrogel. Compared with κC, the swelling ability of κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% is slightly improved because anthocyanins are rich in polyphenols, which can reduce the intermolecular interaction in carrageenan and form hydrogen bonds with the hydrophilic groups of carrageenan, resulting in a decrease in the network cohesion and an increase in swelling.

[0068] Experimental Example 3

[0069] This experiment was to test the pH response characteristics of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% obtained in Examples 1-4. Before the experiment, immerse each hydrogel sample in PBS buffer solutions with pH values of 5, 6, 7, 7.4, 8, and 9, observe the color changes of each hydrogel, and take pictures with a mobile phone of model M2002J9E to record the visible color changes of each hydrogel sample. Figure 3It is the pH-responsive characteristics of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention.

[0070] As Figure 3 shown, when κC was immersed in PBS solutions of different pH values, no color change occurred; while when κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% were immersed in buffer solutions of different pH values, they showed abnormal sensitivity. Each κC-PSPE spontaneously changed color, and the higher the PSPE content added to the κ-carrageenan hydrogel, the deeper the color development. As can be seen from the figure, when each κC-PSPE was immersed in PBS solutions with pH values of 5, 6, and 7 respectively, the color of each κC-PSPE was magenta, and the color became lighter as the pH value increased; when each κC-PSPE was immersed in PBS solutions with pH values of 7.4 and 8 respectively, the color of each κC-PSPE changed to blue; when each κC-PSPE was immersed in PBS solutions with pH value of 9 respectively, the color of each κC-PSPE changed to green. It can be seen from this that the color change of each κC-PSPE matches the pH range required to indicate the state of chronic or infected wounds. As the wound heals, the wound environment develops from an alkaline state to a neutral state and then to an acidic state. Compared with κC, it can be seen that the color change of each κC-PSPE corresponds to the color change of PSPE. Therefore, the κC-PSPE prepared in the present invention can be used as a promising wound dressing to monitor the wound healing process by simple colorimetry.

[0071] Experimental Example 4

[0072] In this experimental example, the antioxidant activities of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% obtained in Examples 1-4 were tested, and the ultraviolet absorption spectra and transmittance of the films were measured using an ultraviolet spectrophotometer. Before the experiment, each hydrogel sample film was cut into strips of 8 mm × 50 mm and directly placed in a quartz cell. The scanning wavelength range was 200-700 nm to obtain the ultraviolet absorption spectra of the film samples, and the transmittance of the films at 280 nm and 660 nm was calculated to evaluate the ultraviolet barrier properties of the films. Figure 4 It is the antioxidant activities of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention, where a is the scavenging activity of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% against DPPH free radicals, and b is the scavenging activity of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% against ABTS free radicals.

[0073] As Figure 4As shown, κC showed low antioxidant activity against DPPH and ABTS free radicals. The addition of PSPE significantly improved the antioxidant activity of κ-carrageenan hydrogel, and the antioxidant ability of κC-PSPE was dose-dependent, that is, the scavenging activities of κC-PSPE against DPPH and ABTS were positively correlated with the polyphenol content in the carrageenan conjugate. When the addition amount of PSPE was 5%, the scavenging abilities against DPPH and ABTS reached 88.6% and 95.8%, respectively.

[0074] Experimental Example 5

[0075] In this experiment, the biocompatibility of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% obtained in Examples 1-4 was tested. Before the experiment, cell culture was carried out, and then the viability of L929 cells was detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method.

[0076] Cell resuscitation: Take out the cryopreservation tube from the ultra-low temperature refrigerator and quickly place it in a 37°C water bath to melt. Transfer the cells in a laminar flow hood, transfer the cells into a cell culture flask containing 4 mL of culture medium, and move it to a CO2 incubator for 4 h. After the cells adhere to the wall, remove the old culture medium, add fresh culture medium, and continue to culture in the CO2 incubator.

[0077] Cell passage: When the bottom of the culture flask is covered with cells, passage culture is carried out; remove the old culture medium in the culture flask, wash it 1-2 times with PBS buffer, add 0.25% trypsin for cell digestion, observe the cell morphology under an inverted microscope. When the cells become round and the intercellular space becomes larger, pour out the trypsin and add 4 mL of fresh medium. Use a pipette to blow back and forth until all the cells slide down. Transfer part of the suspension into a new culture flask, supplement the culture medium to 4 mL, and place it in a CO2 incubator for continued culture.

[0078] Place each hydrogel sample (8 mm in diameter) in a 24-well plate, sterilize it with 75% ethanol for 30 minutes, irradiate it with ultraviolet light for 30 minutes, and wash it twice with PBS buffer; inoculate the L929 cell suspension (4×105 cells / mL) into each well and culture for 24 hours. Then, aspirate the culture medium and add 300 μL of MTT solution to each well, culture at 37°C for 4 hours, remove the culture medium again, and add 300 μL of dimethyl sulfoxide. Mix well with a shaker for 5 minutes and transfer to a 96-well plate. Measure the absorbance at 570 nm using an enzyme-linked immunosorbent assay reader. Figure 5 This is the biocompatibility of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention.

[0079] From Figure 5It can be seen that the κ-carrageenan hydrogel added with PSPE has an obvious proliferative effect on the growth of L929 cells. Compared with the blank control group κC, there were significant differences in the κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% groups after 24 hours (P < 0.05). As the addition amount of PSPE in κC-PSPE increased, the cell viability was higher. Among them, the cell survival rate of κC-PSPE5% was the highest, indicating that the addition of PSPE increased the promoting effect of κ-carrageenan hydrogel on the viability of L929 cells; in addition, it also shows that the addition amount of PSPE is within a safe range.

[0080] Experimental Example 6

[0081] In this experiment, the κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% obtained in Examples 1-4 were used for the L929 cell ROS scavenging test. L929 cells in the logarithmic growth phase were digested with enzymes and inoculated on a 6-well plate (500 μL). After the cells adhered, the medium was removed, and each hydrogel sample and fresh medium were added. The positive control was the diluted ROSUP in the reactive oxygen species detection kit, and the negative control was the addition of fresh medium. After culturing in an incubator at 37°C and 5% for 1.5 h, it was washed twice with PBS, 500 μL of diluted DCFH-DA (diluted with PBS) was added, and it was incubated in the cell culture incubator at 37°C in the dark for 30 min, and measured at 488 nm with a fluorescence spectrophotometer. Figure 6 It is the ability of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention to scavenge intracellular ROS.

[0082] DCFH-DA staining was used to show the generation of intracellular ROS to study the intracellular antioxidant performance of κC-PSPE. The higher the DCF fluorescence density, the more ROS. As Figure 6 shown, the DCF fluorescence density decreased with the increase in the addition amount of PSPE. Introducing PSPE into the κ-carrageenan hydrogel can effectively inhibit the intracellular ROS level. In addition, after treatment with κC-PSPE5%, the DCF fluorescence decreased significantly.

[0083] Experimental Example 7

[0084] In this experiment, the κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% obtained in Examples 1-4 were used for the cell scratch experiment. To detect the migration performance of L929 cells, a line was scratched in the middle of the cells cultured for 24 hours for an in vitro scratch experiment. L929 cells (4×l0 5Cells / mL) were cultured in 24-well plates. When the L929 cells grew to 100%, a linear scratch was created on the back of the wells using a sterile 200 μL pipette tip. The cells were cultured in MEM medium containing 10% FBS. Then, each hydrogel sample (8 mm) was added and cultured in an incubator at 37 °C and 5% CO2 for 24, 48, and 72 hours, and the cell migration was examined under an inverted microscope. Figure 7 This is the cell migration experiment of κC, κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% of the present invention.

[0085] From Figure 7 It can be seen that after treatment with each hydrogel sample for 24 hours, significant cell migration was observed at the scratch edge, and a wider gap was observed in the blank control group; compared with the blank control group, the cell migration in the κC-PSPE1%, κC-PSPE3%, and κC-PSPE5% groups increased significantly. After 48 hours, the gap in the κC-PSPE5% group completely disappeared. After 72 hours, the gap in the κC-PSPE3% group completely disappeared. It can be seen from this that PSPE can significantly promote wound gap closure. Compared with the case of κC, the experimental data show that κC-PSPE can promote the migration of L929 cells.

[0086] It can be seen from the above Experimental Examples 1-7 that κC-PSPE has excellent mechanical properties and biocompatibility. The introduction of anthocyanins not only makes up for the functional defects of κ-carrageenan hydrogels in antioxidant and anti-inflammatory aspects, but also can effectively inhibit the ROS level in cells, has an obvious proliferative effect on cell growth, thereby promoting wound gap closure and shortening the wound healing time.

[0087] The anthocyanins used in the present invention are extracted from purple sweet potatoes, which are harmless to the human body and have good pH responsiveness. Under different pH conditions, the color of anthocyanins changes spontaneously. When the pH value is 5-7, it is purplish red. When the pH value is 7-8, it is blue. When the pH value is greater than 8, it is green. The color change can reach stability within 15-30 s. Therefore, the anthocyanin / κ-carrageenan hydrogel prepared in the present invention is extremely sensitive and very sensitive to environmental acid-base response, and can monitor the pH value of the wound by observing the color change, so as to understand the information on the wound healing status.

[0088] The above is only the preferred embodiment of the present invention, and it does not limit the design of this case. All equivalent changes made according to the key design of this case fall within the protection scope of this case.

Claims

1. A preparation method of a pH-responsive antioxidant hydrogel capable of promoting wound healing, characterized in that: It includes the following steps: Step (1), extraction and purification of purple sweet potato anthocyanins: S1. First, take 50 g of purple sweet potato powder, add it to 1000 mL of 0.1% HCl-ethanol solvent, add a certain amount of cellulase, and ultrasonicate for 30 min at 50 °C to obtain a crude extract of purple sweet potato anthocyanins; S2. Then, load the treated AB-8 resin into a chromatography column, control the flow rate at 30 rpm / min, dilute the crude extract of purple sweet potato anthocyanins, and pass it through the chromatography column at a flow rate of 20 rpm per 500 mL under natural acidity conditions. After the crude extract of purple sweet potato anthocyanins is loaded into the column, wash it with distilled water to remove the adsorbed sugars and other impurities in the chromatography column; S3. Finally, elute the purple sweet potato anthocyanins in the chromatography column with an ethanol solution, centrifugally rotate and evaporate to obtain a concentrated solution, and freeze-dry it for standby; Step (2), preparation of anthocyanin / κ-carrageenan hydrogel: S1. First, disperse the purple sweet potato anthocyanins prepared in step (1) in distilled water to prepare three different concentrations of purple sweet potato anthocyanin solutions, and the mass concentrations of the purple sweet potato anthocyanin solutions are 1% w / v, 3% w / v, and 5% w / v respectively; S2. Secondly, moisten 1 g of κ-carrageenan with 0.5 g of glycerol and dissolve it in 50 mL of deionized water to obtain a 2% κ-carrageenan solution, and stir and heat it to 80 °C for about 30 min in a magnetic stirrer; S3. Thirdly, mix the purple sweet potato anthocyanin solution prepared in S1 and the κ-carrageenan solution prepared in S2 according to a mass concentration ratio of 1:50, and continue to stir the mixture with a magnetic stirrer for 5 minutes, and perform ultrasonic degassing to obtain a pre-gel solution; S4. Next, pour the pre-gel solution into a 55 mm mold, keep it at room temperature for 2 - 5 minutes, and after forming a solid gel, crosslink it with a potassium chloride solution for further stabilization; S5. Finally, wash the solid gel prepared in S4 twice with distilled water to obtain the anthocyanin / κ-carrageenan hydrogel.

2. The preparation method of a pH-responsive antioxidant hydrogel capable of promoting wound healing according to claim 1, wherein: The addition amount of the cellulase in step (1) S1 is 54 U / mL.

3. The preparation method of a pH-responsive antioxidant hydrogel capable of promoting wound healing according to claim 1, characterized in that: The crude extract of purple sweet potato anthocyanins in step (1) S2 is diluted to 1 mg / mL.

4. The preparation method of a pH-responsive antioxidant hydrogel capable of promoting wound healing according to claim 1, characterized in that: The ethanol solution in step (1) S3 is an ethanol solution with a concentration of 60%.

5. The preparation method of a pH-responsive antioxidant hydrogel capable of promoting wound healing according to claim 1, characterized in that: The mold in step (2) S4 is a glass petri dish.

6. The preparation method of a pH-responsive antioxidant hydrogel capable of promoting wound healing according to claim 1, characterized in that: The potassium chloride solution in step (2) S4 is a potassium chloride solution with a concentration of 0.75%.

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