Puerarin hydrogel for promoting wound healing, preparation method and application thereof

Abstract

The present invention discloses a puerarin hydrogel for promoting wound healing, and its preparation method and application. The hydrogel comprises the following raw materials: puerarin and calcium sulfate. The hydrogel is prepared by adding puerarin and calcium sulfate into water and mixing them to obtain a mixed solution. The mixed solution is then stirred at high temperature until clarified, and cooled to room temperature to obtain the hydrogel. The puerarin content is 2wt%, and the calcium sulfate content is 0.08-0.2wt%. The present invention effectively improves the mechanical properties of the hydrogel by combining puerarin and gypsum, and can improve the effect of puerarin in promoting skin wound healing.

Description

Technical Field

[0001] The invention belongs to the field of chronic wound infection treatment, and particularly relates to a puerarin hydrogel with a nanofiber network structure for promoting wound healing, a preparation method thereof, and an application thereof in skin wound healing. Background Art

[0002] Skin defects are common in traumatic injuries and can lead to infection, serious complications, and even death. Conventional wound dressings commonly used in clinical practice include sterile dry gauze, oil-based gauze, and cotton wool. These dressings have varying degrees of exudate absorption capacity to maintain a dry wound environment and prevent the invasion of harmful bacteria and other microorganisms that hinder wound healing. However, skin wound healing is a complex process that requires a certain level of moisture for optimal recovery. Traditional dressings are not fully adapted to this requirement. Fortunately, advances in materials science have led to the development of novel wound dressings, such as 3D-printed scaffolds, sponges, and hydrogels. Hydrogel materials not only create an effective physical barrier within the wound but also create a moist environment conducive to proper healing. Modern hydrogel dressings not only promote effective wound healing but also reduce the frequency of dressing changes, thereby alleviating patient discomfort during these procedures and reducing clinical workload in healthcare.

[0003] The high swelling and breathability of hydrogels provide an optimal water environment for skin wound healing, promote adequate ventilation of skin cells, and offer new possibilities as wound dressings. Hydrogels can effectively form a physical barrier in wounds, creating a moist environment that promotes effective wound healing. In addition, they can also serve as carriers to load other therapeutic drugs, such as antimicrobial agents or cytokines, to achieve multifunctional treatments. Previously, the antimicrobial properties of hydrogel dressings were mainly due to the presence of antibiotics in the hydrogel matrix or the addition of other antimicrobial substances. However, due to their consistent antimicrobial activity, dressings with intrinsic antimicrobial activity are more desirable. Another challenge lies in the development of complex and cost-intensive wound dressings that promote wound healing by combining factors such as growth factors. Therefore, the inherent antimicrobial and antioxidant capabilities of wound dressings become very attractive.

[0004] Puerarin is the main bioactive component of the traditional Chinese medicine Pueraria root, which is composed of hydrophilic glucose and hydrophobic isoflavones. Puerarin has multiple pharmacological activities and can self-assemble into hydrogels without modification, overcoming the limitations caused by its low hydrophilicity and bioavailability. However, the single network structure of puerarin hydrogel makes it mechanically weak and easily disintegrates structurally. At the same time, Chinese herbal medicine (CHM) provides a huge wealth and inspiration for the development of innovative pharmaceutical materials and treatment methods. Inspired by the macroscopic compatibility of Pueraria root and gypsum in CHM, the present invention co-assembles puerarin (PUE) and calcium sulfate (CaSO4) into a two-component molecular hydrogel. Importantly, the two-component gel has good biocompatibility, antibacterial and antioxidant properties, and can effectively promote wound healing. Summary of the Invention

[0005] The purpose of the present invention is to solve the existing problems and provide a puerarin hydrogel for promoting wound healing, a preparation method and an application thereof. The hydrogel of the present invention has high antioxidant and antibacterial properties and the ability to promote wound healing.

[0006] To achieve these objects and other advantages of the present invention, a hydrogel for promoting skin wound healing is provided, characterized in that the hydrogel is prepared by adding puerarin and calcium sulfate to water and mixing them to obtain a mixed solution, then stirring the mixed solution at a high temperature of 85 to 99° C. (more preferably, 88 to 92° C., and most preferably 90° C.) until it becomes clear, and cooling the solution at 20 to 30° C. to obtain the hydrogel.

[0007] Preferably, a hydrogel for promoting skin wound healing is made from the following raw materials in the following weight percentages: 1-5% puerarin; 0.03-0.3% calcium sulfate; the balance water. More preferably, the puerarin content is 1.5-2.5%; the calcium sulfate content is 0.08-0.2%; the balance water. Most preferably, the puerarin content is 2% by weight and the calcium sulfate content is 0.08-0.2% by weight.

[0008] The present invention also provides an application of a hydrogel in promoting skin wound healing, and an application of a hydrogel in preparing a drug for promoting skin wound healing. 2- Hydrogen bonds are formed between them, and a blue shift occurs after adding calcium sulfate, because PUE and SO4 2- The newly formed competitive hydrogen bonds between the PUE molecules may have partially disrupted the hydrogen bonds between them. Furthermore, a blue shift in the stretching vibration frequency of the C=O atoms in PUE was observed after the addition of calcium sulfate. XRD results also showed that the addition of calcium sulfate did not significantly alter the local structure of the PUE assembly. The interaction between PUE and calcium sulfate enhanced the mechanical properties of the puerarin hydrogel and promoted skin wound healing.

[0009] Advantages and benefits of this invention: Inspired by the macroscopic compatibility of kudzu root and gypsum in CHM, puerarin and calcium sulfate were co-assembled into a two-component molecular hydrogel. Compared to single-component PUE gels, the mechanical properties of the two-component PUE gels were enhanced due to the introduction of cross-linking hydrogen bonds between PUE and CaSO₄. Importantly, the two-component gels exhibited excellent biocompatibility and antibacterial and antioxidant properties. Furthermore, the two-component gels exhibited excellent wound healing properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the gelation of hydrogels of Examples 1-4 of the present invention.

[0011] Figure 2 1-4 are SEM images and diameter distributions of the hydrogels of Examples 1-4 of the present invention.

[0012] Figure 3 These are the infrared spectra and X-ray diffraction patterns of the hydrogels of Examples 1-4 of the present invention.

[0013] Figure 4 are G' and G" of the hydrogels of Examples 1-4 of the present invention at different strain amplitudes.

[0014] Figure 5 Graphs of G' and G" at different angular frequencies, viscosity at different shear rates, and syringeability of hydrogels of Examples 1-4 of the present invention are shown.

[0015] Figure 6 These are the cytotoxicity and hemolysis rates of the hydrogels of Examples 1-4 of the present invention.

[0016] Figure 7 This is the antibacterial property of the hydrogels of Examples 1-4 of the present invention.

[0017] Figure 8 is the free radical scavenging ability of the hydrogels of Examples 1-4 of the present invention.

[0018] Figure 9 1-4 hydrogels of the present invention are used to heal wounds in mice.

[0019] Figure 10 This is the histopathological staining analysis of the mouse wound healing of the hydrogels of Examples 1-4 of the present invention.

[0020] Figure 11 The figure is a schematic flow chart of the method for preparing the hydrogel for promoting skin wound healing of the present invention. DETAILED DESCRIPTION

[0021] To further illustrate the present invention, the preparation and application of the supramolecular hydrogel with a nanofiber network structure of the present invention are described in detail below with reference to the examples.

[0022] Example 1 PUE hydrogel

[0023] The hydrogel of this embodiment is prepared from the following raw materials by weight:

[0024] Isoflavones: Puerarin 2wt%;

[0025] The balance is water.

[0026] Preparation method:

[0027] According to the raw material formula, the weighed puerarin was first added into deionized water, stirred at 90°C for 15 minutes until the puerarin was completely dissolved, and then cooled at room temperature of 25°C to form a gel, which was recorded as PUE hydrogel.

[0028] Example 2 PCa-1 hydrogel

[0029] The hydrogel of this embodiment is prepared from the following raw materials by weight:

[0030] Isoflavones: Puerarin 2wt%;

[0031] Calcium sulfate: 0.0825wt%;

[0032] The balance is water.

[0033] Preparation method:

[0034] According to the raw material formula, weighed puerarin and calcium sulfate were first added to deionized water, stirred at 90°C for 15 minutes until the puerarin was completely dissolved, and then cooled at room temperature of 25°C to form a gel, which was recorded as PCa-1 hydrogel.

[0035] Example 3 PCa-2 hydrogel

[0036] The hydrogel of this embodiment is prepared from the following raw materials by weight:

[0037] Isoflavones: Puerarin 2wt%;

[0038] Calcium sulfate: 0.165wt%;

[0039] The balance is water.

[0040] Preparation method:

[0041] According to the raw material formula, weighed puerarin and calcium sulfate were first added to deionized water, stirred at 90°C for 15 minutes until the puerarin was completely dissolved, and then cooled at room temperature of 25°C to form a gel, which was recorded as PCa-2 hydrogel.

[0042] Example 4 PCa-3 hydrogel

[0043] The hydrogel of this embodiment is prepared from the following raw materials by weight:

[0044] Isoflavones: Puerarin 2wt%;

[0045] Calcium sulfate: 0.2wt%;

[0046] The balance is water.

[0047] Preparation method:

[0048] According to the raw material formula, weighed puerarin and calcium sulfate were first added to deionized water, stirred at 90°C for 15 minutes until the puerarin was completely dissolved, and then cooled at room temperature of 25°C to form a gel, which was recorded as PCa-3 hydrogel.

[0049] The gelation results of the supramolecular hydrogel with nanofiber network structure prepared in Examples 1-4 are as follows: Figure 1 shown.

[0050] 1. Experimental Characterization

[0051] 1.1 Fiber morphology

[0052] The effect of calcium sulfate on the gel morphology was observed by scanning electron microscopy. Figure 2 As shown in Figure e (i.e., Example 1), the dry gel of PUE presents a typical dense and fibrous structure with an average diameter of 57 nm. Figure 2 As shown in Figure f (i.e., Example 2), for PCa-1 gel, the addition of a small amount of calcium sulfate causes the nanofibers to become thinner, with a diameter reduced to 36 nm. The reduction in nanofiber diameter can be attributed to the addition of SO4 2- The hydrogen bonds formed between -OH and -OH will affect the bundling of nanofibers during their three-dimensional growth. However, further increase of calcium sulfate leads to thickening of gel fibers, PCa-2 (ie, Example 3) and PCa-3 (ie, Example 4), respectively. Figure 2 g and 2h. The increase in the diameter of the gel nanofibers can be explained by the fact that more SO4 2- More hydrogen bonds are generated, thus connecting more gel nanofiber bundles laterally. In summary, the SEM data showed that the morphology of the gel network remained almost the same, but nanofibers of different diameters were formed depending on the amount of added calcium sulfate.

[0053] 1.2 FTIR spectroscopy analysis

[0054] like Figure 3 As shown, PUE xerogel is at 3320 cm -1 With the increase of calcium sulfate content, the stretching vibration of -OH in PCa-1, PCa-2 and PCa-3 blue-shifts to 3340 cm -1 、3349cm-1 and 3407cm -1 This blue-shift behavior indicates that due to the PUE and SO4 2- The newly formed competitive hydrogen bonds between the PUE molecules may be partially destroyed. In addition, after the addition of calcium sulfate, it was observed that the stretching vibration frequency of C=O in PUE also underwent a blue shift (from 1695cm -1 to 1698 cm in PCa-1, PCa-2, and PCa-3 -1 ). It is reported that the C=O···π interaction is the main driving force for the assembly of PUE molecules into gel networks. Here, the blue shift of the C=O stretching vibration frequency induced by CaSO4 indicates that the -OH of PUE interacts with SO4 2- Hydrogen bonds are formed between them, and the C=O···π interaction between PUE molecules may also be partially disturbed.

[0055] 1.3 X-ray diffraction

[0056] like Figure 3 As shown in Figure 2, for the PUE xerogel sample, a broad diffraction peak centered at 20° indicates the amorphous nature of the PUE gel structure. The corresponding spacing is calculated as This is probably due to the presence of C=O···π interactions. After adding calcium sulfate, PCa-1, PCa-2, and PCa-3 xerogel samples all showed similar diffraction patterns, especially their diffraction spectra were just the overlap of pure PUE and calcium sulfate diffraction. This result indicates that the addition of calcium sulfate did not significantly change the local structure of the PUE assembly, and assuming that SO4 2- are adsorbed on the periphery of the PUE components rather than inserted into the layered and assembled PUE molecules.

[0057] 1.4 Rheological test

[0058] We performed rheological measurements and first used a strain sweep mode to probe the linear viscoelastic region of the hydrogel. The sweep frequency was kept at 1 Hz and the strain range was 0.01–10%. Figure 4 As shown, the storage modulus (G') of the four hydrogels is higher than the loss modulus (G") when the strain does not exceed 10%. Compared with the PUE hydrogel, all gels containing CaSO4 show higher yield strain points.

[0059] At the same time, Figure 5Angular frequency sweeps between 0.01 and 100 rad / s revealed that G' for all gels was also greater than G", demonstrating the gelling properties of these samples. All CaSO4-containing gels exhibited higher G' than the PUE gels. These rheological tests indicate that the addition of calcium sulfate increases the crosslinking density, thereby improving the mechanical properties of the hydrogels. Furthermore, we found that the viscosity of all hydrogels decreased with increasing shear rate, indicating that these hydrogels exhibit shear-thinning properties and can be used as injectable materials. This injectability is believed to be primarily due to the presence of a dynamic, non-covalently interacting gel network.

[0060] 1.5 Biocompatibility

[0061] like Figure 6 As shown in a, the survival rate of L929 cells after incubation with PUE, PCa-1, PCa-2 and PCa-3 hydrogels for 4 days remained above 85%, indicating that all these gels have very low cytotoxicity. Figure 6 Live / dead cell staining experiments in c showed that L929 cells in the control group and hydrogel group showed spindle morphology after 1, 2, and 4 days, with almost no cell death (red), further confirming the good biocompatibility of the hydrogel.

[0062] Blood compatibility is another important prerequisite for the application of biomaterials in wound healing, because wound dressings can directly contact the blood at the wound site. Figure 6 As shown in (b), all light yellow hydrogel groups showed significant color differences from the positive control (deionized water). Quantitative analysis revealed that the hemolysis rates of PCa-1, PCa-2, and PCa-3 hydrogels were below the internationally recognized 5% standard, while the hemolysis rate of the PUE hydrogel exceeded this threshold, indicating that all PCa hydrogels, except the PUE hydrogel, exhibited good hemocompatibility.

[0063] 1.6 In vitro antibacterial and antioxidant efficacy

[0064] The present invention uses Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli as model strains to evaluate the antibacterial activity of the hydrogel. The optical density (OD) of the bacteria at different time points was measured. 600 ) to determine the bacterial growth curve, which provides a basis for understanding the antibacterial properties of hydrogels. Figure 5 As shown in Figures 5b and 5c, compared with the control group, the PUE, PCa-1, PCa-2, and PCa-3 groups inhibited the growth of both strains, while the calcium sulfate group showed no antibacterial activity. Furthermore, the antibacterial properties of the hydrogels were further evaluated by experiments on plates. Compared with the blank group and the calcium sulfate group, the PUE, PCa-1, PCa-2, and PCa-3 gel groups showed antibacterial activity against Staphylococcus aureus and Escherichia coli.

[0065] We then evaluated the antioxidant function of these hydrogels. The antioxidant activity of PUE, PCa-1, PCa-2, and PCa-3 hydrogels was evaluated by measuring the scavenging rates of DPPH and ABTS free radicals. Figure 8 At 1 mg / mL (based on PUE concentration), the hydrogel had weak scavenging abilities for both DPPH and ABTS. However, when the PUE concentration reached 3 mg / mL, more than 80% of DPPH and 91% of ABTS free radicals were scavenged.

[0066] 1.7 In vivo wound healing

[0067] The PCa hydrogel demonstrated excellent in vitro biocompatibility, antibacterial, and antioxidant properties. A full-thickness wound model of E. coli infection in rats was established to further evaluate its in vivo wound healing effect. Wounds in each group were photographed on days 0, 1, 3, 6, and 10, and the wound area was quantified. One day after treatment, yellow pustules appeared at the wound site in the blank group, indicating that the E. coli-infected skin defect model was successfully established. On day 3, the wound area of ​​all six treatment groups decreased. As the healing process extended to day 10, the wounds in the Tegadrem, PUE, PCa-1, PCa-2, and PCa-3 groups were completely closed and healed, while the wound in the blank group remained noticeable and larger. Quantitatively, there were no significant differences among Tegadrem, PUE, PCa-1, PCa-2, and PCa-3 treatments, with wound closure rates of 95.32%, 90.34%, 89.94%, 90.50%, and 91.08%, respectively, which were significantly higher than those in the blank group, indicating that PCa hydrogel exhibited effective wound healing ability.

[0068] 1.8 Histological analysis

[0069] We performed histological and immunological analyses to further understand the process of wound healing, e.g. Figure 10 H&E staining results in Figure a showed that granulation tissue formation was significantly impaired in all groups on day 5. The collagen density in the granulation tissue of the PCa-2 and Tagaderm groups was higher than that of the blank control group, and the number of inflammatory cells was also lower. By day 10, the dermis in the blank control group had clearly not healed, while the epidermis in the PCa-2 and PCa-3 groups had relatively complete regeneration, with visible hair follicles and sebaceous glands near the incision. Figure 10 The Masson staining results in b also showed that the collagen arrangement on the wound surface of the PCa-2 and PCa-3 groups was very similar to that of normal cells, accompanied by more complete epidermal regeneration.

[0070] In summary, PUE / CaSO4 hydrogel has good biocompatibility and antibacterial and antioxidant properties. In addition, the results from the Escherichia coli-infected mouse wound model showed that PUE / CaSO4 hydrogel has the properties of effectively promoting wound healing and has good application prospects.

[0071] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully adapted to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. A puerarin hydrogel for promoting wound healing, characterized in that: Made from the following raw materials in percentage by mass: Puerarin 1-5%; Calcium sulfate 0.03-0.3%; Water balance.

2. The puerarin hydrogel for promoting wound healing according to claim 1, wherein Made from the following raw materials in percentage by mass: Puerarin 1.5-2.5%; Calcium sulfate 0.08-0.2%; Water balance.

3. The method for preparing the puerarin hydrogel for promoting wound healing according to claim 1 or 2, wherein: The following steps are involved: Puerarin and calcium sulfate are added to water and mixed to obtain a mixed solution, and then the mixed solution is stirred at a high temperature of 85-99°C until it becomes clear, and cooled to obtain a puerarin hydrogel that promotes wound healing.

4. The method for preparing the puerarin hydrogel for promoting wound healing according to claim 3, wherein: The mixed solution was stirred at a high temperature of 88-92°C until it became clear.

5. The method for preparing the puerarin hydrogel for promoting wound healing according to claim 3, wherein: Cool at 20~30℃.

6. Use of the puerarin hydrogel for promoting wound healing according to claim 1 or 2 in the preparation of a medicament for promoting the healing of skin wounds infected by Escherichia coli.

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