A sprayable bifunctional gel loaded with propolis extract, its preparation method and application

Abstract

This invention discloses a sprayable bifunctional gel loaded with propolis extract, its preparation method, and its application. The method includes: synthesizing acetylated carboxymethyl chitosan using carboxymethyl chitosan and acetic anhydride as raw materials; reacting acetylated carboxymethyl chitosan and aminophenylboronic acid as raw materials to synthesize a boric acid-modified acetylated carboxymethyl chitosan gel precursor; blending the boric acid-modified acetylated carboxymethyl chitosan gel precursor with water, and self-assembling it into an A-CC-PBA hydrogel through hydrogen bonding and π-π bond interactions; and adding propolis extract to the A-CC-PBA hydrogel to obtain the final product. The gel prepared by this invention exhibits excellent adhesion properties, can stably remain in the oral ulcer site, and can efficiently target and slowly release drugs to achieve highly effective bactericidal and long-lasting anti-inflammatory effects, significantly improving the efficacy of oral ulcer treatment and providing a new formulation type for oral mucosal drug delivery.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a sprayable bifunctional gel loaded with propolis extract, its preparation method, and its application. Background Technology

[0002] Oral ulcers are a common ulcerative lesion of the oral mucosa, and one of the most common diseases in oral mucosal medicine, with a prevalence of 5% to 25%. The causes of oral ulcers are complex, including local trauma, weakened immunity, viral or bacterial infection, and individual susceptibility, with bacterial infection being the most closely related factor. Oral ulcers can affect patients' quality of life and may also impact their mental health. Currently, treatment for oral ulcers is mainly local, aiming to relieve pain, reduce inflammation, and promote ulcer healing. However, antibiotics have drawbacks such as low antibacterial efficiency, low drug concentration due to dilution by saliva in the mouth, and short residence time of drugs on the oral mucosa, which seriously affect the therapeutic effect. Therefore, developing oral ulcer treatment drugs that can effectively kill bacteria at the ulcer site while controlling inflammation is a current research challenge. Simultaneously, the development of a novel bioadhesive oral mucosal drug delivery system for the local treatment of oral ulcers, which can increase the local concentration of active drugs while avoiding the side effects of systemic medication, and produce long-lasting antibacterial and anti-inflammatory effects, is a research hotspot in the clinical treatment of oral ulcers.

[0003] Propolis is a commonly used traditional Chinese medicine. According to the 2020 edition of the Chinese Pharmacopoeia, it is a sticky, solid, colloid formed from plant resin collected by worker bees of the Italian honeybee (Apis mellifera L.) and secretions from their mandibular and wax glands. It is bitter, pungent, and cold in nature, and enters the spleen and stomach meridians. It tonifies deficiency, resolves turbidity and fat, and quenches thirst; externally, it detoxifies, reduces swelling, and promotes tissue regeneration. Propolis contains over 500 chemical components in more than 20 categories, and has been proven to possess broad and significant biological activities, including antibacterial, antioxidant, anti-inflammatory, antitumor, and lipid- and blood-glycemic regulation. However, the most commonly used solvent for propolis extraction is an ethanol-water mixture. The strong irritant properties and low bioavailability of propolis extract hinder its application in the pharmaceutical industry. For example, marketed propolis tinctures for treating oral ulcers have drawbacks such as the strong irritation of ethanol at the ulcer site causing pain, the dilution of the extract by saliva in the mouth shortening its residence time, and the need for frequent administration.

[0004] To address the clinical needs of oral ulcer treatment, the development of a delivery system with good adhesion properties that can stably remain in the local area of ​​oral ulcers and efficiently target and slowly release drugs to kill bacteria and control inflammation is of great significance for the treatment of oral ulcers.

[0005] Sprayable hydrogels, compared to other dosage forms (sheets, fibers, nanoparticles, etc.), are easier to prepare, lower in cost, and have good biocompatibility. They can be easily sprayed and applied to oral ulcers, achieving uniform distribution, making them a relatively ideal local drug delivery system for the oral mucosa. However, most conventional formulations have drawbacks such as poor adhesion to the oral mucosa, high gelation temperature, and long gelation time.

[0006] Therefore, developing a novel bifunctional bioadhesive gel that can be sprayed with propolis extract to produce highly effective bactericidal and long-lasting anti-inflammatory effects on the oral mucosa is an urgent problem to be solved for the effective treatment of oral ulcers, addressing issues such as poor bacterial targeting, poor mucosal adhesion, and high gelation temperature. Summary of the Invention

[0007] To address the aforementioned technical problems, the primary objective of this invention is to provide a method for preparing a sprayable bifunctional gel loaded with propolis extract. The gel prepared by this method exhibits excellent adhesion properties, enabling it to remain stably in the local area of ​​oral ulcers and efficiently target and slowly release drugs to achieve highly effective bactericidal and long-lasting anti-inflammatory effects, thereby significantly improving the therapeutic efficacy for oral ulcers.

[0008] This invention is achieved through the following technical solution:

[0009] A method for preparing a sprayable bifunctional gel loaded with propolis extract includes the following steps:

[0010] (1) Acetylated carboxymethyl chitosan A-CC was synthesized from carboxymethyl chitosan and acetic anhydride;

[0011] (2) Boric acid-modified acetylated carboxymethyl chitosan gel precursor A-CC-PBA was synthesized by reacting acetylated carboxymethyl chitosan and aminophenylboronic acid as raw materials.

[0012] (3) Boric acid-modified acetylated carboxymethyl chitosan gel precursor was blended with water and self-assembled into A-CC-PBA hydrogel through hydrogen bonding and π-π bond interactions.

[0013] (4) Propolis extract was added to A-CC-PBA hydrogel to prepare a sprayable bifunctional gel Pro@A-CC-PBA loaded with propolis extract.

[0014] The reaction formulas involved in the preparation method of the present invention are as follows:

[0015]

[0016] The method of this invention first synthesizes a temperature-sensitive acetylated carboxymethyl chitosan gel precursor A-CC using acetic anhydride as an acylation reagent; then, boric acid groups are modified on the side chains of the A-CC gel precursor. The boric acid groups can specifically bind to the cis-o-dihydroxy groups abundant on bacterial EPS polysaccharides to achieve precise targeted recognition of oral bacterial biofilms by the gel; at the same time, propolis extract with antibacterial and anti-inflammatory activities is encapsulated in the A-CC-PBA hydrogel, which can be slowly released locally in oral ulcers to achieve a long-lasting antibacterial and anti-inflammatory synergistic effect, thereby achieving a highly effective treatment of oral ulcers.

[0017] Preferably, in step (1), the molar ratio of carboxymethyl chitosan to acetic anhydride is 1:10 to 20.

[0018] Preferably, in step (2), the aminophenylboronic acid is 3-aminophenylboronic acid or 4-aminophenylboronic acid.

[0019] To activate the amino group and facilitate the next reaction, aminophenylboronic acid is first activated by adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide before the reaction.

[0020] More preferably, the molar ratio of aminophenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 1:1 to 2:1 to 3.

[0021] By investigating the effect of the degree of substitution of aminophenylboronic acid on the thermosensitive phase transition temperature of the gel, the amount of aminophenylboronic acid added was optimized. When the molar ratio of aminophenylboronic acid to acetylated carboxymethyl chitosan was 1:1 to 3, the phase transition temperature of A-CC-PBA hydrogel was around 36℃. Therefore, the preferred molar ratio of aminophenylboronic acid to acetylated carboxymethyl chitosan was 1:1 to 3.

[0022] Preferably, in step (2), the reaction time is 12 to 24 hours.

[0023] By examining the mass ratio of boric acid-modified acetylated carboxymethyl chitosan gel precursor (A-CC-PBA) to water, a suitable ratio was optimized. When the mass ratio of A-CC-PBA to water was less than 1:0.8, a gel could not be formed; when the mass ratio of A-CC-PBA to water was greater than 1:2, a completely solid was formed. Therefore, in step (3), the preferred mass ratio of the boric acid-modified acetylated carboxymethyl chitosan gel precursor to water was 1:0.8 to 2.

[0024] The propolis extract of this invention can be obtained commercially or prepared by the following method:

[0025] The preparation method of propolis extract according to the present invention specifically includes: using 80-90% ethanol as the extraction solvent, with a material-to-liquid ratio of 1:20-40, and ultrasonic extraction for 20-40 min; purifying with HPD-722 macroporous adsorption resin, eluting with 70-80% ethanol, and collecting the 80% ethanol eluent; then performing Sephadex LH-20 chromatography column chromatography, eluting with methanol, collecting the methanol eluent, concentrating under reduced pressure, and drying to obtain propolis extract.

[0026] This invention also provides a sprayable bifunctional gel loaded with propolis extract, prepared by the above-described method. The sprayable bifunctional gel loaded with propolis extract prepared by this invention exhibits excellent adhesion properties, enabling it to remain stably in the local area of ​​oral ulcers and efficiently target and slowly release drugs to achieve highly effective bactericidal and long-lasting anti-inflammatory effects, significantly improving the therapeutic efficacy for oral ulcers.

[0027] This invention also provides a spray formulation containing propolis extract, comprising the aforementioned sprayable bifunctional gel. When the bifunctional gel of this invention is formulated into an oral spray, it can be sprayed from a spray bottle to produce uniformly sized droplets. Upon contact with the oral mucosa, the droplets connect and form a thin film that uniformly covers the oral ulcer, effectively reducing the amount of medication used and providing a better sustained-release effect.

[0028] The present invention also provides the use of the above-mentioned spray formulation of propolis extract in the preparation of a medicament for treating oral ulcers.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The method of this invention prepares a sprayable bifunctional gel loaded with propolis extract. Acetylation modification controls the gel phase transition temperature, and the boric acid modification group specifically binds to the cis-o-dihydroxy groups abundant on bacterial EPS polysaccharides, targeting oral bacteria. The released propolis extract effectively kills bacteria while controlling inflammation and promoting ulcer healing, providing a new formulation type for oral mucosal drug delivery. Pharmacodynamic results show that the sprayable bifunctional gel prepared by this invention can be sprayed from a spray bottle into uniformly sized droplets that can connect with the oral mucosa at temperature to form a thin film that uniformly covers the oral ulcer. Simultaneously, the boric acid modification group effectively recognizes bacterial EPS polysaccharides, targeting oral bacteria; the slowly released propolis extract achieves a long-lasting antibacterial and anti-inflammatory synergistic effect at the local site of the oral ulcer, effectively treating oral ulcers and providing a new treatment method for patients with oral ulcers. Attached Figure Description

[0031] Figure 1 The above are the hydrogen nuclear magnetic resonance spectra of A-CC and A-CC-PBA in the embodiments of the present invention;

[0032] Figure 2 Figures showing the appearance, injectability, and sprayability of the A-CC-PBA hydrogel of the present invention;

[0033] Figure 3 Thermosensitive and viscosity diagrams of the A-CC-PBA hydrogel and the sprayable bifunctional gel loaded with propolis extract of the present invention.

[0034] Figure 4 The image shows a scanning electron microscope (SEM) image of the A-CC-PBA gel and the sprayable bifunctional gel loaded with propolis extract of the present invention.

[0035] Figure 5 This image shows the in vitro targeted antibacterial effect of the sprayable bifunctional gel loaded with propolis extract of the present invention.

[0036] Figure 6 Figure 1 shows the area of ​​oral ulcers in rats after different days of administration of the sprayable bifunctional gel carrying propolis extract of the present invention.

[0037] Figure 7 This image shows HE and Masson staining of oral ulcer tissue from rats treated with the sprayable bifunctional gel containing propolis extract of the present invention. Detailed Implementation

[0038] The present invention will be further illustrated below through specific embodiments. The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments.

[0039] All raw materials used in the embodiments of this invention are commercially available.

[0040] Preparation of propolis extract

[0041] Propolis extract was extracted using an ethanol concentration of 85%, a solid-liquid ratio of 1:30, and ultrasonic extraction for 30 min. Purification was performed using HPD-722 macroporous adsorption resin with a loading concentration of 2.5 mg / mL, a loading flow rate of 2 BV / h, and a diameter-to-height ratio of 1:9. Elution was performed first with 20% ethanol (3 BV) to remove impurities, followed by 75% ethanol (8 BV) at a flow rate of 1 BV / h. The ethanol eluent was collected, and the ethanol was recovered under reduced pressure. The eluent was then loaded onto a Sephadex LH-20 column and eluted with methanol at a flow rate of 0.75 mL / min. The methanol eluent was collected, concentrated under reduced pressure, and dried to obtain the propolis extract.

[0042] Example 1:

[0043] (1) Synthesis of A-CC

[0044] Weigh 0.5 g of carboxymethyl chitosan and place it in a 100 mL round-bottom flask. Add 25 mL of purified water and stir magnetically at room temperature until completely dissolved. Then add 25 mL of anhydrous methanol and stir magnetically for 20 min. Transfer 10 mL of acetic anhydride and slowly add it dropwise to the above solution. After reacting at room temperature for 12 h, pour the reaction solution into acetone to precipitate the precipitate. Transfer the precipitate to a dialysis bag and dialyze for 48 h (replace the purified water every 8 h). Freeze-dry the dialysate to obtain acetylated carboxymethyl chitosan A-CC.

[0045] (2) Synthesis of A-CC-PBA

[0046] Weigh 0.5 g of 3-aminophenylboronic acid, 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 10 mg of N-hydroxysuccinimide (NHS) into a 100 mL beaker, add 5 mL of anhydrous methanol to dissolve completely, and obtain a 3-aminophenylboronic acid solution. Weigh 0.5 g of A-CC into a 100 mL round-bottom flask, add 20 mL of purified water, and stir magnetically at room temperature until completely dissolved. Add 20 mL of anhydrous methanol, stir magnetically for 20 min, and then transfer 5.3 mL of the 3-aminophenylboronic acid solution and slowly add it dropwise to the above solution. After reacting at room temperature for 12 h, pour the reaction solution into acetone to precipitate, transfer it to a dialysis bag, and dialyze for 48 h (changing the purified water every 8 h). Freeze-dry the dialysate to obtain boric acid-modified acetylated carboxymethyl chitosan gel precursor A-CC-PBA.

[0047] Figure 1 The above are the hydrogen nuclear magnetic resonance spectra of A-CC and A-CC-PBA in embodiments of the present invention; from Figure 1 The chemical shifts of different H atoms on the modified carboxymethyl chitosan product under their corresponding chemical environments can be determined. Figure 1 Image (A) shows the 1H NMR spectrum of A-CC, where 4.63 ppm is the chemical shift of D₂O, and 2.1 ppm and 3-4 ppm correspond to the chemical shifts of the hydrogen atom on the methyl group of the acetyl group and the H₂-H₈ on the glucose ring, respectively. Figure 1 (A) is marked as 9 and 2-8 respectively; Figure 1 Image (B) shows the 1H NMR spectrum of A-CC-PBA, with 7.54 ppm, 7.61 ppm, 7.52 ppm, and 7.78 ppm corresponding to the chemical shifts of the hydrogen atoms on the phenylboronic acid group. Figure 1 (B) are labeled as 1, 2, 3, and 4). These characteristic peaks indicate that amide and phenylboronic acid groups were successfully modified onto the carboxymethyl chitosan molecular chain;

[0048] (3) Preparation of A-CC-PBA hydrogel

[0049] A-CC-PBA and purified water in a mass ratio of 1:1.5 were placed in a vial and magnetically stirred for 5 hours at room temperature to obtain A-CC-PBA hydrogel.

[0050] (4) Preparation of a sprayable bifunctional gel loaded with propolis extract

[0051] Weigh 200 mg of A-CC-PBA hydrogel into a 100 mL round-bottom flask, slowly add 6 mL of propolis extract, and stir magnetically for 8 h at room temperature to obtain a sprayable bifunctional gel Pro@A-CC-PBA loaded with propolis extract.

[0052] Example 2:

[0053] (1) Synthesis of A-CC

[0054] Weigh 1.0 g of carboxymethyl chitosan and place it in a 100 mL round-bottom flask. Add 25 mL of purified water and stir magnetically at room temperature until completely dissolved. Then add 25 mL of anhydrous methanol and stir magnetically for 20 min. Transfer 25 mL of acetic anhydride and slowly add it dropwise to the above solution. After reacting at room temperature for 12 h, pour the reaction solution into acetone to precipitate the precipitate. Transfer the precipitate to a dialysis bag and dialyze for 48 h (replace the purified water every 8 h). Freeze-dry the dialysate to obtain acetylated carboxymethyl chitosan A-CC.

[0055] (2) Synthesis of A-CC-PBA

[0056] Weigh 1.5 g of 3-aminophenylboronic acid, 13 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 25 mg of N-hydroxysuccinimide (NHS) into a 100 mL beaker, add 5 mL of anhydrous methanol to dissolve completely, and obtain a 3-aminophenylboronic acid solution. Weigh 1.0 g of A-CC into a 100 mL round-bottom flask, add 20 mL of purified water, and stir magnetically at room temperature until completely dissolved. Add 20 mL of anhydrous methanol, stir magnetically for 20 min, and then transfer 13.5 mL of the 3-aminophenylboronic acid solution and slowly add it dropwise to the above solution. After reacting at room temperature for 12 h, pour the reaction solution into acetone to precipitate, transfer it to a dialysis bag, and dialyze for 48 h (changing the purified water every 8 h). Freeze-dry the dialysate to obtain boric acid-modified acetylated carboxymethyl chitosan gel precursor A-CC-PBA.

[0057] (3) Preparation of A-CC-PBA hydrogel

[0058] A-CC-PBA and purified water in a mass ratio of 1:0.8 were placed in a vial and magnetically stirred for 5 hours at room temperature to obtain A-CC-PBA hydrogel.

[0059] (4) Preparation of a sprayable bifunctional gel loaded with propolis extract

[0060] Weigh 200 mg of A-CC-PBA hydrogel into a 100 mL round-bottom flask, slowly add 5 mL of propolis extract, and magnetically stir for 6 h at room temperature to obtain a sprayable bifunctional gel Pro@A-CC-PBA loaded with propolis extract.

[0061] Example 3:

[0062] (1) Synthesis of A-CC

[0063] Weigh 0.8 g of carboxymethyl chitosan and place it in a 100 mL round-bottom flask. Add 25 mL of purified water and stir magnetically at room temperature until completely dissolved. Then add 25 mL of anhydrous methanol and stir magnetically for 20 min. Transfer 18 mL of acetic anhydride and slowly add it dropwise to the above solution. After reacting at room temperature for 12 h, pour the reaction solution into acetone to precipitate the precipitate. Transfer the precipitate to a dialysis bag and dialyze for 48 h (replace the purified water every 8 h). Freeze-dry the dialysate to obtain acetylated carboxymethyl chitosan A-CC.

[0064] (2) Synthesis of A-CC-PBA

[0065] Weigh 1.2 g of 4-aminophenylboronic acid, 14 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 26 mg of N-hydroxysuccinimide (NHS) into a 100 mL beaker, add 5 mL of anhydrous methanol to dissolve completely, and obtain a 3-aminophenylboronic acid solution. Weigh 0.6 g of A-CC into a 100 mL round-bottom flask, add 20 mL of purified water, and stir magnetically at room temperature until completely dissolved. Add 20 mL of anhydrous methanol, stir magnetically for 20 min, and then transfer 13 mL of the 4-aminophenylboronic acid solution and slowly add it dropwise to the above solution. After reacting at room temperature for 12 h, pour the reaction solution into acetone to precipitate, transfer it to a dialysis bag, and dialyze for 48 h (changing the purified water every 8 h). Freeze-dry the dialysate to obtain boric acid-modified acetylated carboxymethyl chitosan gel precursor A-CC-PBA.

[0066] (3) Preparation of A-CC-PBA hydrogel

[0067] A-CC-PBA and purified water in a mass ratio of 1:2 were placed in a vial and magnetically stirred for 5 hours at room temperature to obtain A-CC-PBA hydrogel.

[0068] (4) Preparation of a sprayable bifunctional gel loaded with propolis extract

[0069] Weigh 200 mg of A-CC-PBA hydrogel into a 100 mL round-bottom flask, slowly add 4 mL of propolis extract, and stir magnetically for 7 h at room temperature to obtain a sprayable bifunctional gel Pro@A-CC-PBA loaded with propolis extract.

[0070] Comparative Example 1: Preparation of Pro@A-CC

[0071] (1) Synthesis of A-CC

[0072] Weigh 0.5 g of carboxymethyl chitosan and place it in a 100 mL round-bottom flask. Add 25 mL of purified water and stir magnetically at room temperature until completely dissolved. Then add 25 mL of anhydrous methanol and stir magnetically for 20 min. Transfer 10 mL of acetic anhydride and slowly add it dropwise to the above solution. After reacting at room temperature for 12 h, pour the reaction solution into acetone to precipitate the precipitate. Transfer the precipitate to a dialysis bag and dialyze for 48 h (replace the purified water every 8 h). Freeze-dry the dialysate to obtain acetylated carboxymethyl chitosan A-CC.

[0073] (2) Preparation of A-CC hydrogel

[0074] A-CC and purified water in a mass ratio of 1:1.5 were placed in a vial and magnetically stirred for 5 hours at room temperature to obtain A-CC hydrogel.

[0075] (3) Preparation of gel loaded with propolis extract

[0076] Weigh 200 mg of A-CC hydrogel into a 100 mL round-bottom flask, slowly add 6 mL of propolis extract, and stir magnetically for 8 h at room temperature to obtain ordinary hydrogel Pro@A-CC loaded with propolis extract.

[0077] The sprayable bifunctional gel loaded with propolis extract prepared in Example 1 was subjected to the following tests.

[0078] Experimental Example 1: Injectability, Sprayability, and Thermosensitivity of A-CC-PBA Hydrogel

[0079] like Figure 2 As shown in Figure A, the A-CC-PBA hydrogel undergoes a sol-gel transition at 33℃. After inverting the vial for 60 seconds, the hydrogel did not flow, indicating good gelling properties. This demonstrates that the A-CC-PBA hydrogel is thermosensitive.

[0080] like Figure 2 As shown in Figure B, the A-CC-PBA hydrogel can be smoothly extruded using a 2.5 mL syringe and continuously stacked to form the letters "CMCS". The amide bonds break when the hydrogel is squeezed by the needle, allowing it to be extruded, and the amide bonds reform after extrusion, indicating that the A-CC-PBA hydrogel has good injectability.

[0081] like Figure 2As shown in Figure C, the A-CC-PBA hydrogel can be sprayed onto the culture dish using a handheld pump sprayer. The sprayed droplets are of uniform size, and most of the sprayed droplets can connect to form a thin film, indicating that the hydrogel has good sprayability.

[0082] Experimental Example 2: Rheological Properties of Gels

[0083] like Figure 3 As shown in Figure A, the A-CC-PBA hydrogel exhibits a highly significant temperature response, with its elasticity and viscosity changing markedly near the transition temperature, indicating typical thermosensitive phase transition characteristics. Its sol-gel phase transition temperature is 36.7℃.

[0084] like Figure 3 As shown in Figure B, the viscosity of both the A-CC-PBA hydrogel and the sprayable bifunctional gel Pro@A-CC-PBA loaded with propolis extract decreased rapidly with increasing shear rate, exhibiting typical shear-thinning behavior. This shear-thinning characteristic also verifies that the hydrogel is injectable.

[0085] like Figure 4 As shown, the SEM scan results of A-CC-PBA hydrogel and Pro@A-CC-PBA sprayable bifunctional gel loaded with propolis extract show that A-CC-PBA hydrogel has a porous network structure with continuous pores and a loose structure. This structure is conducive to drug loading and sustained release. At the same time, the hydrogel Pro@A-CC-PBA still maintains a porous structure after being loaded with propolis extract.

[0086] Experimental Example 3: In vitro targeted antibacterial effect of a sprayable bifunctional gel loaded with propolis extract

[0087] *Porphyromonas gingivalis* (Pg) was inoculated onto LB solid medium and incubated at 37°C for 24 h. Three bacterial samples were then collected from the solid medium using an inoculation loop, dissolved in liquid medium, and incubated at 37°C for another 24 h. Pg bacteria were co-cultured with 1 mg / mL propolis extract (Pro), a plain blank hydrogel (A-CC Gel), a bifunctional hydrogel group (A-CC-PBA Gel), a plain hydrogel with 1 mg / mL propolis extract (Pro@A-CC Gel), and a sprayable bifunctional hydrogel with 1 mg / mL propolis extract (Pro@A-CC-PBA Gel) for 12 h. 200 μL of bacterial suspension from each group was serially diluted with PBS. 200 μL of the diluted bacterial suspension was evenly spread onto agar plates and incubated for 24 h. Bacterial growth was observed and images were acquired. The bacteria were then stained with live / dead bacterial staining working solution and incubated in a 37°C bacterial incubator in the dark for 10 minutes. They were then washed twice with 0.85% physiological saline and observed and images were acquired using an inverted fluorescence microscope.

[0088] like Figure 5 As shown, the antibacterial activity was studied using the agar plate culture method. Compared with the control group, the bacterial colony counts of propolis extract (Pro), ordinary hydrogel loaded with propolis extract (Pro@A-CC Gel), and sprayable bifunctional gel loaded with propolis extract (Pro@A-CC-PBA Gel) were all reduced. Furthermore, the antibacterial efficacy of the sprayable bifunctional gel loaded with propolis extract (Pro@A-CC-PBA Gel) was significantly higher than that of the ordinary hydrogel loaded with propolis extract (Pro@A-CC Gel), highlighting the excellent antibacterial activity of A-CC-PBA hydrogel against *Porphyromonas gingivalis*. This advantage is mainly attributed to the fact that the PBA groups in the propolis extract-loaded hydrogel can specifically bind to the cis-o-dihydroxy groups abundant in the bacterial wall of *Porphyromonas gingivalis*, enabling targeted recognition of *Porphyromonas gingivalis* by the gel.

[0089] Furthermore, images obtained from live / dead bacterial viability assays showed that, compared to the control group, the sprayable bifunctional gel containing propolis extract (Pro@A-CC-PBA Gel) exhibited the highest number of dead bacteria (red), further validating the specific targeting effect of the PBA group on bacteria.

[0090] Finally, the morphology of the bacteria was observed using scanning electron microscopy. After incubation with the propolis extract-loaded hydrogel, *Porphyromonas gingivalis* lost its physiological structure, resulting in depressions and ruptures. The results indicate that the sprayable bifunctional gel loaded with propolis extract (Pro@A-CC-PBA Gel) possesses good antibacterial activity.

[0091] Experimental Example 4: Pharmacodynamic Results of a Sprayable Bifunctional Gel Loaded with Propolis Extract for the Treatment of Oral Ulcers

[0092] (1) Construction and experimental grouping of rats with oral ulcers:

[0093] Sixty healthy SD rats were purchased and, after one week of acclimatization, anesthetized with 3% sodium pentobarbital solution via intraperitoneal injection at 0.35 mL / 100 g. The labial mucosa of the lower incisors of the SD rats was dried with sterile cotton balls. A 3 mm × 3 mm square filter paper was placed in 50% glacial acetic acid for 5 seconds, then immediately placed on the dried labial mucosa of the lower incisors and maintained for 30 seconds. The filter paper was then removed, and the treated area was wiped with a sterile cotton ball containing physiological saline to remove residual acetic acid, thus establishing a rat oral ulcer model. Rats with successfully established models were randomly divided into four groups: model group, positive control group (Perio), low-dose (1 mg / L), medium-dose (2 mg / L), and high-dose (5 mg / L) sprayable bifunctional gel loaded with propolis extract.

[0094] (2) Drug administration and sample processing:

[0095] The solution was applied to the ulcers of rats using a handheld pump-operated spray bottle, ensuring even coverage, once daily. Rats were deprived of food and water for 30 minutes after each administration, for a total of 10 days. Rats were then sacrificed, and mucosal tissue from the oral ulcer sites of each group was collected and placed in a 4% paraformaldehyde solution for analysis.

[0096] (3) Measurement of oral ulcer area:

[0097] The oral ulcer area of ​​rats was measured using vernier calipers before drug administration and on days 3, 5, 7, and 9 after drug administration, following the method described by Chen Zhuo et al. Calculation: Ulcer area = 3.14 × d1 × d2 × 1 / 4 (d1 and d2 are the maximum transverse and longitudinal diameters of the ulcer surface, respectively). Average feed intake (g) = feed consumption per cage / number of animals per cage.

[0098] like Figure 6 After modeling, the rats exhibited a concave defect in the modeled area, with redness and swelling around the depression, covered by a yellowish-white pseudomembrane. On day 1 of drug administration, there was no significant difference in the ulcer size between the model group and the other drug administration groups. On day 4 of drug administration, the model group showed swelling of the lower lip, obvious oral ulcers, and significant inflammatory edema and congestion in the surrounding tissues; in the other groups, the pseudomembrane thinned, inflammatory edema and congestion decreased, and the ulcer area decreased. On day 7 of drug administration, the symptoms of rats in both the model group and the other drug administration groups were alleviated. In the positive control group and the high-dose group, the pseudomembrane on the ulcer detached, there was no inflammatory edema and congestion, and the ulcer size was significantly reduced; the ulcer size in the low-dose and medium-dose groups showed no significant change compared to the model group. On day 10 of drug administration, compared with the model group, the ulcers in the positive control group and the high-dose group were smooth, without inflammatory edema or congestion, and the ulcers were close to healing.

[0099] (4) Oral mucosal histopathological examination:

[0100] ① HE staining

[0101] Oral mucosal tissue from the ulcer site was routinely embedded in paraffin, sectioned, dewaxed, washed with water, stained with hematoxylin, washed with water, differentiated, blued, and soaked in water, then placed in eosin solution. Routine dehydration, clearing, and mounting were then performed. Changes in the mucosal tissue and the infiltration of inflammatory cells in the epithelial connective tissue were observed under a light microscope.

[0102] ②Masson staining

[0103] Take sections from each group, dewax and hydrate with xylene, and stain with hematoxylin for 5–10 min. Differentiate with acidic ethanol differentiation solution, wash with water. Re-blue with Masson's blue solution; wash with distilled water for 1 min; stain with staining solution for 5–10 min. Wash with weak acid working solution for 1 min. Wash with phosphomolybdic acid solution for 1–2 min. Wash with weak acid working solution for 1 min. Stain with aniline blue staining solution for 1–2 min. Wash with weak acid working solution for 1 min. Dehydrate three times with anhydrous ethanol. Clear three times with xylene. Mount with neutral resin. Observe the distribution of elastic fibers and collagen fibers in the mucosal tissue.

[0104] like Figure 7 As shown, the oral mucosa epithelium of rats in the positive control group and the high-dose group was intact, with loose connective tissue at the base, orderly cell arrangement, and no pathological changes such as vascular hemorrhage or inflammatory cell infiltration. Compared with the former two groups, the model group showed severe oral mucosal damage, loss of squamous epithelium, surface covered with necrotic tissue, large-area inflammatory cell infiltration, and vascular hemorrhage. After 10 days of drug treatment, compared with the model group, the positive control group and the low, medium, and high-dose groups all promoted the healing of acetic acid-induced oral ulcers in rats, with the positive control group and the high-dose group showing the fastest healing effect.

[0105] Masson staining was used to monitor collagen deposition after treatment. Collagen is a basic component for maintaining the function and structure of skin and mucous membranes. The production of collagen in local wounds is also an important marker of the remodeling of damaged mucous membrane tissue. It can be seen that the positive drug and high-dose groups showed more collagen fibers than other groups. A small amount of collagen was visible in the model group, and the arrangement was disordered.

Claims

1. A method for preparing a sprayable bifunctional gel loaded with propolis extract, characterized in that, Includes the following steps: (1) Acetylated carboxymethyl chitosan was synthesized from carboxymethyl chitosan and acetic anhydride; the molar ratio of carboxymethyl chitosan to acetic anhydride was 1:10~20; (2) A boric acid-modified acetylated carboxymethyl chitosan gel precursor is synthesized by reacting acetylated carboxymethyl chitosan and aminophenylboronic acid as raw materials; wherein the aminophenylboronic acid is 3-aminophenylboronic acid or 4-aminophenylboronic acid; and the molar ratio of aminophenylboronic acid to acetylated carboxymethyl chitosan is 1:1~3. (3) Boric acid-modified acetylated carboxymethyl chitosan gel precursor was blended with water and self-assembled into boric acid-modified acetylated carboxymethyl chitosan hydrogel through hydrogen bonding and π-π bond interaction. (4) Add propolis extract to boric acid modified acetylated carboxymethyl chitosan hydrogel to prepare a sprayable bifunctional gel loaded with propolis extract.

2. The method for preparing a sprayable bifunctional gel loaded with propolis extract according to claim 1, characterized in that: In step (2), before the reaction, aminophenylboronic acid is first added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for activation.

3. The method for preparing a sprayable bifunctional gel loaded with propolis extract according to claim 2, characterized in that: In step (2), the molar ratio of aminophenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:1~2:1~3.

4. The method for preparing a sprayable bifunctional gel loaded with propolis extract according to claim 1, characterized in that: In step (2), the reaction time is 12-24 h.

5. The method for preparing a sprayable bifunctional gel loaded with propolis extract according to claim 1, characterized in that: In step (3), the mass ratio of the boric acid-modified acetylated carboxymethyl chitosan gel precursor to water is 1:0.8~2.

6. The method for preparing a sprayable bifunctional gel loaded with propolis extract according to claim 1, characterized in that: In step (4), the preparation method of the propolis extract specifically includes: using 80-90% ethanol as the extraction solvent, with a material-to-liquid ratio of 1:20-40, and ultrasonic extraction for 20-40 min; purifying with HPD-722 macroporous adsorption resin, eluting with 70-80% ethanol, and collecting the 80% ethanol eluent; then performing chromatography with a Sephadex LH-20 column, eluting with methanol, collecting the methanol eluent, concentrating under reduced pressure, and drying to obtain the propolis extract.

7. A sprayable bifunctional gel loaded with propolis extract, prepared by the preparation method according to any one of claims 1-6.

8. A spray formulation containing propolis extract, comprising the sprayable bifunctional gel of claim 7.

9. The use of the spray formulation of propolis extract according to claim 8 in the preparation of a medicament for treating oral ulcers.

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