Oral anti-inflammatory and ulcer auxiliary repair mouthwash and preparation and application thereof

Abstract

The application discloses a mouthwash for oral anti-inflammatory and ulcer auxiliary repair, and preparation and application thereof, and belongs to the technical field of mouthwash. In view of the problems of large side effect of an antibacterial component of an existing mouthwash, lack of mucosa repair capacity, limited biofilm removal effect, and difficulty in giving consideration to anti-inflammatory and anti-calculus and promoting healing, etc., the application adopts farnesol and quaternary ammonium chitooligosaccharide to perform double grafting modification on polymaleic anhydride to prepare double grafting polymaleic anhydride, and the double grafting polymaleic anhydride is compounded with components such as glycerol, cetylpyridinium chloride, sodium fluoride, zinc lactate, etc. to prepare the mouthwash. The double grafting polymaleic anhydride has the functions of antibacterial and anti-inflammatory, biofilm inhibition, mucosa adhesion and tissue repair promotion, and retains excellent calcium ion complexing performance. The mouthwash can effectively inhibit oral pathogenic bacteria and biofilm thereof, relieve inflammatory reaction, assist in repairing oral mucosa ulcer wound, and simultaneously inhibit dental calculus formation, and has high safety, important clinical significance and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of mouthwash technology, and particularly relates to a mouthwash for oral anti-inflammatory and ulcer-aiding repair, its preparation and application. Background Technology

[0002] As people's living standards improve, their demand for oral health is increasing. The human oral cavity has a complex environment with a suitable temperature, constant humidity, and rich nutrients, making it an ideal habitat for various microorganisms to colonize, grow, and reproduce. When the oral flora is imbalanced or the host's immunity declines, various oral diseases can be easily induced, among which oral inflammation (such as gingivitis, periodontitis, and oral mucositis) and oral ulcers are the most common.

[0003] Oral ulcers are the most common type of oral mucosal disease. Essentially, they involve the disruption of the integrity of the mucosal epithelial tissue, with necrosis and sloughing of the superficial tissue forming a localized depressed defect. Depending on the depth of the injury, superficial ulcers affecting only the epithelial layer generally heal without scarring, while deep ulcers that invade the submucosa may leave scar tissue. The typical clinical presentation is a central depression in the ulcer, surrounded by a red halo approximately 1 mm wide, covered with a pseudomembrane, and accompanied by significant burning pain. Oral ulcers are not a single, independent disease, but a common clinical lesion with complex and diverse causes. They may be triggered by single or multiple factors such as local trauma, pathogenic microbial infection, immune dysfunction, digestive tract diseases, genetic predisposition, psychological factors, or environmental factors. Sometimes, they are also a local manifestation of certain systemic diseases. The most common type is recurrent aphthous ulcer, with a prevalence rate as high as 25% in the population, and a recurrence rate of up to 50% within three months. Severe cases result in significant decline in quality of life due to pain affecting daily functions such as eating and speaking.

[0004] Currently, there is no gold standard treatment for oral ulcers; treatment primarily focuses on eliminating potential causes and providing local symptomatic relief. The purpose of topical medication is to reduce inflammation, relieve pain, prevent secondary infections, and promote healing. Mouthwashes are a commonly used dosage form for topical medication. However, existing commercially available mouthwash products generally have the following shortcomings: First, long-term use of some highly effective antibacterial ingredients can lead to side effects such as tooth discoloration, taste disturbances, and oral mucosal peeling, making them unsuitable for daily care or use during the acute phase of ulcers; most mouthwashes only focus on antibacterial or deodorizing functions, lacking the ability to actively repair existing oral ulcer wounds and failing to promote mucosal epithelial healing; their ability to remove oral biofilms is limited, and biofilms are one of the key factors leading to recurrent inflammation and persistent ulcers; few existing products contain components that simultaneously inhibit tartar formation and promote soft tissue repair, making it difficult to meet the comprehensive needs of patients with oral inflammation and ulcers in terms of anti-inflammatory, anti-tartar, and healing-promoting effects.

[0005] Therefore, developing a mouthwash that can effectively inhibit oral pathogens and their biofilms, reduce inflammatory responses, and help repair oral mucosal ulcers, while also being highly safe and having no significant side effects, has important clinical significance and broad application prospects. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention uses farnesol and quaternized chitosan oligosaccharide to double-graft modify polymaleic anhydride, and prepares a mouthwash for oral anti-inflammatory and ulcer-aiding repair.

[0007] To achieve the above objectives, the following technical solution is adopted: On the one hand, the present invention provides a mouthwash for oral anti-inflammatory and ulcer-aiding repair, comprising the following components by mass percentage: glycerin 3.0-5.0%, pH buffer 0.10-0.30%, cetylpyridinium chloride 0.03-0.10%, sodium fluoride 0.02-0.05%, zinc lactate 0.18-0.21%, double-grafted polymaleic anhydride 0.05-0.40%, polyquaternium-10 0.05-0.10%, fragrance 0.05-0.20%, preservative 0.05-0.25%, and the balance being pure water.

[0008] Furthermore, the double-grafted polymaleic anhydride is prepared by the following steps:

[0009] S1. Add farnesol to anhydrous ethanol and stir to dissolve at room temperature. Then add CDI and react at 35-45℃ for 2-4 hours under nitrogen protection. After the reaction is completed, remove the solvent under reduced pressure to obtain activated farnesol for later use.

[0010] S2. Dissolve maleic anhydride in deionized water, adjust the system temperature to 75-95℃, add hydrogen peroxide and sodium hypophosphite, and react for 4-8 hours to obtain a polymaleic anhydride solution; then adjust the pH of the system to 6.0-7.5 with sodium hydroxide solution to cause some anhydride groups to undergo ring-opening hydrolysis to obtain a hydrolyzed polymaleic anhydride solution.

[0011] S3. Add activated farnesol dropwise to the hydrolyzed polymaleic anhydride solution, followed by the addition of DMAP and EDC·HCl; react at 50-70℃ for 6-12 h. After the reaction is complete, precipitate with ethanol, centrifuge, and wash alternately with ethanol and deionized water to obtain the farnesol-grafted polymaleic anhydride intermediate.

[0012] S4. Quaternized chitosan oligosaccharide was dissolved in an aqueous acetic acid solution to obtain a quaternized chitosan oligosaccharide solution. Then, farnesol-grafted polymaleic anhydride was dispersed in deionized water, NHS and EDC·HCl were added, and then the quaternized chitosan oligosaccharide solution was added dropwise. The reaction was carried out at 40-60℃ for 8-24 h. After the reaction was completed, the system was transferred to a dialysis bag and dialyzed in deionized water for 24-72 h. Finally, the system was freeze-dried to obtain double-grafted polymaleic anhydride.

[0013] Farnesol, a natural terpene active substance, possesses hydrophobic segments that enhance the interaction between the polymer and the bacterial biofilm and oral mucosa interface, thus inhibiting bacterial adhesion and biofilm formation. Simultaneously, it retains the complexing ability of polymaleic anhydride to calcium ions, thereby inhibiting plaque mineralization and tartar formation. Quaternized chitosan oligosaccharides contain permanently positively charged quaternary ammonium groups, endowing the polymer with excellent broad-spectrum antibacterial properties and promoting the repair of damaged oral mucosa through the bioactivity of chitosan oligosaccharides themselves. The synergistic grafting of these two functional groups endows polymaleic anhydride with multiple functions, including antibacterial and anti-inflammatory effects, biofilm inhibition, mucosal adhesion, and tissue repair promotion, thereby enhancing its application value in the field of oral care.

[0014] Furthermore, in step S1, the feeding ratio of farnesol, CDI, and anhydrous ethanol is 3.0-4.0g: 2.5-3.5g: 40-60mL.

[0015] Furthermore, in step S2, the feeding ratio of maleic anhydride, hydrogen peroxide, sodium hypophosphite, and deionized water is 10.0-15.0g: 1.0-2.5g: 4.0-6.0g: 80-120mL.

[0016] Further, in step S3, the feeding ratio of activated farnesol, DMAP, EDC·HCl and polymaleic anhydride solution is 3.0-4.0g: 0.3-0.8g: 2.5-4.0g: 80-120mL.

[0017] Further, in step S4, the feed ratio of quaternized chitosan oligosaccharide, aqueous acetic acid solution, farnesol-grafted polymaleic anhydride, deionized water, NHS, and EDC·HCl is 1.5-3.0g: 30-50mL: 2.0-5.0g: 50-80mL: 0.8-1.5g: 1.5-3.0g.

[0018] Further, the quaternized chitosan oligosaccharide is prepared by the following steps: 10-20g of chitosan oligosaccharide is weighed and added to 200-300mL of deionized water. The mixture is magnetically stirred at room temperature for 2 hours. A 20% sodium hydroxide solution is added to the system to adjust the pH to 10-11. The reaction system is heated to 50-60℃. Under nitrogen protection, 30-50mL of iodomethane is slowly added dropwise to the reaction solution, along with 100mL of isopropanol. The reaction temperature is maintained at 55℃, and the mixture is continuously stirred for 12-24 hours. After the reaction is completed, the mixture is cooled to room temperature, concentrated by rotary evaporation under reduced pressure, and then slowly poured into 5 times the volume of anhydrous ethanol to precipitate the product. After standing for 2 hours, the product is centrifuged. The precipitate is washed three times with anhydrous ethanol and twice with acetone. After drying, the quaternized chitosan oligosaccharide is obtained.

[0019] Furthermore, the mouthwash also includes a pH buffer solution to adjust the pH of the mouthwash to 6.4-6.8; the pH buffer solution is made by dissolving sodium dihydrogen phosphate and disodium hydrogen phosphate in pure water.

[0020] Furthermore, the preservative is one of potassium sorbate and sodium benzoate.

[0021] On the other hand, the present invention provides a method for preparing the oral anti-inflammatory and ulcer-aiding mouthwash, comprising the following steps: adding glycerin, sodium fluoride, zinc lactate, double-grafted polymaleic anhydride, and polyquaternium-10 to a portion of pure water according to the specified ratio, stirring and dissolving evenly to obtain a mixture; dissolving cetylpyridinium chloride, fragrance, and preservative in the remaining pure water, and slowly adding them to the mixture; finally, adding pH buffer to adjust the pH to 6.4-6.8, stirring evenly to obtain the mouthwash.

[0022] In another aspect, the present invention also provides the application of an oral anti-inflammatory and ulcer-aiding mouthwash in the preparation of oral care products for treating or aiding in the improvement of oral inflammation and oral ulcers.

[0023] The beneficial effects of this invention are as follows: This invention significantly enhances the bioactivity of polymaleic anhydride by dual functionalizing it with farnesol and quaternized chitosan oligosaccharides, expanding it from a traditional scale-inhibiting polymer into a bioactive material with antibacterial, anti-inflammatory, and tissue repair-promoting functions. The prepared double-grafted polymaleic anhydride exhibits excellent ability to inhibit the adhesion of oral pathogenic bacteria and biofilm formation, helping to reduce the pathogenic microbial load in the oral cavity and improve the oral microecological environment. Furthermore, the double-grafted polymaleic anhydride also has good oral mucosal affinity. Its properties and retention capacity allow it to form a protective layer on ulcer surfaces, reducing the impact of external stimuli on damaged tissues and facilitating the repair of oral ulcers. Furthermore, the double-grafted polymaleic anhydride prepared in this invention retains the excellent calcium ion complexing properties of polymaleic anhydride, inhibiting plaque mineralization and tartar formation, thus combining oral inflammation care with daily oral health maintenance. The synergistic system of double-grafted polymaleic anhydride, cetylpyridinium chloride, and zinc lactate further enhances the antibacterial and anti-inflammatory effects of the mouthwash and strengthens its ability to control oral odor. Attached Figure Description

[0024] Figure 1 Infrared characterization spectra of hydrolyzed polymaleic anhydride and double-grafted polymaleic anhydride prepared in Example 3 of the present invention.

[0025] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0029] Example 1

[0030] A mouthwash for oral inflammation and ulcer repair comprises the following components by weight percentage: 3.0% glycerin, 0.10% pH buffer, 0.03% cetylpyridinium chloride, 0.02% sodium fluoride, 0.18% zinc lactate, 0.05% double-grafted polymaleic anhydride, 0.05% polyquaternium-10, 0.05% fragrance, 0.05% preservative, and the balance being purified water. The preservative is potassium sorbate. The mouthwash also includes a pH buffer to adjust the pH of the mouthwash to 6.4; the pH buffer is prepared by dissolving sodium dihydrogen phosphate and disodium hydrogen phosphate in purified water.

[0031] The double-grafted polymaleic anhydride is prepared by the following steps:

[0032] S1. Add 3.0g of farnesol to 40mL of anhydrous ethanol and stir to dissolve at room temperature. Then add 2.5g of CDI and react at 35℃ for 2h under nitrogen protection. After the reaction is completed, remove the solvent under reduced pressure to obtain activated farnesol for later use.

[0033] S2. Dissolve 10.0g of maleic anhydride in 80mL of deionized water, adjust the system temperature to 75℃, add 1.0g of hydrogen peroxide and 4.0g of sodium hypophosphite, and react for 4h to obtain a polymaleic anhydride solution; then adjust the pH of the system to 6.0 with sodium hydroxide solution to cause some anhydride groups to undergo ring-opening hydrolysis to obtain a hydrolyzed polymaleic anhydride solution.

[0034] S3. Add 3.0 g of activated farnesol dropwise to 80 mL of hydrolyzed polymaleic anhydride solution, then add 0.3 g DMAP and 2.5 g EDC·HCl; react at 50 °C for 6 h, after the reaction is complete, precipitate with ethanol, centrifuge, and wash alternately with ethanol and deionized water to obtain farnesol-grafted polymaleic anhydride intermediate.

[0035] S4. Dissolve 1.5g of quaternized chitosan oligosaccharide in 30mL of acetic acid aqueous solution to obtain quaternized chitosan oligosaccharide solution. Then, disperse 2.0g of farnesol-grafted polymaleic anhydride in 50mL of deionized water, add 0.8g of NHS and 1.5g of EDC·HCl, and then add the quaternized chitosan oligosaccharide solution dropwise. React at 40℃ for 8h. After the reaction is completed, transfer the system to a dialysis bag and dialyze in deionized water for 24h. Finally, freeze-dry to obtain double-grafted polymaleic anhydride.

[0036] The quaternized chitosan oligosaccharide was prepared by the following steps: 10g of chitosan oligosaccharide was weighed and added to 200mL of deionized water. The mixture was magnetically stirred at room temperature for 2h. A 20% sodium hydroxide solution was added to the system to adjust the pH to 10. The reaction system was heated to 50℃. Under nitrogen protection, 30mL of iodomethane was slowly added dropwise to the reaction solution, along with 100mL of isopropanol. The reaction temperature was maintained at 55℃, and the mixture was stirred continuously for 12h. After the reaction was completed, the mixture was cooled to room temperature, concentrated by rotary evaporation under reduced pressure, and then slowly poured into 5 times the volume of anhydrous ethanol to precipitate the mixture. After standing for 2h, the precipitate was centrifuged. The precipitate was washed three times with anhydrous ethanol and twice with acetone. After drying, the quaternized chitosan oligosaccharide was obtained.

[0037] The method for preparing the oral anti-inflammatory and ulcer-aiding mouthwash includes the following steps: adding glycerin, sodium fluoride, zinc lactate, double-grafted polymaleic anhydride, and polyquaternium-10 to a portion of pure water according to the specified ratio, stirring and dissolving evenly to obtain a mixture; dissolving cetylpyridinium chloride, fragrance, and preservative in the remaining pure water, then slowly adding them to the mixture; finally, adding pH buffer to adjust the pH to 6.4, stirring evenly to obtain the mouthwash.

[0038] Example 2

[0039] A mouthwash for oral inflammation and ulcer repair comprises the following components by weight percentage: 5.0% glycerin, 0.30% pH buffer, 0.10% cetylpyridinium chloride, 0.05% sodium fluoride, 0.21% zinc lactate, 0.40% double-grafted polymaleic anhydride, 0.10% polyquaternium-10, 0.20% fragrance, 0.25% preservative, and the balance being purified water. The preservative is sodium benzoate. The mouthwash also includes a pH buffer to adjust the pH of the mouthwash to 6.8; the pH buffer is prepared by dissolving sodium dihydrogen phosphate and disodium hydrogen phosphate in purified water.

[0040] The double-grafted polymaleic anhydride is prepared by the following steps:

[0041] S1. Add 4.0g of farnesol to 60mL of anhydrous ethanol and stir to dissolve at room temperature. Then add 3.5g of CDI and react at 45℃ for 4h under nitrogen protection. After the reaction is completed, remove the solvent under reduced pressure to obtain activated farnesol for later use.

[0042] S2. Dissolve 15.0g of maleic anhydride in 120mL of deionized water, adjust the system temperature to 95℃, add 2.5g of hydrogen peroxide and 6.0g of sodium hypophosphite, and react for 8h to obtain a polymaleic anhydride solution; then adjust the pH of the system to 7.5 with sodium hydroxide solution to cause some anhydride groups to undergo ring-opening hydrolysis to obtain a hydrolyzed polymaleic anhydride solution.

[0043] S3. Add 4.0 g of activated farnesol dropwise to 120 mL of hydrolyzed polymaleic anhydride solution, followed by the addition of 0.8 g DMAP and 4.0 g EDC·HCl; react at 70 °C for 12 h; after the reaction is complete, precipitate with ethanol, centrifuge, and wash alternately with ethanol and deionized water to obtain farnesol-grafted polymaleic anhydride intermediate.

[0044] S4. Dissolve 3.0 g of quaternized chitosan oligosaccharide in 50 mL of acetic acid aqueous solution to obtain quaternized chitosan oligosaccharide solution. Then, disperse 5.0 g of farnesol-grafted polymaleic anhydride in 80 mL of deionized water, add 1.5 g of NHS and 3.0 g of EDC·HCl, and then add the quaternized chitosan oligosaccharide solution dropwise. React at 60 °C for 24 h. After the reaction is completed, transfer the system to a dialysis bag and dialyze in deionized water for 72 h. Finally, freeze-dry to obtain double-grafted polymaleic anhydride.

[0045] The quaternized chitosan oligosaccharide was prepared by the following steps: 20g of chitosan oligosaccharide was weighed and added to 300mL of deionized water. The mixture was magnetically stirred at room temperature for 2h. A 20% sodium hydroxide solution was added to the system to adjust the pH to 11. The reaction system was heated to 60℃. Under nitrogen protection, 50mL of iodomethane was slowly added dropwise to the reaction solution, along with 100mL of isopropanol. The reaction temperature was maintained at 55℃, and the mixture was stirred continuously for 24h. After the reaction was completed, the mixture was cooled to room temperature, concentrated by rotary evaporation under reduced pressure, and then slowly poured into 5 times the volume of anhydrous ethanol to precipitate the mixture. After standing for 2h, the precipitate was centrifuged. The precipitate was washed three times with anhydrous ethanol and twice with acetone. After drying, the quaternized chitosan oligosaccharide was obtained.

[0046] The method for preparing the oral anti-inflammatory and ulcer-aiding mouthwash includes the following steps: adding glycerin, sodium fluoride, zinc lactate, double-grafted polymaleic anhydride, and polyquaternium-10 to a portion of pure water according to the specified ratio, stirring and dissolving evenly to obtain a mixture; dissolving cetylpyridinium chloride, fragrance, and preservative in the remaining pure water, and slowly adding them to the mixture; finally, adding pH buffer to adjust the pH to 6.8, stirring evenly, to obtain the mouthwash.

[0047] Example 3

[0048] A mouthwash for oral inflammation and ulcer repair comprises the following components by weight percentage: 4.0% glycerin, 0.20% pH buffer, 0.065% cetylpyridinium chloride, 0.035% sodium fluoride, 0.195% zinc lactate, 0.225% double-grafted polymaleic anhydride, 0.075% polyquaternium-10, 0.125% fragrance, 0.15% preservative, and the balance being purified water. The preservative is a mixture of potassium sorbate and sodium benzoate in a 1:1 mass ratio. The mouthwash also includes a pH buffer to adjust the pH to 6.6; the pH buffer is prepared by dissolving sodium dihydrogen phosphate and disodium hydrogen phosphate in purified water.

[0049] The double-grafted polymaleic anhydride is prepared by the following steps:

[0050] S1. Add 3.5g of farnesol to 50mL of anhydrous ethanol and stir to dissolve at room temperature. Then add 3.0g of CDI and react at 40℃ for 3h under nitrogen protection. After the reaction is completed, remove the solvent under reduced pressure to obtain activated farnesol for later use.

[0051] S2. Dissolve 12.5g of maleic anhydride in 100mL of deionized water, adjust the system temperature to 85℃, add 1.75g ​​of hydrogen peroxide and 5.0g of sodium hypophosphite, and react for 6h to obtain a polymaleic anhydride solution; then adjust the pH of the system to 6.8 with sodium hydroxide solution to cause some anhydride groups to undergo ring-opening hydrolysis to obtain a hydrolyzed polymaleic anhydride solution.

[0052] S3. Add 3.5g of activated farnesol dropwise to 100mL of hydrolyzed polymaleic anhydride solution, followed by 0.55g of DMAP and 3.25g of EDC·HCl; react at 60℃ for 9h. After the reaction is complete, precipitate with ethanol, centrifuge, and wash alternately with ethanol and deionized water to obtain farnesol-grafted polymaleic anhydride intermediate.

[0053] S4. Dissolve 2.25g of quaternized chitosan oligosaccharide in 40mL of acetic acid aqueous solution to obtain quaternized chitosan oligosaccharide solution. Then, disperse 3.5g of farnesol-grafted polymaleic anhydride in 65mL of deionized water, add 1.15g of NHS and 2.25g of EDC·HCl, and then add the quaternized chitosan oligosaccharide solution dropwise. React at 50℃ for 16h. After the reaction is completed, transfer the system to a dialysis bag and dialyze in deionized water for 48h. Finally, freeze-dry to obtain double-grafted polymaleic anhydride.

[0054] The quaternized chitosan oligosaccharide was prepared by the following steps: 15g of chitosan oligosaccharide was weighed and added to 250mL of deionized water. The mixture was magnetically stirred at room temperature for 2 hours. A 20% sodium hydroxide solution was added to the system to adjust the pH to 10.5. The reaction system was heated to 55°C. Under nitrogen protection, 40mL of iodomethane was slowly added dropwise to the reaction solution, along with 100mL of isopropanol. The reaction temperature was maintained at 55°C, and the mixture was stirred continuously for 18 hours. After the reaction was completed, the mixture was cooled to room temperature, concentrated by rotary evaporation under reduced pressure, and then slowly poured into 5 times the volume of anhydrous ethanol to precipitate the mixture. After standing for 2 hours, the precipitate was centrifuged. The precipitate was washed three times with anhydrous ethanol and twice with acetone. After drying, the quaternized chitosan oligosaccharide was obtained.

[0055] The method for preparing the oral anti-inflammatory and ulcer-aiding mouthwash includes the following steps: adding glycerin, sodium fluoride, zinc lactate, double-grafted polymaleic anhydride, and polyquaternium-10 to a portion of pure water according to the specified ratio, stirring and dissolving evenly to obtain a mixture; dissolving cetylpyridinium chloride, fragrance, and preservative in the remaining pure water, then slowly adding them to the mixture; finally, adding pH buffer to adjust the pH to 6.6, stirring evenly to obtain the mouthwash.

[0056] Comparative Example 1

[0057] A mouthwash with essentially the same composition as in Example 3, except that: no double-grafted polymaleic anhydride is added, and its mass percentage is made up by pure water.

[0058] Comparative Example 2

[0059] A mouthwash with essentially the same composition as in Example 3, except that the double-grafted polymaleic anhydride is replaced with an equal amount of ungrafted polymaleic anhydride.

[0060] Comparative Example 3

[0061] A mouthwash with essentially the same components as in Example 3, except that farnesol monografted polymaleic anhydride is used instead of double-grafted polymaleic anhydride. This monografted product is prepared via steps S1-S3 in Example 3, but step S4 is omitted, i.e., quaternized chitosan oligosaccharide is not grafted.

[0062] Comparative Example 4

[0063] A mouthwash with essentially the same components as in Example 3, except that quaternized chitosan oligosaccharide is used as a single-grafted polymaleic anhydride instead of a double-grafted polymaleic anhydride. This single-grafted product is prepared by the following steps: steps S1 and S3 are omitted, and quaternized chitosan oligosaccharide is directly grafted onto the hydrolyzed polymaleic anhydride prepared in S2 according to method S4.

[0064] Comparative Example 5

[0065] A mouthwash with essentially the same composition as in Example 3, except that in the preparation of double-grafted polymaleic anhydride, the quaternized chitosan oligosaccharide used in step S4 is replaced with an equal mass of unquaternized ordinary chitosan oligosaccharide, while the other grafting conditions remain unchanged.

[0066] Results Analysis

[0067] The hydrolyzed polymaleic anhydride and double-grafted polymaleic anhydride prepared in Example 3 of this invention were characterized by infrared spectroscopy, as shown in the figure. Figure 1 As shown, the upper and lower curves represent hydrolyzed polymaleic anhydride and double-grafted polymaleic anhydride, respectively. Figure 1 It can be seen that, compared to hydrolyzed polymaleic anhydride, the final product has a lower concentration at 1732 cm⁻¹. -1 A distinct characteristic absorption peak for the ester group C=O appears at 2920 cm⁻¹, and at the same time, a peak is observed at 2920 cm⁻¹. -1 and 2850cm -1 The presence of a fatty chain -CH2- stretching vibration peak at 1655 cm⁻¹ indicates that farnesol has been successfully grafted onto the polymaleic anhydride molecular chain via ester bonds; furthermore, a peak is observed at 1655 cm⁻¹. -1 and 1551cm -1 The presence of characteristic peaks for amide I and amide II at 1470 cm⁻¹ indicates that the carboxyl group of polymaleic anhydride undergoes condensation with the amino group of quaternized chitosan oligosaccharide to form an amide bond; -1 Quaternary ammonium salt N appears at [location] + The characteristic absorption peak of (CH3)3 is at 1152 cm⁻¹. -1 The significantly enhanced uptake of nearby glycosidic bonds further confirms that the quaternized chitosan oligosaccharide has been successfully grafted onto the polymaleic anhydride backbone. These results demonstrate that both farnesol and quaternized chitosan oligosaccharide have been successfully introduced into the polymaleic anhydride molecular structure, resulting in the successful acquisition of the target double-grafted polymaleic anhydride.

[0068] Test Example 1: Determination of inhibition rate against Streptococcus mutans

[0069] Referring to the antibacterial test method (agar dilution method) in the "Disinfection Technical Specifications" (2002 edition), the 5-minute antibacterial rate of each mouthwash against Streptococcus mutans (ATCC 25175, purchased from Beijing Huayueyang Biotechnology Co., Ltd.) was determined. The specific steps are as follows:

[0070] (1) Preparation of bacterial suspension: Streptococcus mutans was inoculated onto BHI agar plates and anaerobically cultured at 37°C (80% N2, 10% CO2, 10% H2) for 24 h. Single colonies were picked and inoculated onto BHI liquid medium and cultured at 37°C with shaking (120 rpm) for 18 h until the logarithmic growth phase. The bacterial suspension concentration was adjusted to 1.0 × 10⁻⁶ with sterile PBS. 8 CFU / mL.

[0071] (2) Sample dilution and application: Take a sterile test tube and add 0.2 mL of each mouthwash sample. The positive and negative controls are processed in the same way. Add 0.1 mL of bacterial suspension to each tube (final concentration approximately 3.3 × 10⁻⁶). 7 (CFU / mL), mix well and allow to react accurately in a 25℃ water bath for 5 min.

[0072] (3) Neutralization: After the reaction is complete, immediately add 0.5 mL of neutralizing agent (sterile PBS containing 3% Tween-80, 0.3% lecithin, and 0.5% sodium thiosulfate), mix well and terminate the sterilization reaction. Take 0.1 mL of the mixture and perform a 10-fold serial dilution.

[0073] (4) Culture and counting: Take a dilution of 10 -5 0.1 mL of bacterial suspension was spread onto BHI agar plates, with three plates for each dilution. After anaerobic incubation at 37°C for 48 h, the colony count (CFU) was counted. A positive control group was also provided using PBS instead of the samples.

[0074] (5) Calculation: Antibacterial rate (%) = (CFU of positive control group - CFU of sample group) / CFU of positive control group × 100%.

[0075] Test Example 2: Determination of the inhibition rate of Streptococcus mutans biofilm formation

[0076] The ability of each mouthwash to inhibit initial biofilm formation was evaluated using the crystal violet staining method. The specific steps are as follows:

[0077] (1) Preparation of bacterial suspension: Same as in test example 1, but the concentration of bacterial suspension was adjusted to 1.0 × 10⁻⁶. 7 CFU / mL (diluted in BHI medium).

[0078] (2) Sample preparation and biofilm culture: 100 μL of bacterial suspension was added to each well of a 96-well plate, followed by 100 μL of each mouthwash sample. Equal volumes of chlorhexidine and physiological saline were added to the positive and negative controls, respectively. Each sample was tested in 5 replicates. The plates were anaerobically incubated at 37°C for 24 h without changing the culture medium.

[0079] (3) Staining: Discard the culture medium in the wells and wash gently three times with sterile PBS (200 μL each time). Add 150 μL of 0.1% crystal violet staining solution to each well and stain at room temperature for 30 min. Discard the staining solution and wash three more times with PBS.

[0080] (4) Decolorization and measurement: Add 150 μL of 33% glacial acetic acid to each well and decolorize by shaking at room temperature for 15 min. Measure the absorbance (OD) at 590 nm using a microplate reader. 590 ).

[0081] (5) Calculation: Biomembrane inhibition rate (%) = (OD of negative control group) 590 - Sample group OD 590 ) / Negative control group OD 590 ×100%.

[0082] Test Example 3: Determination of Scavenging Capacity on Mature Biofilms

[0083] Following the ASTM E2871-13 method, the effectiveness of each mouthwash in removing 24-hour mature biofilm was evaluated. The specific steps are as follows:

[0084] (1) Biofilm culture: Place a sterile coverslip (14 mm in diameter) in each well of a 24-well plate, and add 1.8 mL of BHI medium and 0.2 mL of 1.0 × 10⁻⁶ mol / L mol / L to each well. 8 CFU / mL Streptococcus mutans suspension (final concentration 1.0 × 10⁻⁶) 7 CFU / mL). Anaerobic culture at 37℃ (80% N2, 10% CO2, 10% H2) for 24 h, with fresh BHI medium replaced every 12 h to form a mature biofilm.

[0085] (2) Sample preparation: Gently aspirate the culture medium and wash twice with sterile PBS (1 mL each time) to remove airborne bacteria. Add 1 mL of each mouthwash sample to each well, and add 1 mL of physiological saline to the negative control group. Incubate at 37℃ for 5 min.

[0086] (3) Neutralization and elution: Remove the sample, add 1 mL of neutralizing agent (sterile PBS containing 3% Tween-80, 0.3% lecithin, and 0.5% sodium thiosulfate) to each well, and incubate at room temperature for 5 min. Remove the coverslip with sterile forceps, place it in a centrifuge tube containing 2 mL of PBS, sonicate in a water bath (40 kHz) for 5 min, and then vortex for 30 s to completely detach the biofilm.

[0087] (4) Viable cell count: Take the detached bacterial culture and perform a 10-fold serial dilution to 10⁻⁶. -5 Take 100 μL and spread it on a BHI agar plate. Incubate anaerobically at 37°C for 48 h and count the number of colonies (CFU / coverslip).

[0088] (5) Calculation: The mature biofilm removal rate is expressed as the logarithmic reduction in colonies: Mature biofilm removal rate (Log CFUreduction) = Log 10 (Negative control group CFU) - Log10 (sample group CFU).

[0089] Test Example 4: Healing-promoting effect on a rat oral ulcer model

[0090] An acetic acid-induced oral ulcer model was established in rats to evaluate the healing-promoting effects of various mouthwashes on ulcer wounds. The specific steps are as follows:

[0091] (1) Experimental animals: SPF grade SD rats, male, weighing 200-220 g, were acclimatized for 7 days before the experiment. They were fasted for 12 h before the experiment and had free access to water.

[0092] (2) Model establishment: Isoflurane inhalation anesthesia (induction concentration 4%, maintenance concentration 2%). The left buccal mucosa of the rat was dried with a cotton swab, and a circular filter paper (3 mm in diameter) soaked in 50 μL of glacial acetic acid (99.5%) was placed in the center of the mucosa and kept for 30 s to create a circular ulcer with a diameter of about 3 mm. A white pseudomembrane and surrounding congestion were visible to the naked eye, indicating that the model was successfully established.

[0093] (3) Grouping and administration: Rats with successful modeling were randomly divided into 11 groups (Examples 1-3, Comparative Examples 1-5, Positive Control, and Negative Control), with 8 rats in each group. Administration began on the first day after modeling. 0.2 mL of mouthwash was applied directly to the ulcer surface using a micropipette twice daily, morning and evening, maintaining contact for 30 seconds. The negative control group received an equal volume of physiological saline. Administration continued until the ulcer was completely healed.

[0094] (4) Observation indicators: The long and short diameters of the ulcer were measured with electronic calipers at the same time every day, and the ulcer area was calculated (π×long diameter / 2×short diameter / 2). The number of days for complete ulcer healing was recorded (the mucosal epithelium was observed to be intact, without depressions or pseudomembranes). The healing rate on day 5 was calculated as follows: Healing rate (%) = (initial area - area on day 5) / initial area × 100%.

[0095] Test Example 5: Determination of Calcium Ion Complexation Ability

[0096] The specific steps for evaluating the complexing ability of each mouthwash for calcium ions, reflecting its potential to inhibit tartar formation, are as follows:

[0097] (1) Sample pretreatment: Each mouthwash was diluted 5 times with deionized water. Take 5 mL of the diluted solution and add 0.5 mL of 0.1 mol / L CaCl2 solution (final calcium concentration 2 mmol / L). The solution was shaken in a water bath at 37℃ (100 rpm) for 30 min.

[0098] (2) Determination of free calcium: The reaction solution was filtered through a 0.22 μm filter membrane. 0.1 mL of the filtrate was taken, and 3 mL of o-cresolphthalein complex chromogenic reagent was added. The mixture was stirred well and allowed to stand at room temperature for 5 min. The absorbance was measured at 575 nm. Deionized water was used as a blank control instead of the sample. The concentration of free calcium was calculated according to the calcium standard curve (0-2 mmol / L).

[0099] (3) Calculation: Calcium ion complexation rate (%) = (initial total calcium concentration - free calcium concentration) / initial total calcium concentration × 100%.

[0100] Test Example 6: Oral Mucosal Irritation Test

[0101] The short-term irritation of various mouthwashes on the cheek pouch mucosa of golden hamsters was evaluated according to ISO 10993-10:2010 standard. The specific steps are as follows:

[0102] (1) Experimental animals: Syrian hamsters, male, weighing 100-120 g, 6 per group. Adaptation period of 3 days.

[0103] (2) Topical administration: On the day of the experiment, isoflurane was administered by inhalation anesthesia (induction concentration 4%, maintenance concentration 2%). 0.1 mL of each mouthwash solution was applied to the mucosa of the right cheek pouch of golden hamsters using a sterile cotton swab, twice daily (6 hours apart) for 14 consecutive days. The left cheek pouch was left untreated as a self-control. The negative control group received saline solution, and the positive control group received 0.5% sodium dodecyl sulfate (SDS).

[0104] (3) Observation and scoring: Before each day's administration, observe the mucosa for erythema, edema, erosion, etc. On days 1, 7, and 14, 2 hours after administration and 24 hours after the last administration, mucosal irritation is scored according to ISO 10993-10 standard: 0 points = no reaction; 1 point = very mild erythema; 2 points = clear erythema; 3 points = moderate erythema with edema; 4 points = severe erythema, erosion, or ulceration. The average of the total scores over 14 days is taken.

[0105] The test results for the above test cases are shown in Table 1.

[0106] Table 1 Comparison of Test Results for Test Examples 1-6

[0107] sample Antibacterial rate (%) Biomembrane inhibition rate (%) Mature biofilm clearance rate (%) Ulcer healing time (days) Healing rate on day 5 (%) Calcium ion complexation rate (%) Mucosal irritation score (points) Example 1 89.3±2.1 82.5±1.9 2.8±0.2 6.2±0.5 72.4±3.1 58.3±2.0 0.2±0.1 Example 2 94.7±1.8 88.6±1.5 3.4±0.3 5.4±0.4 84.7±2.8 62.7±1.8 0.3±0.1 Example 3 96.2±1.5 91.3±1.2 3.8±0.2 5.1±0.3 88.5±2.5 60.9±1.6 0.2±0.1 Comparative Example 1 72.5±2.4 45.8±2.3 1.2±0.2 9.8±0.7 38.2±3.2 15.3±1.9 0.1±0.1 Comparative Example 2 68.9±2.6 40.2±2.5 1.0±0.1 10.5±0.8 32.5±3.5 55.4±2.1 0.1±0.1 Comparative Example 3 74.8±2.2 68.5±2.0 1.9±0.2 7.9±0.6 54.2±3.0 57.8±1.9 0.2±0.1 Comparative Example 4 92.3±1.7 78.6±1.8 2.6±0.2 6.8±0.5 66.5±2.9 23.6±2.0 0.2±0.1 Comparative Example 5 82.6±2.0 72.3±1.7 2.1±0.2 7.1±0.5 60.2±2.7 56.2±1.8 0.2±0.1 Positive control (0.12% chlorhexidine) 98.5±0.8 94.2±0.9 4.5±0.2 5.8±0.4 78.3±2.2 8.7±1.5 — Negative control (physiological saline) 0 0 0.1±0.1 12.5±1.0 15.2±2.0 4.2±0.8 0 Positive control (0.5% SDS) — — — — — — 3.2±0.3

[0108] Where "—" indicates that the indicator was not detected. In the mucosal irritation test, the positive control was 0.5% SDS, and the remaining samples and negative control were scored according to the ISO 10993-10:2010 standard.

[0109] Based on the experimental results of Test Examples 1-6 above, Examples 1-3 demonstrated excellent performance in antibacterial activity, anti-biofilm activity, ulcer healing promotion, tartar inhibition, and mucosal safety. Example 3 showed an antibacterial rate of 96.2% against Streptococcus mutans, slightly lower than chlorhexidine (98.5%), but significantly higher than all comparative examples (P<0.05); the biofilm inhibition rate was 91.3%, close to chlorhexidine (94.2%), and much higher than comparative examples 1-5; the mature biofilm clearance rate reached 3.8 Log reduction, significantly better than comparative examples 1-5; in the rat oral ulcer model, the ulcers healed completely in only 5.1 days, with a healing rate as high as 88.5% on day 5, both indicators being superior to chlorhexidine and all comparative examples; the calcium ion complexation rate reached 60.9%, much higher than chlorhexidine and comparative examples 1 and 4, and comparable to comparative examples 2, 3, and 5; the mucosal irritation score was only 0.2 points, belonging to the non-irritating level, and its safety was significantly better than traditional antibacterial mouthwashes such as chlorhexidine. This invention introduces farnesol and quaternized chitosan oligosaccharide into the polymaleic anhydride backbone via chemical grafting, achieving intramolecular synergy among the three: quaternized chitosan oligosaccharide provides strong positive charge, enabling broad-spectrum bactericidal activity and mucosal adhesion; the hydrophobic segments of farnesol enhance the penetration of extracellular polymers into biofilms while retaining the calcium ion complexation conformation of polymaleic anhydride, thus maintaining its anti-tartar function; the polymaleic anhydride backbone serves as a multifunctional framework, contributing calcium ion complexation sites and stably covalently linking the two active groups. The modified design of this invention achieves a balance in antibacterial, anti-biofilm, healing-promoting, tartar-preventing, and non-irritating properties across all embodiments, which cannot be simultaneously met by single grafting or non-chemical grafting methods. In summary, the oral anti-inflammatory and ulcer-aiding mouthwashes prepared in Examples 1-3 of this invention are significantly superior to the comparative examples in terms of in vitro antibacterial activity, anti-biofilm activity, removal of mature biofilms, promotion of ulcer healing in animals, inhibition of tartar, and mucosal safety. Their overall performance is close to or partially exceeds that of commercially available chlorhexidine mouthwashes, while avoiding the mucosal irritation and tooth discoloration side effects of chlorhexidine. They have good prospects for clinical application and industrialization.

[0110] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0111] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A mouthwash for oral inflammation and ulcer repair, characterized in that: The product comprises the following components by weight percentage: glycerol 3.0-5.0%, cetylpyridinium chloride 0.03-0.10%, sodium fluoride 0.02-0.05%, zinc lactate 0.18-0.21%, double-grafted polymaleic anhydride 0.05-0.40%, polyquaternium-10 0.05-0.10%, fragrance 0.05-0.20%, preservative 0.05-0.25%, and the balance being purified water; the double-grafted polymaleic anhydride is prepared by the following steps: S1. Add farnesol to anhydrous ethanol and stir to dissolve at room temperature. Then add CDI and react at 35-45℃ for 2-4 hours under nitrogen protection. After the reaction is completed, remove the solvent under reduced pressure to obtain activated farnesol for later use. S2. Dissolve maleic anhydride in deionized water, adjust the system temperature to 75-95℃, add hydrogen peroxide and sodium hypophosphite, and react for 4-8 hours to obtain a polymaleic anhydride solution; then adjust the pH of the system to 6.0-7.5 with sodium hydroxide solution to cause some anhydride groups to undergo ring-opening hydrolysis to obtain a hydrolyzed polymaleic anhydride solution. S3. Add activated farnesol dropwise to the hydrolyzed polymaleic anhydride solution, followed by the addition of DMAP and EDC·HCl; react at 50-70℃ for 6-12 h. After the reaction is complete, precipitate with ethanol, centrifuge, and wash alternately with ethanol and deionized water to obtain the farnesol-grafted polymaleic anhydride intermediate. S4. Quaternized chitosan oligosaccharide was dissolved in an aqueous acetic acid solution to obtain a quaternized chitosan oligosaccharide solution. Then, farnesol-grafted polymaleic anhydride was dispersed in deionized water, NHS and EDC·HCl were added, and then the quaternized chitosan oligosaccharide solution was added dropwise. The reaction was carried out at 40-60℃ for 8-24 h. After the reaction was completed, the system was transferred to a dialysis bag and dialyzed in deionized water for 24-72 h. Finally, the system was freeze-dried to obtain double-grafted polymaleic anhydride.

2. The mouthwash for oral anti-inflammatory and ulcer-aiding repair according to claim 1, characterized in that: In step S1, the feeding ratio of farnesol, CDI, and anhydrous ethanol is 3.0-4.0g: 2.5-3.5g: 40-60mL.

3. The mouthwash for oral anti-inflammatory and ulcer-aiding repair according to claim 2, characterized in that: In step S2, the feeding ratio of maleic anhydride, hydrogen peroxide, sodium hypophosphite, and deionized water is 10.0-15.0g: 1.0-2.5g: 4.0-6.0g: 80-120mL.

4. The mouthwash for oral anti-inflammatory and ulcer-aiding repair according to claim 3, characterized in that: In step S3, the feeding ratio of activated farnesol, DMAP, EDC·HCl and polymaleic anhydride solution is 3.0-4.0g: 0.3-0.8g: 2.5-4.0g: 80-120mL.

5. The mouthwash for oral anti-inflammatory and ulcer-aiding repair according to claim 4, characterized in that: In step S4, the feeding ratio of quaternized chitosan oligosaccharide, aqueous acetic acid solution, farnesol-grafted polymaleic anhydride, deionized water, NHS and EDC·HCl is 1.5-3.0g: 30-50mL: 2.0-5.0g: 50-80mL: 0.8-1.5g: 1.5-3.0g.

6. The mouthwash for oral anti-inflammatory and ulcer-aiding repair according to claim 5, characterized in that: The quaternized chitosan oligosaccharide is prepared by the following steps: 10-20 g of chitosan oligosaccharide is weighed and added to 200-300 mL of deionized water. The mixture is magnetically stirred at room temperature for 2 h. A 20% sodium hydroxide solution is added to the system to adjust the pH to 10-11. The reaction system is heated to 50-60°C. Under nitrogen protection, 30-50 mL of iodomethane is slowly added dropwise to the reaction solution, along with 100 mL of isopropanol. The reaction temperature is maintained at 55°C, and the mixture is stirred continuously for 12-24 h. After the reaction is completed, the mixture is cooled to room temperature, concentrated by rotary evaporation under reduced pressure, and then slowly poured into 5 times the volume of anhydrous ethanol to precipitate the mixture. After standing for 2 h, the precipitate is centrifuged. The precipitate is washed three times with anhydrous ethanol and twice with acetone. After drying, the quaternized chitosan oligosaccharide is obtained.

7. The mouthwash for oral anti-inflammatory and ulcer-aiding repair according to claim 6, characterized in that: The mouthwash also includes a pH buffer solution to adjust the pH of the mouthwash to 6.4-6.8; the pH buffer solution is made by dissolving sodium dihydrogen phosphate and disodium hydrogen phosphate in pure water.

8. The mouthwash for oral anti-inflammatory and ulcer-aiding repair according to claim 7, characterized in that: The preservative is one of potassium sorbate and sodium benzoate.

9. A method for preparing a mouthwash for oral anti-inflammatory and ulcer-aiding repair as described in any one of claims 1-8, characterized in that: Includes the following steps: Glycerin, sodium fluoride, zinc lactate, double-grafted polymaleic anhydride, and polyquaternium-10 were added to a portion of pure water according to the specified ratio and stirred until dissolved to obtain a mixture. Cetylpyridinium chloride, fragrance, and preservative were dissolved in the remaining pure water and slowly added to the mixture. Finally, pH buffer was added to adjust the pH to 6.4-6.8, and the mixture was stirred until dissolved to obtain the mouthwash.

10. The use of the oral anti-inflammatory and ulcer-aiding mouthwash as described in any one of claims 1-8 in the preparation of oral care products for treating or aiding in the improvement of oral inflammation and oral ulcers.

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