Citrus flavonoid loaded hydrogel wound dressing and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a citrus flavonoid loaded hydrogel wound dressing and a preparation method thereof. The citrus flavonoid loaded hydrogel wound dressing provided by the invention is prepared by taking carboxymethyl chitosan and oxidized sodium alginate as matrixes and loading citrus flavonoid micromolecular active substances. The hydrogel wound dressing prepared by the invention has better mechanical property and good biocompatibility, also has the effect of continuously releasing citrus flavonoid substances, can meet the requirements of wound healing in different periods on antibacterial, anti-inflammatory, cell growth and the like, accelerates wound healing, and has the advantages of no toxic or side effect and low cost. Meanwhile, formation of wound scar tissues can be reduced, and the hydrogel wound dressing is a novel hydrogel wound dressing.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to a hydrogel wound dressing loaded with citrus flavonoids and a preparation method thereof. Background Art

[0002] As the largest organ in the human body, the skin is in direct contact with the external environment. However, the skin is relatively soft and inevitably suffers varying degrees of damage in daily life. Although the skin has a relatively good regenerative ability, severe wounds or complications caused by chronic diseases are difficult to recover on their own. During the wound healing process, wound dressings can act as a barrier against external microorganisms and help damaged skin regenerate quickly, thereby promoting wound healing. Traditional wound dressings such as gauze are difficult to achieve a sealed environment and are prone to adhesion during dressing changes, resulting in secondary damage. New wound dressings can act as a barrier to protect against the invasion of foreign pathogens, and can also promote wound healing by promoting cell attachment, proliferation and migration.

[0003] Hydrogels, a novel wound dressing with a three-dimensional mesh structure, have garnered widespread attention. Hydrogels, with their high water absorption and moisture retention, can absorb tissue permeate while providing a moist environment at the wound site. This moist wound environment can accelerate the release of growth factors, promoting the softening, dissolution, and elimination of necrotic tissue and scabs. At the same time, hydrogels can also create a hypoxic or anaerobic environment, better stimulating macrophages to release growth factors and promoting the growth of fibroblasts and capillaries. Self-healing hydrogels can rapidly cross-link in a short period of time, allowing chemical bonds to be repaired at the breakage site. This allows for better environmental adaptability and extends the life of wound dressings, reducing the frequency of changes and alleviating pain for patients, thus better meeting consumer requirements.

[0004] Patent document CN104069537A discloses a sodium alginate-sodium carboxymethyl cellulose-chitosan wound dressing. This wound dressing is mainly made of raw materials such as sodium alginate, sodium carboxymethyl cellulose, chitosan, glycerol and water. Specifically, sodium alginate and sodium carboxymethyl cellulose are added to water, and then chitosan and acetic acid are added. After obtaining a uniform solution, it is poured into a culture dish and freeze-dried for the first time; then it is sequentially placed in a CaCl2 solution and a NaOH solution for immersion, and after a second freeze-drying, it is immersed in a glycerol aqueous solution and then freeze-dried for a third time to obtain the product. This wound dressing has high absorbability and can maintain a moist wound environment. It also has the characteristics of low adhesion and easy replacement, which is conducive to wound healing.

[0005] Patent document CN118490873A discloses a hydrogel wound dressing and its preparation method. This hydrogel wound dressing utilizes methacryloylated gelatin and carboxymethyl chitosan as raw materials, which are physically cross-linked through multiple hydrogen bonds to create a composite hydrogel wound dressing with wound repair and angiogenic properties. This hydrogel wound dressing not only ensures the long-lasting effect and effective controlled release of growth factors in a physiological environment, but also maintains the excellent biocompatibility, high availability, and good bioactivity of the hydrogel material.

[0006] However, current hydrogel dressings can promote the wound healing process, but a single gel component cannot meet the antibacterial, anti-inflammatory and cell growth needs of wounds at different stages, nor can it meet the therapeutic effects of different types of wounds. Summary of the Invention

[0007] In order to solve the defects of the prior art, the present invention provides a hydrogel wound dressing loaded with citrus flavonoid active substances. The hydrogel wound dressing provided by the present invention is made of carboxymethyl chitosan and oxidized sodium alginate as a matrix, and loaded with citrus flavonoid small molecule active substances. The citrus flavonoids used in the present invention have multiple active functions such as antibacterial, anti-inflammatory, antioxidant, anti-apoptosis, anti-tumor, improvement of myocardial damage and liver damage, and regulation of blood lipids. They are loaded in a three-dimensional carboxymethyl chitosan / oxidized sodium alginate hydrogel matrix and have a sustained release effect. They can meet the needs of wounds of different periods or different types for antibacterial, anti-inflammatory and cell growth, accelerate wound healing, and reduce the formation of scar tissue on the wound surface. It not only expands the application path of citrus flavonoids, but also provides a new research idea for new wound dressing loading materials.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] The present invention provides a method for preparing a hydrogel wound dressing loaded with citrus flavonoids, comprising the following steps:

[0010] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 2500-3500 rpm for 1-3 hours to obtain a carboxymethyl chitosan solution;

[0011] Step S2, dissolving oxidized sodium alginate in deionized water, and stirring at a rotation speed of 2500-3500 rpm for 25-35 minutes to obtain an oxidized sodium alginate solution;

[0012] Step S3, dissolving the citrus flavonoids in deionized water and stirring evenly to obtain a citrus flavonoid solution;

[0013] Step S4: mixing the carboxymethyl chitosan solution obtained in step S1 with the oxidized sodium alginate solution obtained in step S2, stirring evenly to obtain a mixed solution I; adding the citrus flavonoid solution obtained in step S3 to the mixed solution I, stirring evenly to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 1 to 3 minutes, and demolding to obtain the product.

[0014] Furthermore, the carboxymethyl chitosan concentration of the carboxymethyl chitosan solution in step S1 is 3-6 mg / mL, and more preferably 4 mg / mL.

[0015] Furthermore, the preparation method of oxidized sodium alginate in step S2 is:

[0016] Sodium alginate was oxidized and modified with sodium periodate as an oxidant for 6 hours, anhydrous ethanol was added for precipitation, and vacuum filtration was performed to obtain a precipitate; the precipitate was washed with deionized water, impurities were removed with anhydrous ethanol, and the ethanol was removed by rotary evaporation, and dialyzed with distilled water to obtain a dialysate; and the precipitate was freeze-dried to obtain the product.

[0017] Furthermore, the specific preparation method of the oxidized sodium alginate is:

[0018] Step A: suspending 10 g of sodium alginate in 50 mL of anhydrous ethanol to obtain a sodium alginate suspension; dissolving 8 g of sodium periodate in 50 mL of deionized water to obtain a sodium periodate solution; introducing the sodium periodate solution into the sodium alginate suspension, stirring the mixture magnetically at room temperature in the dark for 6 h, then adding 3 mL of ethylene glycol, and stirring the mixture for another 1 h to completely terminate the chemical reaction, thereby obtaining a mixed solution;

[0019] Step B: In order to purify the product, 150 mL of anhydrous ethanol was introduced to completely precipitate the mixed solution prepared in step A to obtain a precipitate. The precipitate was vacuum filtered and then washed with deionized water. Anhydrous ethanol was repeatedly added three times under vigorous stirring to effectively eliminate any residual unreacted impurities. The obtained white product was collected, deionized water was introduced into the purified white product, and then rotary evaporated at a temperature of 60° C. to remove ethanol. The product was dialyzed with distilled water for 24 hours, with the water changed every 12 hours (MW cutoff = 3500 kDa) to remove unreacted sodium periodate and small molecular impurities such as ethylene glycol every 12 hours to obtain a dialysate. The liquid in the dialysis bag was freeze-dried for 48 hours to finally obtain oxidized sodium alginate.

[0020] Furthermore, the concentration of oxidized sodium alginate in the oxidized sodium alginate solution in step S2 is 3-6 mg / mL, and more preferably 4 mg / mL.

[0021] Furthermore, the citrus flavonoids in step S3 include but are not limited to naringin, naringenin, hesperidin and hesperetin, and naringin is more preferred.

[0022] Furthermore, the concentration of citrus flavonoids in the citrus flavonoid solution in step S3 is 0.5-2 mmol / L, and more preferably 1 mmol / L.

[0023] Furthermore, in step S4, the volume ratio of the carboxymethyl chitosan solution to the oxidized sodium alginate solution is (1-7):(1-3), and more preferably the volume ratio is 1:1.

[0024] Furthermore, in step S4, the amount of the citrus flavonoid solution added is 8-15% (v / v) of the total volume of the mixed solution I, and more preferably 10% (v / v) of the total volume of the mixed solution I.

[0025] At the same time, the present invention also claims protection for the citrus flavonoid-loaded hydrogel wound dressing prepared by the preparation method of the citrus flavonoid-loaded hydrogel wound dressing.

[0026] In addition, the citrus flavonoid-loaded hydrogel wound dressing provided by the present invention can also contain ferulic acid in addition to the citrus flavonoid substance as an active ingredient. The specific steps are: dissolving ferulic acid in deionized water and stirring evenly to prepare a ferulic acid solution with a concentration of 1 mmol / L, wherein the volume ratio of the ferulic acid solution to the naringin solution is (1-2):3.

[0027] The citrus flavonoid-loaded hydrogel wound dressing provided by the present invention comprises sodium alginate modified by sodium periodate oxidation to oxidized sodium alginate, and the oxidized sodium alginate solution and carboxymethyl chitosan solution are mixed uniformly in a specific ratio to prepare a hydrogel precursor solution. Subsequently, an active small molecule citrus flavonoid solution is added, stirred evenly, poured into a mold, and allowed to stand for 2 minutes. After demolding, a carboxymethyl chitosan / oxidized sodium alginate / citrus flavonoid hydrogel patch is obtained. The citrus flavonoid-loaded hydrogel wound dressing prepared by the present invention has the advantages of short preparation time, high transparency, good adhesion, and easy replacement.

[0028] Furthermore, the inventors studied the micromorphology, mechanical properties, basic physical and chemical properties of the hydrogel wound dressing loaded with citrus flavonoids, and the influence of the citrus flavonoids on its release performance, and confirmed that the hydrogel wound dressing loaded with citrus flavonoids prepared by the present invention has the advantages of strong self-healing ability, good adhesion performance, and good sustained release effect of active ingredients. The hydrogel wound dressing can be directly and firmly adhered to the wound surface of the tissue to keep the wound clean and moist and prevent external infection. At the same time, the hydrogel wound dressing also has the function of continuously releasing citrus flavonoid substances, which can meet the needs of wound healing in different stages in terms of antibacterial, anti-inflammatory and cell growth, accelerate wound healing, and reduce the formation of scar tissue on the wound surface. It is a new type of hydrogel wound dressing.

[0029] Furthermore, the inventors established an animal wound infection model to evaluate the therapeutic efficacy of the citrus flavonoid-loaded hydrogel wound dressing on wounds of mice infected with Staphylococcus aureus. Experiments demonstrated that wounds of injured mice treated with the citrus flavonoid-loaded hydrogel wound dressing gradually healed by day 7, and by day 14, the wounds were completely closed with virtually no scarring.

[0030] Furthermore, in-depth research has revealed that combining naringin and ferulic acid in a specific ratio can synergistically accelerate wound healing and shorten wound healing time. Experiments have shown that compared to hydrogel wound dressings containing naringin alone, wounds covered with the naringin-ferulic acid hydrogel dressing began to gradually heal on day 6, and by day 11, the wounds were completely closed without scarring, effectively shortening wound healing time.

[0031] Compared with the prior art, the citrus flavonoid-loaded hydrogel wound dressing provided by the present invention has the following advantages:

[0032] (1) The citrus flavonoid-loaded hydrogel wound dressing provided by the present invention has the advantages of natural and safe ingredients, good biocompatibility and degradability. It also has a short preparation time and can be formed after standing for 2 minutes. It does not require the addition of any chemical cross-linking agent and has high promotion potential.

[0033] (2) The citrus flavonoid-loaded hydrogel wound dressing provided by the present invention achieves the effects of slow release, sustained release, and sequential delivery of the active ingredients of the citrus flavonoids, meeting the wound's needs for antibacterial, anti-inflammatory, and cell growth at different stages, promoting wound cell adhesion and migration, accelerating skin regeneration, preventing scab formation, and promoting wound healing. This not only expands the application of citrus flavonoids but also provides a new research approach for novel wound dressing loading materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The infrared spectra of sodium alginate (SA) and oxidized sodium alginate (OSA).

[0035] Figure 2 This is a diagram showing the self-healing results of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4.

[0036] Figure 3 This is an electron micrograph of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4.

[0037] Figure 4 This is a graph showing the adhesion test results of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 on a bent finger.

[0038] Figure 5This is a graph showing the adhesion test results of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 on pig skin.

[0039] Figure 6 This is a frequency scanning test chart of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4, Example 6, Example 7 and Example 8.

[0040] Figure 7 This is a temperature scanning test chart of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4, Example 6, Example 7 and Example 8.

[0041] Figure 8 This is a graph showing the hardness results of the citrus flavonoid-loaded hydrogel wound dressings prepared in Examples 4, 6, 7, and 8.

[0042] Figure 9 This is a graph showing the swelling performance results of the citrus flavonoid-loaded hydrogel wound dressings prepared in Examples 4, 6, 7, and 8.

[0043] Figure 10 This is a graph showing the drying rate results of the citrus flavonoid-loaded hydrogel wound dressings prepared in Examples 4, 6, 7, and 8.

[0044] Figure 11 This is a graph showing the in vitro release rate results of the citrus flavonoid-loaded hydrogel wound dressings prepared in Examples 4, 6, 7, and 8.

[0045] Figure 12 This is a graph showing the wound healing results of mice using the citrus flavonoid-loaded hydrogel wound dressings prepared in Examples 2, 3, 4, and 5. DETAILED DESCRIPTION

[0046] The present invention will be further described below by way of specific embodiments, but this is not intended to limit the present invention. Those skilled in the art may make various modifications or improvements based on the basic concept of the present invention, but as long as they do not depart from the basic concept of the present invention, they are all within the scope of the present invention. The raw materials involved in the present invention can be obtained by commercially available or conventional techniques in the art.

[0047] Example 1. Preparation of oxidized sodium alginate

[0048] 1. Preparation steps of oxidized sodium alginate:

[0049] Step A: suspending 10 g of sodium alginate in 50 mL of anhydrous ethanol to obtain a sodium alginate suspension; dissolving 8 g of sodium periodate in 50 mL of deionized water to obtain a sodium periodate solution; introducing the sodium periodate solution into the sodium alginate suspension, stirring the mixture magnetically at room temperature in the dark for 6 h, then adding 3 mL of ethylene glycol, stirring the mixture for another 1 h to terminate the chemical reaction, and obtaining a mixed solution;

[0050] Step B: 150 mL of anhydrous ethanol is introduced into the mixed solution prepared in step A to obtain a precipitate. The precipitate is vacuum filtered, washed with deionized water, and then anhydrous ethanol is repeatedly added three times to remove impurities. The resulting white product is collected, deionized water is introduced into the purified white product, and then rotary evaporated at a temperature of 60° C.; dialyzed with distilled water for 24 hours, changing the water every 12 hours to obtain a dialysate; and the dialysate is freeze-dried to finally obtain oxidized sodium alginate.

[0051] 2. Detection of oxidized sodium alginate:

[0052] Sodium alginate (SA) and oxidized sodium alginate (OSA) were used as test samples and the infrared spectra were measured using a Fourier transform infrared spectrometer. Figure 1 As shown, the FTIR spectrum shows that the broad absorption peak around 3440 cm⁻¹ is the hydroxyl stretching vibration peak of SA and OSA. Compared with the SA spectrum, the OSA spectrum shows a strong and sharp absorption peak around 1725 cm⁻¹, indicating that the C=O absorption peak vibration is significantly enhanced, the aldehyde group is generated, and the SA is effectively oxidized.

[0053] Example 2: Preparation of a hydrogel wound dressing loaded with citrus flavonoids

[0054] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 3000 rpm for 2 h, and preparing a carboxymethyl chitosan solution with a concentration of 4 mg / mL;

[0055] Step S2: dissolving the oxidized sodium alginate prepared in Example 1 in deionized water, stirring at a rotation speed of 3000 rpm for 30 minutes, and preparing an oxidized sodium alginate solution with a concentration of 4 mg / mL;

[0056] Step S3, dissolving naringin in deionized water, stirring evenly, to prepare a naringin solution with a concentration of 1 mmol / L;

[0057] Step S4: mixing the carboxymethyl chitosan solution prepared in step S1 and the oxidized sodium alginate solution prepared in step S2 in a volume ratio of 7:3, and stirring uniformly to obtain a mixed solution I; adding the naringin solution prepared in step S3 to the mixed solution I, wherein the amount of the naringin solution added is 10% (v / v) of the total volume of the mixed solution I, and stirring uniformly to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 2 minutes, and demolding to obtain the product.

[0058] Example 3: Preparation of a hydrogel wound dressing loaded with citrus flavonoids

[0059] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 3000 rpm for 2 h, and preparing a carboxymethyl chitosan solution with a concentration of 4 mg / mL;

[0060] Step S2: dissolving the oxidized sodium alginate prepared in Example 1 in deionized water, stirring at a rotation speed of 3000 rpm for 30 minutes, and preparing an oxidized sodium alginate solution with a concentration of 4 mg / mL;

[0061] Step S3, dissolving naringin in deionized water, stirring evenly, to prepare a naringin solution with a concentration of 1 mmol / L;

[0062] Step S4: mixing the carboxymethyl chitosan solution prepared in step S1 and the oxidized sodium alginate solution prepared in step S2 in a volume ratio of 3:2, and stirring uniformly to obtain a mixed solution I; adding the naringin solution prepared in step S3 to the mixed solution I, wherein the amount of the naringin solution added is 10% (v / v) of the total volume of the mixed solution I, and stirring uniformly to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 2 minutes, and demolding to obtain the product.

[0063] Example 4: Preparation of a hydrogel wound dressing loaded with citrus flavonoids

[0064] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 3000 rpm for 2 h, and preparing a carboxymethyl chitosan solution with a concentration of 4 mg / mL;

[0065] Step S2: dissolving the oxidized sodium alginate prepared in Example 1 in deionized water, stirring at a rotation speed of 3000 rpm for 30 minutes, and preparing an oxidized sodium alginate solution with a concentration of 4 mg / mL;

[0066] Step S3, dissolving naringin in deionized water, stirring evenly, to prepare a naringin solution with a concentration of 1 mmol / L;

[0067] Step S4: mixing the carboxymethyl chitosan solution prepared in step S1 and the oxidized sodium alginate solution prepared in step S2 in a volume ratio of 1:1, and stirring uniformly to obtain a mixed solution I; adding the naringin solution prepared in step S3 to the mixed solution I, wherein the amount of the naringin solution added is 10% (v / v) of the total volume of the mixed solution I, and stirring uniformly to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 2 minutes, and demolding to obtain the product.

[0068] Example 5: Preparation of a hydrogel wound dressing loaded with citrus flavonoids

[0069] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 3000 rpm for 2 h, and preparing a carboxymethyl chitosan solution with a concentration of 4 mg / mL;

[0070] Step S2: dissolving the oxidized sodium alginate prepared in Example 1 in deionized water, stirring at a rotation speed of 3000 rpm for 30 minutes, and preparing an oxidized sodium alginate solution with a concentration of 4 mg / mL;

[0071] Step S3, dissolving naringin in deionized water, stirring evenly, to prepare a naringin solution with a concentration of 1 mmol / L;

[0072] Step S4: mixing the carboxymethyl chitosan solution prepared in step S1 and the oxidized sodium alginate solution prepared in step S2 in a volume ratio of 2:3, stirring uniformly, to obtain a mixed solution I; adding the naringin solution prepared in step S3 to the mixed solution I, wherein the amount of the naringin solution added is 10% (v / v) of the total volume of the mixed solution I, stirring uniformly, to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 2 minutes, and demolding to obtain the product.

[0073] Example 6: Preparation of a hydrogel wound dressing loaded with citrus flavonoids

[0074] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 3000 rpm for 2 h, and preparing a carboxymethyl chitosan solution with a concentration of 4 mg / mL;

[0075] Step S2: dissolving the oxidized sodium alginate prepared in Example 1 in deionized water, stirring at a rotation speed of 3000 rpm for 30 minutes, and preparing an oxidized sodium alginate solution with a concentration of 4 mg / mL;

[0076] Step S3, dissolving naringenin in deionized water and stirring evenly to prepare a naringenin solution with a concentration of 1 mmol / L;

[0077] Step S4: mixing the carboxymethyl chitosan solution prepared in step S1 and the oxidized sodium alginate solution prepared in step S2 in a volume ratio of 1:1 and stirring uniformly to obtain a mixed solution I; adding the naringenin solution prepared in step S3 to the mixed solution I, wherein the amount of the naringenin solution added is 10% (v / v) of the total volume of the mixed solution I, and stirring uniformly to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 2 minutes, and demolding to obtain the product.

[0078] Example 7: Preparation of a hydrogel wound dressing loaded with citrus flavonoids

[0079] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 3000 rpm for 2 h, and preparing a carboxymethyl chitosan solution with a concentration of 4 mg / mL;

[0080] Step S2: dissolving the oxidized sodium alginate prepared in Example 1 in deionized water, stirring at a rotation speed of 3000 rpm for 30 minutes, and preparing an oxidized sodium alginate solution with a concentration of 4 mg / mL;

[0081] Step S3, dissolving hesperidin in deionized water, stirring evenly, to prepare a hesperidin solution with a concentration of 1 mmol / L;

[0082] Step S4: mixing the carboxymethyl chitosan solution prepared in step S1 and the oxidized sodium alginate solution prepared in step S2 in a volume ratio of 1:1, and stirring uniformly to obtain a mixed solution I; adding the hesperidin solution prepared in step S3 to the mixed solution I, wherein the amount of the hesperidin solution added is 10% (v / v) of the total volume of the mixed solution I, and stirring uniformly to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 2 minutes, and demolding to obtain the product.

[0083] Example 8: Preparation of a hydrogel wound dressing loaded with citrus flavonoids

[0084] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 3000 rpm for 2 h, and preparing a carboxymethyl chitosan solution with a concentration of 4 mg / mL;

[0085] Step S2: dissolving the oxidized sodium alginate prepared in Example 1 in deionized water, stirring at a rotation speed of 3000 rpm for 30 minutes, and preparing an oxidized sodium alginate solution with a concentration of 4 mg / mL;

[0086] Step S3, dissolving hesperetin in deionized water, stirring evenly, to prepare a hesperetin solution with a concentration of 1 mmol / L;

[0087] Step S4: mixing the carboxymethyl chitosan solution prepared in step S1 and the oxidized sodium alginate solution prepared in step S2 in a volume ratio of 1:1 and stirring uniformly to obtain a mixed solution I; adding the hesperetin solution prepared in step S3 to the mixed solution I, wherein the amount of the hesperetin solution added is 10% (v / v) of the total volume of the mixed solution I, and stirring uniformly to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 2 minutes, and demolding to obtain the product.

[0088] Example 9: Preparation of a hydrogel wound dressing loaded with citrus flavonoids

[0089] Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 3000 rpm for 2 h, and preparing a carboxymethyl chitosan solution with a concentration of 4 mg / mL;

[0090] Step S2: dissolving the oxidized sodium alginate prepared in Example 1 in deionized water, stirring at a rotation speed of 3000 rpm for 30 minutes, and preparing an oxidized sodium alginate solution with a concentration of 4 mg / mL;

[0091] Step S3, dissolving naringin in deionized water and stirring evenly to prepare a naringin solution with a concentration of 1 mmol / L; dissolving ferulic acid in deionized water and stirring evenly to prepare a ferulic acid solution with a concentration of 1 mmol / L; then mixing the naringin solution and the ferulic acid solution in a volume ratio of 3:2 to obtain an active ingredient solution;

[0092] Step S4: mixing the carboxymethyl chitosan solution prepared in step S1 and the oxidized sodium alginate solution prepared in step S2 in a volume ratio of 1:1, stirring evenly, to obtain a mixed solution I; adding the active ingredient solution prepared in step S3 to the mixed solution I, wherein the amount of the active ingredient solution added is 10% (v / v) of the total volume of the mixed solution I, stirring evenly, to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 2 minutes, and demolding to obtain the product.

[0093] Experimental Example 1: Self-healing experiment of hydrogel wound dressing loaded with citrus flavonoids

[0094] 1. Experimental methods:

[0095] The citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 was dyed into two different colors. The two circular hydrogels with different colors were cut into two parts, and the differently dyed semicircular hydrogels were tightly placed together to heal, and the healing process was recorded by taking photos at different time points.

[0096] 2. Experimental results:

[0097] The experimental results are as follows Figure 2 shown.

[0098] Figure 2 This is a self-healing result diagram of the hydrogel wound dressing loaded with citrus flavonoids prepared in Example 4. Figure 2 It can be seen that the healing hydrogel did not fall due to gravity when picked up by hand, which proves that the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 of the present invention successfully healed itself.

[0099] Experimental Example 2: Structural morphology experiment of hydrogel wound dressing loaded with citrus flavonoids

[0100] 1. Experimental methods:

[0101] The structural morphology of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 was examined using a scanning electron microscope (SEM).

[0102] 2. Experimental results:

[0103] The experimental results are as follows Figure 3 shown.

[0104] Figure 3 This is an electron microscope image of the hydrogel wound dressing loaded with citrus flavonoids prepared in Example 4. Figure 3 It can be seen that: from the scanning electron microscopy image, the internal structure of the hydrogel appears to be a uniform and compact three-dimensional network aggregation. The densely cross-linked network structure can provide higher mechanical strength to avoid structural fracture, and can be subsequently suitable for drug encapsulation and release.

[0105] Experimental Example 3: Adhesion Test of Hydrogel Wound Dressing Loaded with Citrus Flavonoids

[0106] 1. Experimental methods:

[0107] 1.1. The citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 was adhered to a bent finger joint and observed for tight adhesion under inverted, twisted, and moving conditions.

[0108] 1.2. The citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 was adhered to pig skin. The pig skin was stretched and bent to observe whether the gel fell off and whether it was tightly adhered.

[0109] 2. Experimental results:

[0110] The experimental results are as follows Figure 4 and Figure 5 shown.

[0111] Figure 4 This is a graph showing the adhesion test results of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 on a bent finger; Figure 5The figure shows the results of the pig skin adhesion test of the hydrogel wound dressing loaded with citrus flavonoids prepared in Example 4. Figure 4 and Figure 5 It can be seen that the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4 of the present invention has good adhesion whether on a bent finger or on pig skin.

[0112] Experimental Example 4: Rheological Properties of Hydrogel Wound Dressing Loaded with Citrus Flavonoids

[0113] 1. Experimental methods:

[0114] The hydrogel wound dressings loaded with citrus flavonoids prepared in Example 4 (naringin--YPG), Example 6 (naringenin--YPS), Example 7 (hesperidin--CPG) and Example 8 (hesperetin--CPS) were placed on parallel plates with a diameter of 50 mm and a gap of 1 mm, and the hydrogel wound dressing without citrus flavonoids loading was used as a blank dressing. The rheometer was used in an oscillation mode at 25°C, and a frequency sweep test was performed in the frequency range of 0.1-10 Hz with a strain amplitude of 2% to determine the storage modulus (G') and loss modulus (G") of the hydrogel wound dressing. Next, a temperature test was performed at a strain amplitude of 2%, with the temperature increasing from 25°C to 70°C to evaluate the changes in G' and G".

[0115] 2. Experimental results:

[0116] The experimental results are as follows Figure 6 and Figure 7 shown.

[0117] Figure 6 This is a frequency scanning test graph of the citrus flavonoid-loaded hydrogel wound dressing prepared in Example 4, Example 6, Example 7, and Example 8; Figure 7 The temperature scanning test graph of the hydrogel wound dressing loaded with citrus flavonoids prepared in Examples 4, 6, 7 and 8. Figure 6 and Figure 7 As can be seen from the test graphs of different frequency scans, the storage modulus (G') of the hydrogel encapsulating citrus flavonoids is slightly higher than that of the blank hydrogel matrix, indicating good elasticity. Furthermore, in the test graphs of different temperature scans, the storage modulus (G') of all gel groups is higher than the loss modulus (G"), indicating that the hydrogel encapsulating citrus flavonoids has good mechanical properties.

[0118] Experimental Example 5: Hardness Test of Hydrogel Wound Dressing Loaded with Citrus Flavonoids

[0119] 1. Experimental methods:

[0120] The citrus flavonoid-loaded hydrogel wound dressings prepared in Example 4 (naringin), Example 6 (naringenin), Example 7 (hesperidin), and Example 8 (hesperetin) with a size of 1 cm × 1 cm and uniform thickness were placed on parallel plates along with a blank hydrogel wound dressing without citrus flavonoid loading. A texture test was performed using a texture analyzer at a testing speed of 1 mm / sec, 20% strain, and a trigger force of 5 g to determine the hardness of the hydrogel wound dressing.

[0121] 2. Experimental results:

[0122] The experimental results are as follows Figure 8 shown.

[0123] Figure 8 The hardness results of the hydrogel wound dressing loaded with citrus flavonoids prepared in Examples 4, 6, 7 and 8 are shown in FIG. Figure 8 It can be seen that the four citrus flavonoid hydrogels used in the present invention have similar gel hardness. At the same time, the gel hardness of the hydrogel loaded with citrus flavonoids is slightly reduced compared with the blank hydrogel matrix, but the difference is not significant.

[0124] Experimental Example 6: Swelling Performance Experiment of Hydrogel Wound Dressing Loaded with Citrus Flavonoids

[0125] 1. Experimental methods:

[0126] The citrus flavonoid-loaded hydrogel wound dressings prepared in Example 4 (naringin), Example 6 (naringenin), Example 7 (hesperidin), and Example 8 (hesperetin), along with a blank hydrogel wound dressing without citrus flavonoid loading, were weighed and immersed in pH 7.2-7.4 PBS and placed in a 37°C environment. At designated time points, excess PBS on the surface of the hydrogel was removed from the hydrogel using absorbent paper and reweighed. The hydrogel expansion ratio was calculated (W0: initial hydrogel mass, W1: swollen hydrogel mass):

[0127]

[0128] 2. Experimental results:

[0129] The experimental results are as follows Figure 9 shown.

[0130] Figure 9 The swelling performance results of the hydrogel wound dressing loaded with citrus flavonoids prepared in Examples 4, 6, 7 and 8 are shown in FIG. Figure 9The results show that all hydrogel groups showed a significant initial increase in swelling performance, which is related to the hydrogel's three-dimensional network structure. After 1 hour, the swelling rate of the hydrogel partially loaded with citrus flavonoids began to decrease, and after 3 hours, the swelling rate of all hydrogel groups showed a downward trend. This may be related to the oxidized sodium alginate in the hydrogel matrix. Sodium alginate is highly hydrophilic and may degrade after absorbing water and swelling, resulting in a decrease in swelling rate.

[0131] Experimental Example 7: Drying Rate Experiment of Hydrogel Wound Dressing Loaded with Citrus Flavonoids

[0132] 1. Experimental methods:

[0133] The citrus flavonoid-loaded hydrogel wound dressings prepared in Example 4 (naringin), Example 6 (naringenin), Example 7 (hesperidin), and Example 8 (hesperetin) were prepared, along with a blank dressing without the citrus flavonoid-loaded hydrogel wound dressing. The freshly prepared hydrogels were weighed and placed in a drying oven at 37°C. At designated time points, the hydrogels were taken out and weighed, and the drying rates of the hydrogels at different time points were calculated (W0: initial hydrogel mass, Wt: hydrogel mass at different time points):

[0134]

[0135] 2. Experimental results:

[0136] The experimental results are as follows Figure 10 shown.

[0137] Figure 10 The figure shows the drying rate results of the hydrogel wound dressing loaded with citrus flavonoids prepared in Examples 4, 6, 7 and 8. Figure 10 The results show that the hydrogel dried rapidly within the first two hours and gradually decreased over time. The drying rate of the hydrogel loaded with citrus flavonoids was slightly lower than that of the blank hydrogel matrix, but the difference was not significant. This may be related to the density of the hydrogel after loading with citrus flavonoids, which also alleviated the degree of water loss.

[0138] Experimental Example 8: In vitro sustained-release experiment of hydrogel wound dressing loaded with citrus flavonoids

[0139] 1. Experimental methods:

[0140] The cumulative release of naringenin (YPS), hesperetin (CPS), naringin (YPG), and hesperidin (CPG) was determined using a UV-visible spectrophotometer. UV absorption spectra of YPS, CPS, YPG, and CPG solutions at varying concentrations were measured using PBS as the solvent, and the absorbance values corresponding to the maximum absorption peaks at 290 nm, 285 nm, 284 nm, and 285 nm, respectively, were fitted to a standard curve.

[0141] Citrus flavonoid-loaded hydrogel wound dressing samples prepared in Examples 4 (naringin), 6 (naringenin), 7 (hesperidin), and 8 (hesperetin) were cut into approximately 1g pieces and placed in 20mL of PBS. The samples were then incubated in a constant-temperature shaker at 37°C with an oscillation speed of 100 rpm. 3mL of the release solution was periodically aspirated from the sample solution and replaced with 3mL of fresh PBS to maintain a constant volume. Finally, the absorbance of each 3mL of the release solution at its maximum absorption peak was measured using a UV spectrophotometer. The cumulative release amounts of naringenin (YPS), hesperetin (CPS), naringin (YPG), and hesperidin (CPG) over different time periods were calculated using a standard curve and the measured absorbance values.

[0142] 2. Experimental results:

[0143] The experimental results are as follows Figure 11 shown.

[0144] Figure 11 The in vitro release rate results of the hydrogel wound dressing loaded with citrus flavonoids prepared in Example 4, Example 6, Example 7 and Example 8 are shown. Figure 11 It can be seen that the hydrogel loaded with citrus flavonoids has a rapid initial release rate, which begins to slowly increase after 6 hours and then gradually slows down after 12 hours. Among the several citrus flavonoids used in this invention, the release rate of most reaches approximately 40% within 24 hours, with naringenin having a relatively high release rate of approximately 60%, which may be related to the good encapsulation efficiency of the loaded naringenin.

[0145] Experimental Example 9: Application of hydrogel wound dressing loaded with citrus flavonoids

[0146] 1. Experimental methods:

[0147] Five 7-week-old male BALB / c mice were selected and adapted for one week before constructing a wound infection model. The mouse skin was completely excised with a punch, and the wound size was 6 mm in diameter. The mice were infected with Staphylococcus aureus. Drug treatment began 24 hours after infection. The model group did not receive any treatment. The other treatment groups were covered with the hydrogel wound dressing loaded with citrus flavonoids prepared in Example 2, Example 3, Example 4 and Example 5, respectively. The patch was replaced every 24 hours, and the mouse wounds were photographed and recorded at different time points. The experimental groups were recorded as control (model group), 70% CMC (Example 2), 60% CMC (Example 3), 50% CMC (Example 4) and 40% CMC (Example 5).

[0148] 2. Experimental results:

[0149] The experimental results are as follows Figure 12 shown.

[0150] Figure 12 The figure shows the wound healing results of mice using the hydrogel wound dressing loaded with citrus flavonoids prepared in Examples 2, 3, 4 and 5. Figure 12 It was found that, in the screening of carboxymethyl chitosan and oxidized sodium alginate as hydrogel matrices, different groups of hydrogel patches loaded with naringin were used to treat a wound infection model. The conclusion is that the degree of cross-linking in the hydrogel is correlated with the sustained release of the drug and the rate of hydrogel degradation, both of which are closely related to the wound healing rate. Furthermore, the wound infection animal model demonstrated that the hydrogel can serve as a matrix for loading citrus flavonoids and can be successfully applied in the field of novel wound dressings.

[0151] Experimental Example 10: Application of hydrogel wound dressing loaded with citrus flavonoids

[0152] 1. Experimental methods:

[0153] Three 7-week-old male BALB / c mice were selected and acclimated for one week before establishing a wound infection model. Complete excision of the mouse skin was performed using a punch, leaving a 6 mm diameter wound. The mice were then infected with Staphylococcus aureus. Dosing began 24 hours after infection. The model group received no treatment. The remaining treatment groups were covered with the citrus flavonoid-loaded hydrogel wound dressings prepared in Examples 4 and 9, respectively. The patches were replaced every 24 hours, and the time it took for the mice's wounds to begin healing and complete closure was observed.

[0154] 2. Experimental results:

[0155] The experimental results are shown in Table 1.

[0156] Table 1 Application experiment of hydrogel wound dressing loaded with citrus flavonoids

[0157] Group Wound healing time Complete wound closure time Model Group Day 9 Day 17 Example 4 Day 7 Day 14 Example 9 Day 6 Day 11

[0158] As shown in Table 1, the naringin-ferulic acid hydrogel wound dressing prepared in Example 9 gradually healed on day 6, and on day 11, the wound was completely closed without leaving a scar. This indicates that the synergistic interaction between ferulic acid and naringin, when mixed in a specific ratio, can further promote wound healing and shorten the time it takes for the wound to close.

[0159] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for preparing a hydrogel wound dressing loaded with citrus flavonoids, characterized in that: The following steps are involved: Step S1, dissolving carboxymethyl chitosan in deionized water, stirring at a speed of 2500-3500 rpm for 1-3 hours to obtain a carboxymethyl chitosan solution; Step S2, dissolving oxidized sodium alginate in deionized water, and stirring at a rotation speed of 2500-3500 rpm for 25-35 minutes to obtain an oxidized sodium alginate solution; Step S3, dissolving the citrus flavonoids in deionized water and stirring evenly to obtain a citrus flavonoid solution; Step S4: mixing the carboxymethyl chitosan solution obtained in step S1 with the oxidized sodium alginate solution obtained in step S2, stirring evenly to obtain a mixed solution I; adding the citrus flavonoid solution obtained in step S3 to the mixed solution I, stirring evenly to obtain a mixed solution II; pouring the mixed solution II into a mold, letting it stand for 1 to 3 minutes, and demolding to obtain the product.

2. The method for preparing the hydrogel wound dressing loaded with citrus flavonoids according to claim 1, wherein: The carboxymethyl chitosan concentration of the carboxymethyl chitosan solution in step S1 is 3-6 mg / mL.

3. The method for preparing the hydrogel wound dressing loaded with citrus flavonoids according to claim 1, wherein: The preparation method of oxidized sodium alginate in step S2 is: Sodium alginate was oxidized and modified with sodium periodate as an oxidant for 6 hours, anhydrous ethanol was added for precipitation, and vacuum filtration was performed to obtain a precipitate; the precipitate was washed with deionized water, impurities were removed with anhydrous ethanol, and the ethanol was removed by rotary evaporation, and dialyzed with distilled water to obtain a dialysate; and the precipitate was freeze-dried to obtain the product.

4. The method for preparing the hydrogel wound dressing loaded with citrus flavonoids according to claim 1, wherein: The concentration of the oxidized sodium alginate solution in step S2 is 3-6 mg / mL.

5. The method for preparing the hydrogel wound dressing loaded with citrus flavonoids according to claim 1, wherein: The citrus flavonoids in step S3 include but are not limited to naringin, naringenin, hesperidin and hesperetin.

6. The method for preparing the hydrogel wound dressing loaded with citrus flavonoids according to claim 1, wherein: The concentration of citrus flavonoids in the citrus flavonoid solution in step S3 is 0.5-2 mmol / L.

7. The method for preparing the hydrogel wound dressing loaded with citrus flavonoids according to claim 1, wherein: In step S4, the volume ratio of the carboxymethyl chitosan solution to the oxidized sodium alginate solution is (1-7):(1-3).

8. The method for preparing the hydrogel wound dressing loaded with citrus flavonoids according to claim 7, wherein: In step S4, the volume ratio of the carboxymethyl chitosan solution to the oxidized sodium alginate solution is 1:

1.

9. The method for preparing the hydrogel wound dressing loaded with citrus flavonoids according to claim 1, wherein: In step S4, the amount of the citrus flavonoid solution added is 8-15% of the total volume of the mixed solution I. 10 . The citrus flavonoid-loaded hydrogel wound dressing prepared by the method for preparing a citrus flavonoid-loaded hydrogel wound dressing according to any one of claims 1 to 9 .

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