Sprayable bifunctional gel carrying propolis extract as well as preparation method and application of sprayable bifunctional gel

Abstract

The invention discloses a propolis extract-loaded sprayable bifunctional gel as well as a preparation method and application thereof. The preparation method comprises the following steps: synthesizing acetylated carboxymethyl chitosan by taking carboxymethyl chitosan and acetic anhydride as raw materials; the preparation method comprises the following steps: reacting acetylated carboxymethyl chitosan and aminophenylboronic acid serving as raw materials to synthesize a boric acid modified acetylated carboxymethyl chitosan gel precursor; the preparation method comprises the following steps: blending a boric acid modified acetylated carboxymethyl chitosan gel precursor with water, and self-assembling into A-CC-PBA hydrogel under the action of a hydrogen bond and a pi-pi bond; and adding the propolis extract into the A-CC-PBA hydrogel, so as to obtain the A-CC-PBA hydrogel. The gel prepared by the invention has good adhesion performance, can stably retain in the local part of the oral ulcer and can realize efficient targeted recognition and slow release of drugs to realize efficient sterilization and long-acting anti-inflammatory effects, the curative effect on the oral ulcer is obviously improved, and a new preparation type is provided for oral mucosa administration.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to a sprayable dual-function gel carrying propolis extract, and a preparation method and application thereof. Background Art

[0002] Oral ulcer is a common ulcerative lesion that occurs in the oral mucosa. It is one of the common diseases of oral mucosa, with a prevalence of 5% to 25%. The etiology of oral ulcer is relatively complex, including local trauma, low immunity, viral or bacterial infection and individual susceptibility, among which oral ulcer is most closely related to bacterial infection. Oral ulcer can affect the patient's quality of life and may also affect the patient's mental health. At present, the treatment of oral ulcer is mainly local treatment, aiming to relieve pain, reduce inflammation, promote ulcer healing, etc. However, there are disadvantages such as low antibacterial efficiency of antibiotics, low drug concentration under the dilution of saliva in the mouth, and drug residence time on the oral mucosa, which seriously affect the effect of drug treatment. Therefore, the current research difficulty is to develop oral ulcer treatment drugs that can effectively kill bacteria at the ulcer site and control inflammation at the same time. At the same time, developing a local mucosal drug delivery system for oral ulcers, which can increase the local concentration of active drugs while avoiding the side effects of systemic medication, and produce a new type of bioadhesive oral mucosal drug delivery system with long-lasting antibacterial and anti-inflammatory effects is a research hotspot in the clinical treatment of oral ulcers.

[0003] Propolis is one of the commonly used Chinese medicines in clinical practice of traditional Chinese medicine. The 2020 edition of the Chinese Pharmacopoeia states that this product is a sticky solid colloid formed by the mixture of plant resin collected by the worker bees of the family Apis mellifera L and their secretions from the upper jaw gland and wax gland. It is bitter, pungent, and cold in nature. It enters the spleen and stomach meridians, replenishes weakness, removes turbid fat, and stops thirst. It can be used externally to detoxify and reduce swelling, and astringe and promote tissue regeneration. Propolis contains more than 500 chemical components from more than 20 categories, and has been proven to have a wide range of significant biological activities such as antibacterial, antioxidant, anti-inflammatory, anti-tumor, and regulation of blood lipids and blood sugar. However, the most commonly used solvent for propolis extraction is an ethanol-water mixture. The strong irritation and low bioavailability of propolis extracts hinder their application in the pharmaceutical industry. For example, the propolis tincture on the market for the treatment of oral ulcers has the disadvantages that ethanol is highly irritating at the ulcer site, causing pain in patients, and the extract is washed away by saliva in the mouth to dilute the drug concentration, shorten the residence time, and require frequent administration.

[0004] In response to the clinical treatment needs of oral ulcers, it is of great significance to develop a delivery system with good adhesion properties that can stably retain locally in oral ulcers and can efficiently target and identify, slowly release drugs to kill bacteria, and control inflammation.

[0005] Compared with other dosage forms (films, fibers, nanoparticles, etc.), sprayable hydrogels are easy to prepare, low in cost, and have good biosafety. They can be easily sprayed and cover the oral ulcer surface to achieve uniform distribution, so they are ideal oral mucosal local drug delivery systems. However, most conventional preparations have disadvantages such as poor oral mucosal adhesion, high gelation temperature, and long time.

[0006] Therefore, it is an urgent problem to be solved for the efficient treatment of oral ulcers to develop a new type of bioadhesive bifunctional gel that can be sprayed with propolis extract to produce efficient bactericidal and long-lasting anti-inflammatory effects in the oral mucosa to solve the above-mentioned problems such as bacterial targeting, poor mucosal adhesion, and high gelation temperature. Summary of the invention

[0007] In order to solve the above technical problems, the primary purpose of the present invention is to provide a method for preparing a sprayable dual-functional gel loaded with propolis extract. The gel prepared by this method has good adhesion properties, can be stably retained locally in oral ulcers, and can efficiently target and identify, and slowly release drugs to achieve efficient bactericidal and long-lasting anti-inflammatory effects, thereby significantly improving the efficacy of oral ulcers.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for preparing a sprayable dual-function gel containing propolis extract comprises the following steps:

[0010] (1) Acetylated carboxymethyl chitosan A-CC was synthesized using carboxymethyl chitosan and acetic anhydride as raw materials;

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

[0012] (3) The boric acid-modified acetylated carboxymethyl chitosan gel precursor was mixed with water and self-assembled into A-CC-PBA hydrogel through hydrogen bonding and π-π bonding;

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

[0014] The reaction formula involved in the above-mentioned preparation method of the present invention is as follows:

[0015]

[0016] The method of the present invention first uses acetic anhydride as an acylating agent to synthesize a temperature-sensitive acetylated carboxymethyl chitosan gel precursor A-CC; then, a boric acid group is modified on the side chain of the A-CC gel precursor, and the boric acid group can specifically bind to the cis-vicinal dihydroxyl group rich in bacterial EPS polysaccharide, so as to achieve the precise targeted recognition of the oral bacterial biofilm by the gel; at the same time, a propolis extract with antibacterial and anti-inflammatory activity is encapsulated in the A-CC-PBA hydrogel, which can be slowly released locally in the oral ulcer to achieve a long-term antibacterial and anti-inflammatory synergistic effect, thereby achieving the effect of efficiently treating the oral ulcer.

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

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

[0019] In order to activate the amino group and facilitate the next reaction, before the reaction, aminophenylboronic acid was first added with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for activation.

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

[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 1:1-3, the phase transition temperature of A-CC-PBA hydrogel was around 36°C. Therefore, the preferred molar ratio of aminophenylboronic acid to acetylated carboxymethyl chitosan was 1:1-3.

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

[0023] By examining the mass ratio of the boric acid-modified acetylated carboxymethyl chitosan gel precursor (A-CC-PBA) to water, an appropriate ratio is preferably selected. When the mass ratio of A-CC-PBA to water is lower than 1:0.8, no gel can be formed, and when the mass ratio of A-CC-PBA to water is higher than 1:2, a complete solid is formed. Therefore, in the preferred step (3), the mass ratio of the boric acid-modified acetylated carboxymethyl chitosan gel precursor to water is 1:0.8 to 2.

[0024] The propolis extract of the present invention can be obtained by commercial purchase, or can be prepared by the following preparation method:

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

[0026] The present invention also provides a sprayable dual-function gel loaded with propolis extract, which is prepared by the above-mentioned preparation method. The sprayable dual-function gel loaded with propolis extract prepared by the present invention has good adhesion performance, can be stably retained locally in oral ulcers, and can efficiently target and identify, slowly release drugs to achieve efficient bactericidal and long-term anti-inflammatory effects, and significantly improve the efficacy of oral ulcers.

[0027] The present invention also provides a spray preparation containing propolis extract, comprising the above-mentioned sprayable dual-function gel. After the dual-function gel of the present invention is made into an oral spray, it can be sprayed into droplets of uniform size through a spray bottle, and the temperature response of the contact with the oral mucosa can be connected to form a thin film that evenly covers the oral ulcer, effectively reducing the amount of drug used and having a better drug sustained release effect.

[0028] The present invention also provides the use of the above-mentioned spray preparation containing propolis extract in preparing medicine for treating oral ulcer.

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

[0030] The sprayable bifunctional gel loaded with propolis extract prepared by the method of the present invention has the advantages that acetylation modification can control the phase transition temperature of the gel, the boric acid modification group can specifically bind to the cis-vicinal dihydroxyl group rich in bacterial EPS polysaccharide, target and identify oral bacteria, and the released propolis extract can efficiently kill bacteria, and can also control inflammation and promote ulcer surface healing, providing a new type of preparation for oral mucosal administration. Pharmacodynamic results show that the sprayable bifunctional gel prepared by the present invention can spray droplets of uniform size through a spray bottle and can connect to form a thin film uniformly covering the oral ulcer in response to the temperature of contact with the oral mucosa, and the boric acid modification group can efficiently identify bacterial EPS polysaccharide and target oral bacteria; the slowly released propolis extract is slowly released locally in the oral ulcer to achieve a long-term antibacterial and anti-inflammatory synergistic effect, and effectively treats oral ulcers, providing a new means for the treatment of patients with oral ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0033] Figure 3 The temperature sensitivity and viscosity diagram of the A-CC-PBA hydrogel and the sprayable dual-functional gel loaded with propolis extract of the present invention;

[0034] Figure 4 The scanning electron micrographs of the A-CC-PBA gel and the sprayable dual-functional gel loaded with propolis extract of the present invention;

[0035] Figure 5 This is a diagram showing the in vitro targeted antibacterial effect of the sprayable dual-functional gel loaded with propolis extract of the present invention;

[0036] Figure 6 A diagram showing the area of ​​oral ulcers in rats after administration of the sprayable dual-function gel containing propolis extract of the present invention on different days;

[0037] Figure 7 The HE and Masson staining images of oral ulcer tissues of rats with oral ulcers treated with the sprayable dual-function gel loaded with propolis extract of the present invention. DETAILED DESCRIPTION

[0038] The present invention is further described below by means of specific implementation methods. The following examples are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0039] The raw materials used in the embodiments of the present invention are all commercially available.

[0040] Preparation of propolis extract

[0041] The propolis extract was extracted by adopting an ethanol concentration of 85%, a solid-liquid ratio of 1:30, and an ultrasonic 30-min process; HPD-722 macroporous adsorption resin was used for purification, the loading solution concentration was 2.5 mg / mL, the loading flow rate was 2BV / h, and the diameter-to-height ratio was 1:9; during elution, 3BV of 20% ethanol was first used for impurity removal, and then 8BV of 75% ethanol was used for elution, and the elution flow rate was 1BV / h; the ethanol eluate was collected, the ethanol was recovered under reduced pressure, and the sample was loaded on a Sephadex LH-20 chromatography column, eluted with methanol, and the elution flow rate was 0.75mL / min, and the methanol eluate was collected, concentrated under reduced pressure, and dried to obtain the propolis extract.

[0042] Embodiment 1:

[0043] (1) Synthesis of A-CC

[0044] Weigh 0.5 g of carboxymethyl chitosan into 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, stir magnetically for 20 min, transfer 10 mL of acetic anhydride, slowly dropwise add to the above solution, react at room temperature for 12 h, pour the reaction solution into acetone for precipitation, transfer to a dialysis bag and dialyze for 48 h (purified water is replaced every 8 h), and freeze-dry the dialyzate 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, stir magnetically at room temperature until completely dissolved, add 20 mL of anhydrous methanol, stir magnetically for 20 min, then transfer 5.3 mL of 3-aminophenylboronic acid solution and slowly add it dropwise to the above solution, react at room temperature for 12 h, pour the reaction solution into acetone for precipitation, and then transfer it to a dialysis bag for 48 h (purified water is replaced every 8 h), and freeze-dry the dialyzate to obtain the boric acid-modified acetylated carboxymethyl chitosan gel precursor A-CC-PBA;

[0047] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of A-CC and A-CC-PBA in the embodiment of the present invention; Figure 1 The chemical shifts of different H atoms on the modified carboxymethyl chitosan product in their corresponding chemical environments can be learned. Figure 1 (A) shows the H NMR spectrum of A-CC, where 4.63 ppm is the chemical shift of D2O, 2.1 ppm and 3-4 ppm correspond to the chemical shifts of the hydrogen atoms on the methyl group of the acetyl group and H2-H8 on the glucose ring, respectively (in Figure 1 in (A), marked as 9 and 2-8, respectively); Figure 1 (B) shows the H NMR spectrum of A-CC-PBA, 7.54ppm, 7.61ppm, 7.52ppm and 7.78ppm correspond to the chemical shifts of hydrogen atoms on the phenylboronic acid group ( Figure 1 In (B), they are marked as 1, 2, 3 and 4). These characteristic peaks indicate that the amide group and phenylboronic acid group 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 h at room temperature to obtain A-CC-PBA hydrogel;

[0050] (4) Preparation of sprayable dual-function gel containing 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 the sprayable bifunctional gel Pro@A-CC-PBA loaded with propolis extract.

[0052] Embodiment 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 it is completely dissolved, then add 25 mL of anhydrous methanol, stir magnetically for 20 min, then transfer 25 mL of acetic anhydride and slowly drop it into the above solution, react at room temperature for 12 h, pour the reaction solution into acetone for precipitation, transfer it to a dialysis bag and dialyze it for 48 h (purified water is replaced every 8 h), and freeze-dry the dialyzate 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 and place it in a 100 mL round-bottom flask, add 20 mL of purified water and stir magnetically at room temperature until it is completely dissolved, add 20 mL of anhydrous methanol, stir magnetically for 20 min, then transfer 13.5 mL of 3-aminophenylboronic acid solution and slowly add it dropwise to the above solution, react at room temperature for 12 h, pour the reaction solution into acetone for precipitation, and then transfer it to a dialysis bag for 48 h (purified water is replaced every 8 h), and freeze-dry the dialyzate to obtain the 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 h at room temperature to obtain A-CC-PBA hydrogel;

[0059] (4) Preparation of sprayable dual-function gel containing 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 stir magnetically for 6 h at room temperature to obtain the sprayable bifunctional gel Pro@A-CC-PBA loaded with propolis extract.

[0061] Embodiment 3:

[0062] (1) Synthesis of A-CC

[0063] Weigh 0.8 g of carboxymethyl chitosan into 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, stir magnetically for 20 min, transfer 18 mL of acetic anhydride, slowly dropwise add to the above solution, react at room temperature for 12 h, pour the reaction solution into acetone for precipitation, transfer to a dialysis bag and dialyze for 48 h (purified water is replaced every 8 h), and freeze-dry the dialyzate to obtain acetylated carboxymethyl chitosan A-CC;

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

[0065] 1.2 g of 4-aminophenylboronic acid, 14 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 26 mg of N-hydroxysuccinimide (NHS) were weighed into a 100 mL beaker, and 5 mL of anhydrous methanol was added to completely dissolve to obtain a 3-aminophenylboronic acid solution; 0.6 g of A-CC was weighed and placed in a 100 mL round-bottom flask, 20 mL of purified water was added, and magnetic stirring was performed at room temperature until it was completely dissolved, 20 mL of anhydrous methanol was added, and magnetic stirring was performed for 20 min. 13 mL of 4-aminophenylboronic acid solution was transferred and slowly added dropwise to the above solution, and the reaction was reacted at room temperature for 12 h, and the reaction solution was poured into acetone for precipitation and then transferred to a dialysis bag for 48 h (purified water was replaced every 8 h), and the dialyzate was freeze-dried to obtain the 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 h at room temperature to obtain A-CC-PBA hydrogel;

[0068] (4) Preparation of sprayable dual-function gel containing 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 the 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 into 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, stir magnetically for 20 min, transfer 10 mL of acetic anhydride, slowly dropwise add to the above solution, react at room temperature for 12 h, pour the reaction solution into acetone for precipitation, transfer to a dialysis bag and dialyze for 48 h (purified water is replaced every 8 h), and freeze-dry the dialyzate 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 h at room temperature to obtain A-CC hydrogel;

[0075] (3) Preparation of gel containing 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 the common hydrogel Pro@A-CC loaded with propolis extract.

[0077] The sprayable dual-function gel loaded with propolis extract prepared in Example 1 was subjected to the following test.

[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°C, and the hydrogel does not flow when the vial is inverted for 60 seconds, indicating good gelation, indicating that the A-CC-PBA hydrogel is temperature-sensitive.

[0080] like Figure 2 As shown in B, the A-CC-PBA hydrogel can be smoothly extruded through a 2.5 mL syringe and continuously stacked to write the English letters "CMCS". When the hydrogel is squeezed by the needle, the amide bond breaks and can be extruded, and the amide bond is re-formed after the hydrogel is squeezed by the needle. The results show that the A-CC-PBA hydrogel has good injectability.

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

[0082] Test Example 2: Rheological properties of gel

[0083] like Figure 3 As shown in Figure A, the A-CC-PBA hydrogel responds significantly to temperature, and its elasticity and viscosity change significantly near the transition temperature, indicating that it has typical thermosensitive phase transition characteristics. Its sol-gel phase transition temperature is 36.7°C.

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

[0085] like Figure 4 As shown in the SEM scanning results of A-CC-PBA hydrogel and sprayable bifunctional gel Pro@A-CC-PBA loaded with propolis extract, it can be observed that A-CC-PBA hydrogel has a porous network structure with coherent pores and loose structure. This structure is conducive to the loading and sustained release of drugs. At the same time, after loading propolis extract, the hydrogel Pro@A-CC-PBA still maintains a porous structure.

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

[0087] Porphyromonas gingivalis (Pg) was inoculated on LB solid medium and cultured in a 37°C bacterial incubator for 24 hours. The bacteria were taken three times on the solid medium using an inoculation loop, dissolved in a liquid medium, and cultured in a 37°C bacterial incubator for 24 hours. 1 mg / mL propolis extract (Pro), ordinary blank hydrogel (A-CC Gel), bifunctional hydrogel group (A-CC-PBA Gel), 1 mg / mL ordinary hydrogel loaded with propolis extract (Pro@A-CC Gel), and 1 mg / mL sprayable bifunctional gel loaded with propolis extract (Pro@A-CC-PBA Gel) were co-cultured with Pg bacteria for 12 hours, and 200 μL of bacterial suspension was taken from each group and diluted with PBS in a gradient manner. 200 μL of the diluted bacterial suspension was evenly spread on an agar plate and cultured for 24 hours. The bacterial growth was observed and images were collected. Then stain with live / dead bacteria staining working solution, stain for 10 min in a 37°C bacterial incubator away from light, wash twice with 0.85% saline, and observe and collect images using an inverted fluorescence microscope.

[0088] like Figure 5 As shown in the figure, the antibacterial activity was studied by 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; and the antibacterial efficacy of sprayable bifunctional gel loaded with propolis extract (Pro@A-CC-PBA Gel) was significantly higher than that of ordinary hydrogel loaded with propolis extract (Pro@A-CC Gel), highlighting that A-CC-PBA hydrogel has good antibacterial activity against Porphyromonas gingivalis. This advantage is mainly attributed to the fact that the PBA group in the propolis extract hydrogel can specifically bind to the cis-ortho-dihydroxyl group rich in the bacterial wall of Porphyromonas gingivalis, which can achieve targeted recognition of Porphyromonas gingivalis by the gel.

[0089] In addition, through the images of live / dead bacterial viability assay, the most dead bacteria (red) were observed by fluorescence microscopy in the sprayable bifunctional gel group loaded with propolis extract (Pro@A-CC-PBA Gel) compared with the control group, which also verified the specific targeting effect of the PBA group on bacteria.

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

[0091] Experimental Example 4: Pharmacodynamic results of sprayable dual-function gel loaded with propolis extract in the treatment of oral ulcers

[0092] (1) Construction of oral ulcer model rats and experimental grouping:

[0093] 60 healthy SD rats were purchased. After adaptive feeding for 1 week, 3% sodium pentobarbital solution was intraperitoneally injected at 0.35mL / 100g for anesthesia. The labial mucosa of the lower incisor of SD rats was dried with sterile cotton balls. A 3mm×3mm square filter paper was placed in 50% glacial acetic acid for 5s and then removed. It was immediately placed on the dried labial mucosa of the lower incisor of the rats. After 30s, the filter paper was removed and the treated area was wiped with a sterile cotton ball containing physiological saline to remove residual acetic acid. In this way, the rat oral ulcer model was established. The rats with successful modeling were randomly divided into: model group, positive drug group (Paleo), low-dose (1mg / L), medium-dose group (2mg / L), and high-dose group (5mg / L) of the sprayable dual-function gel containing propolis extract.

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

[0095] Spray the rat ulcer with a handheld pump spray bottle to evenly cover the ulcer surface, once a day. After each administration, the rats were deprived of water and food for 30 minutes, and the administration was continued for 10 days. The rats were killed, and the mucosal tissues of the oral ulcers in each group were taken and placed in 4% paraformaldehyde solution for testing;

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

[0097] The oral ulcer area of ​​rats was measured with a vernier caliper before administration and on the 3rd, 5th, 7th and 9th days after administration according to the method of Chen Zhuo et al. Calculation: ulcer area = 3.14 × d1 × d2 × 1 / 4 (d1 and d2 are the maximum transverse diameter and maximum longitudinal diameter of the ulcer surface, respectively). Average food intake (g) = feed consumption per cage / number of animals per cage.

[0098] like Figure 6 After modeling, the modeling area of ​​the rats showed a sunken defect, the surrounding tissues of the sunken area were red and swollen, and the surface was covered with a yellow-white pseudomembrane. On the first day of administration, there was no significant difference in the ulcer surface between the model group and the rats in the drug administration groups. On the fourth day of administration, the model group showed swelling of the lower lip, obvious oral ulcer wounds, and obvious inflammatory edema and congestion of the surrounding tissues; the pseudomembranes of the other groups became thinner, the inflammatory edema and congestion were alleviated, and the ulcer area was reduced. On the seventh day of administration, the symptoms of the rats in the model group and the drug administration groups were alleviated. Among them, the pseudomembranes of the ulcer wounds in the positive drug group and the high-dose group faded, there was no inflammatory edema and congestion, and the ulcer surface was significantly reduced; the ulcer surface of the low-dose and medium-dose groups had no significant changes compared with the model group. On the tenth day of administration, compared with the model group, the ulcer surface of the rats in the positive drug group and the high-dose group was flat, without inflammatory edema, no congestion, and the ulcer surface was close to healing.

[0099] (4) Oral mucosal pathological examination:

[0100] ①HE staining

[0101] The oral mucosal tissue at the ulcer site was routinely paraffin-embedded, sliced, dewaxed, washed, stained with hematoxylin, washed, differentiated, blued, immersed in water, and placed in eosin solution. Routine dehydration, transparency, and sealing were performed. Changes in mucosal tissue and inflammatory cell infiltration in epithelial connective tissue were observed under an optical microscope.

[0102] ②Masson staining

[0103] Take sections from each group, dewax and hydrate with xylene, and stain with hematoxylin for 5-10 minutes. Differentiate with acidic ethanol differentiation solution and wash with water. Re-blue with Masson blue solution; wash with distilled water for 1 minute; stain with staining solution for 5-10 minutes. Wash with weak acid working solution for 1 minute. Wash with phosphomolybdic acid solution for 1-2 minutes. Wash with weak acid working solution for 1 minute. Stain with aniline blue staining solution for 1-2 minutes. Wash with weak acid working solution for 1 minute. Dehydrate with anhydrous ethanol 3 times. Transparent with xylene 3 times. Seal with neutral gum. Observe the distribution of elastic fibers and collagen fibers in mucosal tissue.

[0104] like Figure 7 As shown, the oral mucosal epithelium of rats in the positive drug and high-dose groups was intact, with loose connective tissue at the bottom, orderly arrangement of cells, and no pathological changes such as vascular hemorrhage and inflammatory cell infiltration. Compared with the previous two groups, the model group had severe damage to the oral mucosa, loss of squamous epithelium, necrotic tissue on the surface, large-area inflammatory cell infiltration and vascular hemorrhage. After 10 days of drug treatment, compared with the model group, the positive drug and low, medium and high dose groups all promoted the healing of rat oral ulcers induced by glacial acetic acid, among which the positive drug and high dose group had 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 mucosa. The production of collagen in local wounds is also an important sign of remodeling of damaged mucosal tissue. It can be seen that the positive drug and high-dose groups showed more collagen fibers than other groups, while a small amount of collagen was seen in the model group, and the arrangement was disordered.

Claims

1. A method for preparing a sprayable dual-function gel containing propolis extract, characterized in that: The steps include: (1) Synthesizing acetylated carboxymethyl chitosan using carboxymethyl chitosan and acetic anhydride as raw materials; (2) A boric acid-modified acetylated carboxymethyl chitosan gel precursor was synthesized by reacting acetylated carboxymethyl chitosan and aminophenylboronic acid as raw materials; (3) The boric acid-modified acetylated carboxymethyl chitosan gel precursor is mixed with water to self-assemble through hydrogen bonding and π-π bonding. Assembled into A-CC-PBA hydrogel; (4) Propolis extract was added to the A-CC-PBA hydrogel to prepare a sprayable dual-functional gel loaded with propolis extract.

2. The method for preparing the sprayable dual-function gel containing propolis extract according to claim 1, characterized in that: In step (1), the molar ratio of the carboxymethyl chitosan to acetic anhydride is 1:10-20.

3. The method for preparing the sprayable dual-function gel containing propolis extract according to claim 1, characterized in that: In step (2), the aminophenylboronic acid is 3-aminophenylboronic acid or 4-aminophenylboronic acid; before the reaction, the aminophenylboronic acid is first activated by adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

4. The method for preparing the sprayable dual-function gel containing propolis extract according to claim 3, 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; and the molar ratio of aminophenylboronic acid to acetylated carboxymethyl chitosan is 1:1-3.

5. The method for preparing the sprayable dual-function gel containing propolis extract according to claim 1, characterized in that: In step (2), the reaction time is 12 to 24 hours.

6. The method for preparing the sprayable dual-function gel containing 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.

7. The method for preparing the sprayable dual-function gel containing propolis extract according to claim 1, characterized in that: In step (4), the preparation method of the propolis extract specifically includes: using ethanol with a volume concentration of 80-90% as the extraction solvent, the solid-liquid ratio is 1:20-40, and ultrasonic extraction is performed for 20-40 min; and using HPD-722 macroporous adsorption resin for purification, eluting with 70-80% ethanol, and collecting the 80% ethanol eluate; and then using Sephadex LH-20 chromatography column chromatography, eluting with methanol, collecting the methanol eluate, and concentrating under reduced pressure and drying to obtain the propolis extract.

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

9. A spray preparation containing propolis extract, comprising the sprayable dual-functional gel according to claim 8.

10. Use of the spray preparation containing propolis extract according to claim 9 in the preparation of a medicine for treating oral ulcers.

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