Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a hydrogel wound auxiliary material loaded with citrus flavonoid active substances. The hydrogel wound dressing provided by the invention is prepared by taking carboxymethyl chitosan and oxidized sodium alginate as matrixes and loading citrus flavonoid small-molecule active substances. The citrus flavonoid adopted by the invention has various active functions of resisting bacteria, resisting inflammation, resisting oxidization, resisting apoptosis, resisting tumors, improving myocardial injury, improving liver injury, conditioning blood fat and the like, has a sustained and slow release effect when being loaded in a three-dimensional carboxymethyl chitosan/oxidized sodium alginate hydrogel matrix, can meet the requirements of different times or different types of wounds on resisting bacteria, resisting inflammation and cell growth, accelerates the healing of the wounds, can reduce the formation of scar tissues of wound surfaces, expands the application path of the citrus flavonoid, and provides a new research thought for novel wound dressing loading substances.
In order to achieve the above object, the technical scheme of the present invention is as follows:
the invention provides a preparation method of a hydrogel wound dressing loaded with citrus flavonoids, which comprises the following steps:
step S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 1-3 hours under the condition that the rotating speed is 2500-3500 rpm to obtain carboxymethyl chitosan solution;
s2, dissolving oxidized sodium alginate in deionized water, and stirring for 25-35 min under the condition that the rotating speed is 2500-3500 rpm to obtain oxidized sodium alginate solution;
S3, dissolving the citrus flavonoid substances in deionized water, and uniformly stirring to obtain a citrus flavonoid solution;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 with the oxidized sodium alginate solution prepared in the step S2, stirring uniformly to obtain a mixed solution I, adding the citrus flavonoid solution prepared in the step S3 into the mixed solution I, stirring uniformly to obtain a mixed solution II, pouring the mixed solution II into a mould, standing for 1-3 min, and demoulding to obtain the finished product.
Further, the concentration of carboxymethyl chitosan in the carboxymethyl chitosan solution in the step S1 is 3-6 mg/mL, and more preferably the concentration is 4mg/mL.
Further, the preparation method of the oxidized sodium alginate in the step S2 comprises the following steps:
Oxidizing and modifying sodium alginate for 6h by taking sodium periodate as an oxidant, adding absolute ethyl alcohol for precipitation, vacuum-filtering to obtain a precipitate, washing the precipitate by adopting deionized water, removing impurities by adopting absolute ethyl alcohol, removing the ethanol by rotary evaporation, dialyzing by adopting distilled water to obtain a dialyzate, and freeze-drying to obtain the sodium alginate.
Further, the specific preparation method of the oxidized sodium alginate comprises the following steps:
step A, suspending 10g of sodium alginate in 50mL of absolute ethyl alcohol to obtain a sodium alginate suspension, dissolving 8g of sodium periodate in 50mL of deionized water to obtain a sodium periodate solution, introducing the sodium periodate solution into the sodium alginate suspension, magnetically stirring for 6h under a room temperature and light-shielding environment, adding 3mL of ethylene glycol, and stirring for 1h to completely terminate chemical reaction to obtain a mixed solution;
And B, in order to purify the product, introducing 150mL of anhydrous ethanol for completely precipitating the mixed solution prepared in the step A to obtain a precipitate, performing vacuum filtration treatment on the precipitate, washing the precipitate with deionized water, repeatedly adding the anhydrous ethanol three times under intense stirring to effectively eliminate any residual unreacted impurities, collecting the obtained white product, introducing the deionized water into the purified white product, performing rotary evaporation at 60 ℃ to remove the ethanol, dialyzing with distilled water for 24 hours, changing water every 12 hours (interception MW=3500 kDa), removing unreacted sodium periodate, ethylene glycol and other small molecular impurities every 12 hours to obtain a dialysate, and performing freeze drying on the dialysate in a dialysis bag for 48 hours to finally obtain oxidized sodium alginate.
Further, the concentration of oxidized sodium alginate in the oxidized sodium alginate solution in the step S2 is 3-6 mg/mL, and more preferably the concentration is 4mg/mL.
Further, the citrus flavonoid in step S3 includes, but is not limited to, naringin, naringenin, hesperidin and hesperetin, and more preferably naringin.
Further, the concentration of the citrus flavonoid substance in the citrus flavonoid solution in the step S3 is 0.5 to 2mmol/L, and more preferably 1mmol/L.
Further, in the 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.
Further, the addition amount of the citrus flavonoid solution in the step S4 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.
Meanwhile, the invention also claims the citrus flavonoid-loaded hydrogel wound dressing prepared by the preparation method of the citrus flavonoid-loaded hydrogel wound dressing.
In addition, the wound dressing of the present invention may contain ferulic acid in addition to the citrus flavonoid substance as an active ingredient. The method comprises the specific steps of dissolving ferulic acid in deionized water, uniformly stirring to prepare a ferulic acid solution with the concentration of 1mmol/L, wherein the volume ratio of the ferulic acid solution to naringin solution is (1-2): 3.
The hydrogel wound dressing loaded with citrus flavonoids is prepared by oxidizing sodium alginate into oxidized sodium alginate through sodium periodate, uniformly mixing the oxidized sodium alginate solution and carboxymethyl chitosan solution according to a specific proportion to prepare a hydrogel precursor solution, then adding the active micromolecular citrus flavonoids solution, uniformly stirring, pouring into a mould, standing for 2min, and demoulding to obtain the carboxymethyl chitosan/oxidized sodium alginate/citrus flavonoids hydrogel patch. The hydrogel wound dressing loaded with the citrus flavonoid has the advantages of short preparation time, high transparency, good adhesion performance, easy replacement and the like.
Further, the inventor confirms that the hydrogel wound dressing loaded with the citrus flavonoid has the advantages of strong self-healing capacity, good adhesion performance and good sustained release effect of active ingredients by researching the micro-morphology, mechanical property, physical and chemical basic properties and the influence of the release performance of the hydrogel wound dressing loaded with the citrus flavonoid. The hydrogel wound dressing can be directly and firmly adhered to the wound surface of tissue, and is used for keeping the wound clean and moist and preventing external infection. Meanwhile, the hydrogel wound dressing has the effect of continuously releasing citrus flavonoid substances, can meet the demands of wound healing in different periods on the aspects of antibacterial, anti-inflammatory, cell growth and the like, quickens the wound healing, can reduce the formation of wound scar tissues, and is a novel hydrogel wound dressing.
Further, the inventors have also established an animal model of wound infection for evaluating the therapeutic effect of the citrus flavonoid-loaded hydrogel wound dressing on a wound of a staphylococcus aureus-infected mouse. Experiments prove that the wound of the injured mice treated by the hydrogel wound dressing loaded with the citrus flavonoid gradually heals on the 7 th day, and the wound is completely closed and basically no scar is left on the 14 th day.
In addition, the inventor finds that naringin and ferulic acid are mixed according to a specific proportion in intensive research, and the naringin and the ferulic acid cooperate to further accelerate the healing capacity of the wound surface and shorten the time for healing the wound. Experiments show that compared with the hydrogel wound dressing added with naringin alone, the wound covered with the naringin-ferulic acid hydrogel wound dressing starts to heal gradually on the 6 th day, and the wound is completely closed without scars on the 11 th day, so that the time for healing the wound can be effectively shortened.
Compared with the prior art, the hydrogel wound dressing loaded with the citrus flavonoid has the following advantages:
(1) The hydrogel wound dressing loaded with the citrus flavonoid has the advantages of natural and safe components, good biocompatibility and degradability, short preparation time, and high popularization, and can be molded after standing for 2min without adding any chemical cross-linking agent.
(2) The hydrogel wound dressing loaded with the citrus flavonoid provided by the invention realizes the effects of slow release, sustained release and sequential delivery of the active ingredients of the citrus flavonoid substances, meets the requirements of the wound on antibacterial, anti-inflammatory and cell growth in different periods, helps the adhesion and migration of wound cells, enables the skin to regenerate faster, prevents the formation of crusts and promotes the wound healing. Not only expands the application way of citrus flavonoid, but also provides a new thought for researching novel wound dressing loading substances.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention without departing from the basic idea of the invention. The starting materials referred to in the present invention may be obtained by means of commercially available or conventional techniques in the art.
Example 1 preparation of oxidized sodium alginate
1. The preparation method of the oxidized sodium alginate comprises the following steps:
Step A, suspending 10g of sodium alginate in 50mL of absolute ethyl alcohol to obtain a sodium alginate suspension, dissolving 8g of sodium periodate in 50mL of deionized water to obtain a sodium periodate solution, introducing the sodium periodate solution into the sodium alginate suspension, magnetically stirring for 6h under a room temperature and light-shielding environment, adding 3mL of ethylene glycol, and stirring for 1h to terminate a chemical reaction to obtain a mixed solution;
And B, introducing 150mL of absolute ethyl alcohol into the mixed solution prepared in the step A to obtain a precipitate, vacuum filtering the precipitate, washing the precipitate with deionized water, repeatedly adding the absolute ethyl alcohol for three times to remove impurities, collecting the obtained white product, introducing the deionized water into the purified white product, performing rotary evaporation at the temperature of 60 ℃, dialyzing with distilled water for 24 hours, changing water every 12 hours to obtain a dialysate, and freeze-drying the dialysate to finally obtain oxidized sodium alginate.
2. And (3) detecting oxidized sodium alginate:
Sodium Alginate (SA) and Oxidized Sodium Alginate (OSA) were used as samples to be measured, and infrared spectra were measured using a fourier infrared spectrometer. As a result, the FTIR spectrum showed that the broad absorption peak around 3440cm-1 was the hydroxyl stretching vibration peak of SA and OSA. The SA spectrum showed a strong and sharp absorption peak near 1725cm "1 compared to the OSA spectrum, demonstrating that the c=o absorption peak vibration is significantly enhanced and aldehyde groups are generated, SA being effectively oxidized.
EXAMPLE 2 preparation of a Citrus flavonoid-loaded hydrogel wound dressing
S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 2 hours at a rotating speed of 3000rpm to prepare carboxymethyl chitosan solution with a concentration of 4 mg/mL;
S2, dissolving the oxidized sodium alginate prepared in the embodiment 1 in deionized water, and stirring for 30min at a rotating speed of 3000rpm to prepare an oxidized sodium alginate solution with a concentration of 4 mg/mL;
s3, dissolving naringin in deionized water, and uniformly stirring to prepare naringin solution with the concentration of 1 mmol/L;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 and the oxidized sodium alginate solution prepared in the step S2 according to a volume ratio of 7:3, stirring uniformly to obtain a mixed solution I, adding the naringin solution prepared in the step S3 into the mixed solution I, wherein the addition amount of the naringin solution 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 mould, standing for 2min, and demoulding to obtain the sodium alginate gel.
Example 3 preparation of a Citrus flavonoid-loaded hydrogel wound dressing
S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 2 hours at a rotating speed of 3000rpm to prepare carboxymethyl chitosan solution with a concentration of 4 mg/mL;
S2, dissolving the oxidized sodium alginate prepared in the embodiment 1 in deionized water, and stirring for 30min at a rotating speed of 3000rpm to prepare an oxidized sodium alginate solution with a concentration of 4 mg/mL;
s3, dissolving naringin in deionized water, and uniformly stirring to prepare naringin solution with the concentration of 1 mmol/L;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 and the oxidized sodium alginate solution prepared in the step S2 according to a volume ratio of 3:2, stirring uniformly to obtain a mixed solution I, adding the naringin solution prepared in the step S3 into the mixed solution I, wherein the addition amount of the naringin solution 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 mould, standing for 2min, and demoulding to obtain the sodium alginate gel.
EXAMPLE 4 preparation of a Citrus flavonoid-loaded hydrogel wound dressing
S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 2 hours at a rotating speed of 3000rpm to prepare carboxymethyl chitosan solution with a concentration of 4 mg/mL;
S2, dissolving the oxidized sodium alginate prepared in the embodiment 1 in deionized water, and stirring for 30min at a rotating speed of 3000rpm to prepare an oxidized sodium alginate solution with a concentration of 4 mg/mL;
s3, dissolving naringin in deionized water, and uniformly stirring to prepare naringin solution with the concentration of 1 mmol/L;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 and the oxidized sodium alginate solution prepared in the step S2 according to a volume ratio of 1:1, stirring uniformly to obtain a mixed solution I, adding the naringin solution prepared in the step S3 into the mixed solution I, wherein the addition amount of the naringin solution 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 mould, standing for 2min, and demoulding to obtain the sodium alginate gel.
Example 5 preparation of a Citrus flavonoid-loaded hydrogel wound dressing
S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 2 hours at a rotating speed of 3000rpm to prepare carboxymethyl chitosan solution with a concentration of 4 mg/mL;
S2, dissolving the oxidized sodium alginate prepared in the embodiment 1 in deionized water, and stirring for 30min at a rotating speed of 3000rpm to prepare an oxidized sodium alginate solution with a concentration of 4 mg/mL;
s3, dissolving naringin in deionized water, and uniformly stirring to prepare naringin solution with the concentration of 1 mmol/L;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 and the oxidized sodium alginate solution prepared in the step S2 according to a volume ratio of 2:3, stirring uniformly to obtain a mixed solution I, adding the naringin solution prepared in the step S3 into the mixed solution I, wherein the addition amount of the naringin solution 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 mould, standing for 2min, and demoulding to obtain the sodium alginate gel.
EXAMPLE 6 preparation of a Citrus flavonoid-loaded hydrogel wound dressing
S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 2 hours at a rotating speed of 3000rpm to prepare carboxymethyl chitosan solution with a concentration of 4 mg/mL;
S2, dissolving the oxidized sodium alginate prepared in the embodiment 1 in deionized water, and stirring for 30min at a rotating speed of 3000rpm to prepare an oxidized sodium alginate solution with a concentration of 4 mg/mL;
s3, dissolving naringenin in deionized water, and uniformly stirring to prepare naringenin solution with the concentration of 1 mmol/L;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 and the oxidized sodium alginate solution prepared in the step S2 according to a volume ratio of 1:1, stirring uniformly to obtain a mixed solution I, adding naringenin solution prepared in the step S3 into the mixed solution I, wherein the addition amount of naringenin solution 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 mould, standing for 2min, and demoulding.
EXAMPLE 7 preparation of a Citrus flavonoid-loaded hydrogel wound dressing
S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 2 hours at a rotating speed of 3000rpm to prepare carboxymethyl chitosan solution with a concentration of 4 mg/mL;
S2, dissolving the oxidized sodium alginate prepared in the embodiment 1 in deionized water, and stirring for 30min at a rotating speed of 3000rpm to prepare an oxidized sodium alginate solution with a concentration of 4 mg/mL;
S3, dissolving hesperidin in deionized water, and uniformly stirring to prepare a hesperidin solution with the concentration of 1 mmol/L;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 and the oxidized sodium alginate solution prepared in the step S2 according to a volume ratio of 1:1, stirring uniformly to obtain a mixed solution I, adding the hesperidin solution prepared in the step S3 into the mixed solution I, wherein the addition amount of the hesperidin solution 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 mould, standing for 2min, and demoulding to obtain the chitosan.
Example 8 preparation of a Citrus flavonoid-loaded hydrogel wound dressing
S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 2 hours at a rotating speed of 3000rpm to prepare carboxymethyl chitosan solution with a concentration of 4 mg/mL;
S2, dissolving the oxidized sodium alginate prepared in the embodiment 1 in deionized water, and stirring for 30min at a rotating speed of 3000rpm to prepare an oxidized sodium alginate solution with a concentration of 4 mg/mL;
s3, dissolving the hesperetin in deionized water, and uniformly stirring to prepare a hesperetin solution with the concentration of 1 mmol/L;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 and the oxidized sodium alginate solution prepared in the step S2 according to a volume ratio of 1:1, stirring uniformly to obtain a mixed solution I, adding the hesperetin solution prepared in the step S3 into the mixed solution I, wherein the addition amount of the hesperetin solution 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 mould, standing for 2min, and demoulding to obtain the chitosan.
Example 9 preparation of a Citrus flavonoid-loaded hydrogel wound dressing
S1, dissolving carboxymethyl chitosan in deionized water, and stirring for 2 hours at a rotating speed of 3000rpm to prepare carboxymethyl chitosan solution with a concentration of 4 mg/mL;
S2, dissolving the oxidized sodium alginate prepared in the embodiment 1 in deionized water, and stirring for 30min at a rotating speed of 3000rpm to prepare an oxidized sodium alginate solution with a concentration of 4 mg/mL;
s3, dissolving naringin in deionized water, stirring uniformly to prepare naringin solution with the concentration of 1mmol/L, dissolving ferulic acid in the deionized water, stirring uniformly to prepare ferulic acid solution with the concentration of 1mmol/L, and mixing the naringin solution and the ferulic acid solution according to the volume ratio of 3:2 to obtain an active ingredient solution;
And S4, mixing the carboxymethyl chitosan solution prepared in the step S1 and the oxidized sodium alginate solution prepared in the step S2 according to a volume ratio of 1:1, stirring uniformly to obtain a mixed solution I, adding the active ingredient solution prepared in the step S3 into the mixed solution I, wherein the addition amount of the active ingredient solution 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 mould, standing for 2min, and demoulding.
Experimental example one self-healing experiment of a Citrus flavonoid-loaded hydrogel wound dressing
1. The experimental method comprises the following steps:
The citrus flavonoid-loaded hydrogel wound dressing prepared in example 4 was stained in two different colors, two round hydrogels with different colors were cut into two parts, and the differently stained semicircular hydrogels were tightly put together to heal, and the healing process was recorded by photographing at different time points.
2. Experimental results:
The experimental results are shown in FIG. 2.
FIG. 2 is a graph showing the self-healing results of the citrus flavonoid-loaded hydrogel wound dressing prepared in example 4. From fig. 2, it can be seen that the healing hydrogel was lifted by hand and not dropped by gravity, which demonstrates the success of self-healing of the citrus flavonoid-loaded hydrogel wound dressing prepared in example 4 of the present invention.
Experimental example two structural morphology experiments of Citrus flavonoid-loaded hydrogel wound dressing
1. The experimental method comprises the following steps:
the morphology of the citrus flavonoid-loaded hydrogel wound dressing prepared in example 4 was examined using a Scanning Electron Microscope (SEM).
2. Experimental results:
the experimental results are shown in FIG. 3.
FIG. 3 is an electron micrograph of the citrus flavonoid-loaded hydrogel wound dressing prepared in example 4. From fig. 3, the internal structure of the hydrogel is observed from a scanning electron microscope, the microstructure of the hydrogel is in a uniform and compact three-dimensional network aggregation shape, and the crosslinked and compact network structure can provide higher mechanical strength to avoid structural fracture and can be suitable for packaging and releasing medicines subsequently.
Experimental example III adhesion experiment of Citrus flavonoid-loaded hydrogel wound dressing
1. The experimental method comprises the following steps:
the citrus flavonoid-loaded hydrogel wound dressing prepared in example 4 was adhered to a curved knuckle and observed for tight adhesion under inverted, twisted, and active conditions, respectively.
1.2, The citrus flavonoid-loaded hydrogel wound dressing prepared in example 4 was adhered to pigskin, and the pigskin was stretched and bent to observe whether the gel was closely adhered when dropped.
2. Experimental results:
the experimental results are shown in fig. 4 and 5.
FIG. 4 is a graph showing the results of the test for adhesion to curved fingers of a wound dressing comprising a citrus flavonoid-loaded hydrogel prepared in example 4, and FIG. 5 is a graph showing the results of the test for adhesion to pigskin of a wound dressing comprising a citrus flavonoid-loaded hydrogel prepared in example 4. As can be seen from FIGS. 4 and 5, the citrus flavonoid-loaded hydrogel wound dressing prepared in example 4 of the present invention had good adhesion to both the curved finger and the pigskin.
Experiment example IV rheological Property experiments of Citrus flavonoid-loaded hydrogel wound dressing
1. The experimental method comprises the following steps:
The citrus flavonoid-loaded hydrogel wound dressings prepared in example 4 (naringin-YPG), example 6 (naringenin-YPS), example 7 (hesperidin-CPG) and example 8 (hesperetin-CPS) were placed using parallel plates having a diameter of 50 mm with a gap of 1mm, and the hydrogel wound dressing without the citrus flavonoid being loaded was used as a blank dressing. Frequency sweep testing was performed using a rheometer at an oscillation mode of 25 ℃ at a strain amplitude of 2% over a frequency range of 0.1-10 hz to determine the storage modulus (G') and loss modulus (G ") of the hydrogel wound dressing. Next, temperature testing was performed at a strain amplitude of 2% with the temperature increasing from 25 ℃ to 70 ℃ to evaluate the change in G' and G ".
2. Experimental results:
The experimental results are shown in fig. 6 and 7.
Fig. 6 is a frequency scan test chart of the citrus flavonoid-loaded hydrogel wound dressings prepared in examples 4,6, 7 and 8, and fig. 7 is a temperature scan test chart of the citrus flavonoid-loaded hydrogel wound dressings prepared in examples 4,6, 7 and 8. From fig. 6 and 7, it can be seen that the storage modulus (G') of the entrapped citrus flavonoid hydrogel is slightly higher than that of the blank hydrogel matrix, and the entrapped citrus flavonoid hydrogel has better elasticity. And in different temperature scanning test charts, the storage modulus (G ') of all gel groups is higher than the loss modulus (G'), which shows that the hydrogel for encapsulating the citrus flavonoid has good mechanical properties.
Experiment example five, hardness test of hydrogel wound dressing loaded with citrus flavonoid
1. The experimental method comprises the following steps:
the citrus flavonoid-loaded hydrogel wound dressings prepared in examples 4 (naringin), 6 (naringenin), 7 (hesperidin) and 8 (hesperetin) having a uniform thickness of 1cm×1cm were placed on parallel plates as blank dressing without the citrus flavonoid-loaded hydrogel wound dressing, and texture test was performed using a texture analyzer at a test speed of 1mm/sec under conditions of 20% strain and 5g trigger force to determine the hardness of the hydrogel wound dressing.
2. Experimental results:
The experimental results are shown in FIG. 8.
FIG. 8 is a graph showing the hardness results of the citrus flavonoid-loaded hydrogel wound dressings prepared in examples 4, 6, 7 and 8. From FIG. 8, it can be seen that the four citrus flavonoid hydrogels used in the present invention have similar gel hardness, while the citrus flavonoid-loaded hydrogels have slightly reduced gel hardness but not significantly different compared to the blank hydrogel matrix.
Experiment example six swelling Property experiment of Citrus flavonoid-loaded hydrogel wound dressing
1. The experimental method comprises the following steps:
The citrus flavonoid-loaded hydrogel wound dressings prepared in example 4 (naringin), example 6 (naringin), example 7 (hesperidin) and example 8 (hesperetin) were weighed and immersed in pH 7.2 to 7.4pbs as a blank dressing for a hydrogel wound dressing without the citrus flavonoid, and placed in an environment of 37 ℃. At the indicated time points, the hydrogel surface excess PBS was wrapped from the hydrogel with absorbent paper and weighed again. The hydrogel expansion ratio (W0: initial hydrogel mass, W1: swollen hydrogel mass) was calculated:
2. experimental results:
The experimental results are shown in FIG. 9.
FIG. 9 is a graph showing the swelling performance results of the citrus flavonoid-loaded hydrogel wound dressings prepared in examples 4, 6, 7 and 8. From FIG. 9, it is clear that all hydrogel groups have a significant increase in initial swelling properties, which is related to the three-dimensional network structure of the hydrogels. The swelling ratio of the hydrogel partially loaded with citrus flavonoids starts to decrease after 1h, and the swelling ratio of all hydrogel groups tends to decrease after 3h, which may be related to oxidized sodium alginate in the hydrogel matrix, which has a strong hydrophilicity, and may degrade after swelling with water, resulting in a decrease in swelling ratio.
Experiment seven drying Rate experiment of Citrus flavonoid-loaded hydrogel wound dressing
1. The experimental method comprises the following steps:
The citrus flavonoid-loaded hydrogel wound dressings prepared in example 4 (naringin), example 6 (naringenin), example 7 (hesperidin) and example 8 (hesperetin) were weighed as a blank dressing without the citrus flavonoid-loaded hydrogel wound dressing, placed in a drying oven at 37 ℃ after the freshly prepared hydrogel was weighed, taken out at the designated time point, weighed and the dryness of the hydrogel at various time points (W0: initial hydrogel mass, wt: hydrogel mass at various time points) was calculated:
2. experimental results:
The experimental results are shown in FIG. 10.
FIG. 10 is a graph showing the results of the drying rate of the citrus flavonoid-loaded hydrogel wound dressings prepared in examples 4, 6, 7 and 8. From fig. 10, it can be seen that the hydrogel dried faster in the first 2 hours and gradually began to decrease in drying rate over time. The drying rate of the citrus flavonoid-loaded hydrogel was slightly lower than that of the blank hydrogel matrix, but the difference was not significant, which may be related to the degree of densification after loading the citrus flavonoid, and the degree of water loss was also somewhat alleviated.
Experiment example eight in vitro sustained Release experiments of Citrus flavonoid-loaded hydrogel wound dressing
1. The experimental method comprises the following steps:
The cumulative release amounts of naringenin (YPS), hesperetin (CPS), naringin (YPG) and hesperidin (CPG) were measured by an ultraviolet-visible spectrophotometer. Ultraviolet absorption spectra of the YPS, CPS, YPG, CPG solutions with different concentrations are respectively measured by using PBS as a solvent, and absorbance values corresponding to maximum absorption peaks of 290nm, 285nm, 284nm and 285nm are respectively fitted into standard curves.
The citrus flavonoid-loaded hydrogel wound dressing samples prepared in example 4 (naringin), example 6 (naringin), example 7 (hesperidin) and example 8 (hesperetin) were cut to a size of about 1g, placed in 20mLPBS, and then incubated at 37 ℃ in a constant temperature shaking box at an oscillation speed of 100 rpm. 3mL of the release solution was aspirated from the sample solution at regular intervals, and replaced by adding 3mL of fresh PBS to keep the volume constant. Finally, the absorbance of the 3mL of the release solution at each of the maximum absorption peaks was measured by an ultraviolet spectrophotometer. Cumulative release of naringenin (YPS), hesperetin (CPS), naringin (YPG), hesperidin (CPG) over different time periods was calculated from the standard curve and the measured absorbance values.
2. Experimental results:
The experimental results are shown in FIG. 11.
FIG. 11 is a graph showing the results of the in vitro release rate of the citrus flavonoid-loaded hydrogel wound dressings prepared in examples 4, 6, 7 and 8. From FIG. 11, it is seen that the citrus flavonoid-loaded hydrogel was released at a relatively high initial rate, and the release rate began to rise slowly after 6 hours, and became gradually gentle after 12 hours. Of the several citrus flavones used in the present invention, most release rates reached around 40% within 24 hours, with higher naringenin release rates reaching around 60%, which may be related to good encapsulation of naringenin loading.
Experiment example nine application experiment of hydrogel wound dressing loaded with citrus flavonoid
1. The experimental method comprises the following steps:
5 male BALB/c mice 7 weeks were selected and adapted for one week, and then a wound infection model was constructed. The skin of the mice was completely excised with a punch, the size of the wound was 6mm in diameter, the mice were infected with staphylococcus aureus, the administration treatment was started 24 hours after infection, the model group did not perform any treatment, the other treatments were covered with the wound dressing of citrus flavonoid-loaded hydrogel prepared in example 2, example 3, example 4 and example 5, respectively, the patches were replaced every 24 hours, and the wounds of the mice were photographed 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), respectively.
2. Experimental results:
The experimental results are shown in FIG. 12.
Fig. 12 is a graph showing wound healing results in mice of the citrus flavonoid-loaded hydrogel wound dressings prepared in example 2, example 3, example 4 and example 5. From FIG. 12, it can be seen that in the screening of carboxymethyl chitosan and oxidized sodium alginate of the hydrogel matrix, naringin-loaded hydrogel patches were used to treat wound infection models in different groups. The conclusion shows that the crosslinking degree of the hydrogel is related to the slow release effect of the medicine and the degradation speed of the hydrogel, which are closely related to the wound healing speed. Meanwhile, animal models of wound infection prove that the hydrogel can be used as a matrix for loading citrus flavonoids and can be successfully applied to the field of novel wound dressings.
Experimental example ten application experiments of hydrogel wound dressing loaded with citrus flavonoid
1. The experimental method comprises the following steps:
3 male BALB/c mice 7 weeks were selected and adapted for one week, and then a wound infection model was constructed. The skin of the mice was completely excised with a punch, the wound size was 6mm in diameter, the mice were infected with staphylococcus aureus, the administration treatment was started 24 hours after infection, the model group did not perform any treatment, the remaining treatment groups covered the wound with the citrus flavonoid-loaded hydrogel wound dressing prepared in example 4 and example 9, respectively, the patch was replaced every 24 hours, and the time at which the wound of the mice began to heal and the time at which it was completely closed were observed.
2. Experimental results:
The experimental results are shown in table 1.
Table 1 application experiments on citrus flavonoid-loaded hydrogel wound dressing
| Group of | Time to wound onset healing | Time to complete wound closure |
| Model group | Day 9 | Day 17 |
| Example 4 | Day 7 | Day 14 |
| Example 9 | Day 6 | Day 11 |
As is clear from Table 1, the naringin-ferulic acid hydrogel wound dressing prepared in example 9 gradually healed on day 6, and the wound surface was completely closed without leaving any scar on day 11. The method shows that the ferulic acid and naringin are mixed according to a specific proportion to use the synergistic interaction of the ferulic acid and naringin, so that the healing of wounds can be further promoted, and the time for closing the wounds is shortened.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.