Dihydrochalcone-based oral spray and its preparation method

Abstract

This invention discloses a dihydrochalcone-based oral spray and its preparation method, belonging to the field of oral pharmaceutical formulations. The spray uses 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, isolated and purified from *Sarcandra glabra*, as active ingredients. It is solubilized using a medium-chain triglyceride / polyoxyethylene hydrogenated castor oil / propylene glycol self-microemulsification delivery system, and hydroxypropyl methylcellulose is introduced to enhance mucosal adhesion. This spray can spontaneously form nanoemulsion droplets in oral saliva, selectively inhibiting *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, and is suitable for adjunctive treatment of gingivitis and periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of oral pharmaceutical formulation technology, specifically relating to an oral spray with dihydrochalcone monomer compounds derived from *Sarcandra glabra* as the active ingredient and its preparation method. The spray achieves solubilization of the active ingredient and efficient delivery to the oral mucosa through a self-microemulsification delivery system, and can be used as an adjunct treatment for gingivitis and periodontitis. Background Technology

[0002] Gingivitis and periodontitis are the most common infectious diseases in dentistry, with a global prevalence exceeding 50% in adults, severely impacting patients' quality of life and overall health. *Porphyromonas gingivalis* (… Porphyromonas gingivalis ) and Fusobacterium nucleatum ( Fusobacterium nucleatum Porphyromonas gingivalis is a recognized key pathogen of periodontal disease. Among them, Porphyromonas gingivalis is considered a key pathogen of periodontitis, capable of destroying periodontal tissues by secreting virulence factors such as gingival protease and lipopolysaccharide. Fusobacterium nucleatum acts as a bridging bacterium, mediating the co-aggregation between early colonizing bacteria and late pathogenic bacteria, promoting the maturation of subgingival plaque biofilm. Currently, clinical oral medications for treating gingivitis and periodontitis mainly include chlorhexidine mouthwash, metronidazole gel, and cetylpyridinium chloride spray. Although these chemical drugs have strong broad-spectrum antibacterial activity, long-term use can lead to problems such as tooth discoloration, altered taste, dysbiosis, and increased drug resistance, limiting their continued application in oral health care.

[0003] *Sarcandra glabra* (Thunb.) Nakai is a perennial evergreen subshrub belonging to the genus *Sarcandra* in the family Chloranthaceae. It is abundant in Guizhou, Jiangxi, and Fujian provinces of my country. *Sarcandra glabra* has a long history of medicinal use, possessing properties such as clearing heat and detoxifying, dispelling wind and promoting blood circulation, reducing swelling and relieving pain. Clinically, it is widely used in the treatment of oral diseases, as seen in compound *Sarcandra glabra* lozenges and other pharmaceutical products. However, existing *Sarcandra glabra* oral preparations mostly use crude extracts, resulting in unclear active ingredients, difficulties in quality control, and low concentrations of effective components, making it difficult to achieve precise inhibition of specific oral pathogens.

[0004] Chinese invention CN1596924A discloses a traditional Chinese and Western medicine compound for treating oral and pharyngeal diseases, which uses crude extract of *Sarcandra glabra* combined with chemical antibacterial agents such as diquinalium chloride to prepare an oral spray. However, this solution essentially relies on chemical antibacterial agents to exert its antibacterial effect, with the crude extract of *Sarcandra glabra* only playing an auxiliary role in clearing heat. The specific active ingredients are not clearly identified, and there is a risk of adverse reactions caused by chemical drugs. Chinese invention CN116492369B discloses an oral care composition containing *Sarcandra glabra* polysaccharides. Its active ingredient is a polysaccharide, which belongs to a completely different category of chemical substances from the small-molecule dihydrochalcone compounds involved in this invention, and its antibacterial mechanism is also completely different.

[0005] Dihydrochalcones are a class of natural flavonoids with a 1,3-diphenyl-1-propanone skeleton. Recent studies have revealed that various dihydrochalcones possess significant antibacterial, anti-inflammatory, and antioxidant activities. However, dihydrochalcones generally suffer from poor water solubility and limited oral mucosal permeability, making direct preparation of sprays in aqueous solution a technical bottleneck due to insufficient solubility and difficulty in ensuring effective concentration. Therefore, developing a spray formulation that uses dihydrochalcone monomers derived from *Sarcandra glabra* as the active ingredient, while ensuring safety and possessing both good solubilizing effects and oral mucosal retention, is a pressing technical challenge. Summary of the Invention

[0006] To address the technical problems in existing oral preparations such as unclear active ingredients, adverse reactions from long-term use of chemical antibacterial agents, and difficulties in developing oral preparations due to the poor water solubility of dihydrochalcone compounds, the present invention aims to provide an oral spray containing dihydrochalcone compounds and its preparation method. The spray uses 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, isolated and purified from *Sargassum fusiforme*, as its core active ingredients. A self-microemulsifying delivery system is employed to achieve rapid solubilization and release of the active ingredients in the oral environment. Furthermore, a mucosal adhesive polymer is introduced to prolong the drug's retention time on the oral mucosa, thereby achieving highly effective inhibition of *Porphyromonas gingivalis* and *Fusobacterium nucleatum*.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A dihydrochalcone oral spray comprising the following components in weight percentages: 0.1%-0.5% 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, 0.05%-0.3% 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, 1%-3% medium-chain triglycerides, 2%-5% polyoxyethylene hydrogenated castor oil, 10%-20% propylene glycol, 0.1%-0.5% hydroxypropyl methylcellulose, 0.2%-0.8% citrate-sodium citrate buffer, 0.05%-0.2% menthol, 0.02%-0.1% steviol glycosides, 0.1%-0.15% methylparaben, and purified water to 100%, wherein the pH of the spray is 5.5-6.5.

[0009] The molecular formula of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone is C 16 H 16O4, with a molecular weight of 272.30. The molecular formula of the 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone is C. 17 H 18 O5, with a molecular weight of 302.32. Both compounds mentioned above are derived from the ethanol extract of the whole plant of Sarcandra glabra.

[0010] Preferably, the spray further contains 0.02%-0.1% of 2',6'-dihydroxy-4'-methoxydihydrochalcone and / or 0.02%-0.1% of 2',4'-dihydroxy-4,6'-dimethoxydihydrochalcone as an auxiliary antibacterial active ingredient.

[0011] Preferably, the medium-chain triglyceride has a carbon chain length of C8-C9. 10 The HLB value of the polyoxyethylene hydrogenated castor oil is 12-16.

[0012] This invention also provides a method for preparing the above-mentioned dihydrochalcone compound oral spray, comprising the following steps: Step 1, Preparation of active ingredients: Take dried and pulverized whole herb of *Sargassum fusiforme*, add 8 times the amount of 90% ethanol and reflux for 45 min, filter and collect the filtrate, recover the ethanol to obtain the alcohol extract; disperse the alcohol extract with an equal amount of purified water, and extract successively with petroleum ether, dichloromethane, ethyl acetate and n-butanol, collect the dichloromethane extract; load the dichloromethane extract onto a D101 macroporous resin column, elute successively with 0%, 30% and 90% ethanol, collect the 90% ethanol eluent; load the 90% ethanol eluent onto a silica gel column, elute with a petroleum ether-ethyl acetate gradient, collect the eluent fraction with a petroleum ether-ethyl acetate volume ratio of 10:1; further load this fraction onto a silica gel column, elute with a petroleum ether-ethyl acetate volume ratio of 8:1 to 6:1 and crystallize to purify, respectively, to obtain 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone monomers.

[0013] Step 2, Preparation of self-microemulsion concentrate: Dissolve 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone in medium-chain triglycerides and stir at 40-50 °C until completely dissolved; add polyoxyethylene hydrogenated castor oil and continue stirring until uniform and transparent to form a self-microemulsion concentrate.

[0014] Step 3, Preparation of oral spray: Add propylene glycol to the microemulsion concentrate and mix thoroughly; separately, take purified water and dissolve hydroxypropyl methylcellulose, citric acid, sodium citrate, steviol glycosides, and methylparaben. After complete dissolution, slowly add the propylene glycol-microemulsion mixture to the aqueous phase and stir continuously to form a homogeneous transparent or semi-transparent solution; add an ethanol solution of menthol, add purified water to the total volume, adjust the pH to 5.5-6.5, filter through a 0.22 μm microporous membrane, fill into a metered spray pump bottle, and seal with the valve to obtain the oral spray.

[0015] The beneficial effects of this invention are as follows: First, this invention is the first to apply the monomeric compounds 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone isolated and purified from *Sarcandra glabra* to oral spray formulations. Compared with the use of crude extracts of *Sarcandra glabra* in existing technologies, the active ingredients are clearly defined, the quality is controllable, and the batch-to-batch reproducibility is good. Second, the self-microemulsification delivery system allows dihydrochalcone compounds with extremely poor water solubility to spontaneously form nanoscale microemulsion droplets with a particle size of 20-80 nm in the oral saliva environment, significantly improving the solubility of the active ingredients and the permeability to the oral mucosa. Third, hydroxypropyl methylcellulose endows the spray with good mucosal adhesion properties, extending the retention time of the active ingredients on the oral mucosa surface to more than 30 minutes, thus continuously exerting its antibacterial effect. Fourth, the combination of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone exhibits a synergistic antibacterial effect against *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, with a combined inhibition index (FICI) of 0.375-0.5, falling within the synergistic range. Simultaneously, it shows no significant inhibitory activity against *Streptococcus mutans* in the normal oral flora, indicating that this spray has good antibacterial selectivity. Fifth, all excipients used in this invention are commonly used in oral preparations, ensuring high safety and a simple preparation process suitable for industrial production. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the technical solutions of the present invention and do not limit the scope of protection of the present invention.

[0017] I. Source and structural identification of active ingredients.

[0018] The dihydrochalcone active compounds used in this invention are all derived from the whole plant of Sarcandra glabra (Thunb.) Nakai. Sarcandra glabra was collected from Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou Province, and identified as a plant belonging to the Sarcandra genus of the Chloranthus family. After collection, it was naturally air-dried and then pulverized for later use.

[0019] The extraction and separation process of the active ingredients is as follows: Weigh the dried and pulverized *Sarcandra glabra*, add 8 times its mass of 90% ethanol (volume fraction), and reflux extract in an 80 °C water bath for 45 min. Filter and collect the filtrate. Repeat the extraction once more with the residue. Combine the two filtrates, and recover the ethanol and water under reduced pressure to obtain the *Sarcandra glabra* ethanol extract. Add an equal mass of purified water to the ethanol extract, disperse evenly by ultrasonication, and then perform liquid-liquid extraction sequentially with 3 times, 2 times, and 1 volume of petroleum ether (boiling range 60-90 °C). Combine the petroleum ether layers and recover the solvent to obtain the petroleum ether extract. The aqueous layer after petroleum ether extraction is further extracted sequentially with dichloromethane, ethyl acetate, and saturated water with n-butanol, and collect each extract separately.

[0020] Using *Porphyromonas gingivalis* (ATCC 33277) as the indicator strain, the antibacterial activity of each extract was evaluated using the microdilution method. The results showed that the dichloromethane extract exhibited the strongest inhibitory activity against *Porphyromonas gingivalis*. The dichloromethane extract was loaded onto a D101 macroporous adsorption resin column (column diameter to height ratio 1:8), and eluted sequentially with 0% ethanol (i.e., purified water), 30% ethanol, 60% ethanol, and 90% ethanol. The eluents were collected, and the solvents were recovered under reduced pressure to obtain eluents of different polarities. The MIC (minimum inhibitory concentration) of each eluent was determined. The results showed that the 60% ethanol eluent (MIC = 12.5 ug / mL, MIC50 = 3.87 ug / mL) and the 90% ethanol eluent (MIC = 100 ug / mL, MIC50 = 15.05 ug / mL) had significant inhibitory effects on *Porphyromonas gingivalis*.

[0021] The 60% and 90% ethanol eluates were combined and further separated. They were then loaded onto a normal-phase silica gel column (200-300 mesh) and eluted with a petroleum ether-ethyl acetate system in gradient ratios of 20:1, 15:1, 10:1, 5:1, and 0:1 (volume ratio). The MICs of each eluent were determined, showing that the petroleum ether-ethyl acetate (10:1) eluent exhibited the strongest antibacterial activity, with an MIC of 0.5 ug / mL and an MIC50 of 0.37 ug / mL against *Porphyromonas gingivalis*. This most active eluent was further loaded onto a silica gel column and eluted with fine gradients of petroleum ether-ethyl acetate at (8:1), (7:1), and (6:1) ratios. Through repeated crystallization purification, five monomeric compounds were obtained.

[0022] Nuclear magnetic resonance spectroscopy (NMR) technique 1 H-NMR and 13Five compounds were identified by C-NMR (using a Bruker AVANCE III 500MHz instrument). Compound 1 was identified as 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, a colorless needle-like crystal with the molecular formula C. 17 H 18 O5, its 1 The H-NMR characteristic signals include one active hydrogen signal (δH 14.14, s), five hydrogen signals from the 1-substituted benzene ring (7.32–7.21, 5H, m, overlap), and two methoxy signals (δH 14.14, s). H 3.90, 3H, s; δ H 3.82, 3H, s), and a pair of mutually coupled methylene signals (δ H 3.32, 2H, m; δ H 3.00, 2H, m). Compound 2 was identified as 2',6'-dihydroxy-4'-methoxydihydrochalcone, a white powder. Compound 3 was identified as 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, a colorless needle-like crystal with the molecular formula C. 16 H 16 O4, its characteristic signal includes one active hydrogen signal ( δ H 13.95, s), 5 hydrogen signals of 1-substituted benzene ring (7.32-7.19, 5H, m, overlap), 1 hydrogen signal of meta-coupled benzene ring [( δ H 6.00, 1H, d, J =2.0 Hz), δ H 5.91, 1H, d J =2.0 Hz), 1 methoxy signal ( δ H 3.84, 3H, s), and a pair of mutually coupled methylene signals ( δ H 3.32, 2H, m; δ H 2.99, 2H, m). Compound 4 was identified as 2',4'-dihydroxy-6',4-dimethoxydihydrochalcone, a colorless needle-like crystal. Compound 5 was identified as linoleic acid, a colorless flaky crystal.

[0023] All five compounds were isolated from *Sarcandra glabra* for the first time. The structures of four dihydrochalcone compounds (compounds 1 to 4) were determined by comparing them one by one with NMR data from published literature. Compound 5 (linoleic acid) is a common octadecadienoic acid fatty acid. The purity of each compound was confirmed to be greater than 95% by silica gel thin-layer chromatography and high-performance liquid chromatography, meeting the purity requirements for pharmacological activity evaluation and formulation preparation.

[0024] II. Evaluation of the antibacterial activity of monomeric compounds against oral pathogens.

[0025] The effects of five monomeric compounds on Porphyromonas gingivalis (Porphyromonas) were determined. Porphyromonas gingivalis ATCC 33277 ), Fusobacterium nucleatum ( Fusobacterium nucleatum ATCC 25586 ) and Streptococcus mutans ( Streptococcus mutans ATCC 25175 The minimum inhibitory concentration (MIC) and median inhibitory concentration (MIC50) of the bacteria.

[0026] Culture conditions: *Porphyromonas gingivalis* and *Fusobacterium nucleatum* were cultured on BHI+ liquid medium in an anaerobic environment of 80% N2, 10% CO2, and 10% H2 at a constant temperature of 37 °C for 48 h. *Streptococcus mutans* was cultured on BHI+ liquid medium in an aerobic environment of 37 °C for 48 h. All strains underwent resuscitation and subculturing activation treatment before use to ensure they were in the logarithmic growth phase.

[0027] MIC determination method: Each compound was dissolved in DMSO to prepare a 10 mg / mL stock solution, which was then serially diluted twofold with BHI+ medium to 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μmol / L. The activated bacterial solution was adjusted to a concentration of 1 x 10^7 CFU / mL. 100 μL of drug diluent and 100 μL of bacterial solution were added to each well of a 96-well plate. A blank control group, a solvent control group (1% DMSO), and a positive control group (metronidazole) were established. After incubating the 96-well plate at 37 °C for 48 h, the turbidity of each well was observed against a dark background. Wells with clear, bright, and turbid content were considered to indicate sterile growth, and the corresponding concentration was the MIC. Simultaneously, the absorbance at 600 nm was measured using an ELISA reader. The inhibition rate was calculated and the MIC50 value was fitted using the following formula: Inhibition rate (%) = [(A(solvent) - A(blank)) - (A(sample) - A(sample background))] / (A(solvent) - A(blank)) x 100%. Three parallel wells were set for each concentration, and the experiment was independently repeated three times.

[0028] The experimental results showed that the five compounds had varying degrees of inhibitory activity against Porphyromonas gingivalis and Fusobacterium nucleatum, but none showed inhibitory activity against Streptococcus mutans within the tested concentration range (up to 128 μmol / L). The inhibitory activity against *Porphyromonas gingivalis*, from strongest to weakest, was as follows: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone (compound 3, MIC = 16 μmol / L, MIC50 = 10.28 μmol / L) > Compound 4 (MIC = 32 μmol / L, MIC50 = 18.72 μmol / L) ≈ Compound 2 (MIC = 32 μmol / L, MIC50 = 16.10 μmol / L) ≈ Linoleic acid (compound 5, MIC = 32 μmol / L, MIC50 = 18.56 μmol / L) > 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone (compound 1, MIC = 64 μmol / L, MIC50 = 29.73 μmol / L). The inhibitory activity against *Fusobacterium nucleatum*, from strongest to weakest, was as follows: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone (compound 3, MIC = 16 μmol / L, MIC50 = 6.86 μmol / L) ≈ linoleic acid (compound 5, MIC = 16 μmol / L, MIC50 = 3.27 μmol / L) > compound 4 (MIC = 32 μmol / L, MIC50 = 14.35 μmol / L) > compound 2 (MIC = 64 μmol / L, MIC50 = 28.69 μmol / L) > 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone (compound 1, MIC = 128 μmol / L, MIC50 = 56.71 μmol / L). The MIC of the positive control metronidazole against Porphyromonas gingivalis and Fusobacterium nucleatum was 0.16 μmol / L.

[0029] Notably, all five compounds showed no inhibitory activity against *Streptococcus mutans* (MIC > 128 μmol / L). This selective antibacterial characteristic is significant for the development of oral formulations. *Streptococcus mutans* is one of the most prevalent *Streptococcus* species in the natural oral flora. This bacterium produces acidic substances by metabolizing sugars, which can lead to enamel demineralization and caries formation, making it one of the most common cariogenic bacteria. The active compounds of this invention selectively inhibit periodontal pathogens, but showed no effect against cariogenic bacteria.

[0030] To further verify the reliability of the above antibacterial effect, time-kill kinetics experiments were conducted on the most active compound, 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone. *Porphyromonas gingivalis* was inoculated into BHI+ medium containing 1x MIC (16 μmol / L), 2x MIC (32 μmol / L), and 4x MIC (64 μmol / L) of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, and cultured anaerobically at 37 °C. Viable cell counts (CFU / mL) were performed at 0, 2, 4, 8, 12, 24, and 48 h. The results showed that 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone at a concentration of 1x MIC reduced the viable count of *Porphyromonas gingivalis* by approximately two log10 orders of magnitude within 24 hours, while a concentration of 2x MIC reduced it by more than three log orders of magnitude within 12 hours. A concentration of 4x MIC reduced the viable count to below the detection limit (< 10 CFU / mL) within 8 hours. In contrast, metronidazole at the same concentration reduced the viable count below the detection limit in just 4 hours under 2x MIC conditions. This indicates that although 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone has lower absolute antibacterial activity than metronidazole, it still exhibits a clear concentration-dependent bactericidal effect and is suitable as an active ingredient for topical oral medications.

[0031] In addition, the minimum bactericidal concentration (MBC) of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone was determined. 100 μL of culture from each well in the MIC assay was transferred to drug-free BHI+ agar plates and anaerobically incubated at 37°C for 72 h before colony counting. The results showed that the MBC of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone against *Porphyromonas gingivalis* was 32 μmol / L (2x MIC), with an MBC / MIC ratio of 2. This indicates that 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone exhibits bactericidal rather than merely bacteriostatic activity against *Porphyromonas gingivalis*, a characteristic that is more advantageous for the clinical application of oral sprays.

[0032] III. Evaluation of the synergistic antibacterial effect of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone.

[0033] Based on the antibacterial activity results of the monomeric compounds, the combined antibacterial effect of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone (compound 3), which exhibits the strongest antibacterial activity, and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone (compound 1) was evaluated. The combined inhibitory index (FICI) against *Porphyromonas gingivalis* when the two compounds were used in combination was determined using the checkerboard dilution method. Concentration gradients of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone were set at 1, 2, 4, 8, and 16 μmol / L, and concentration gradients of 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone were set at 4, 8, 16, 32, and 64 μmol / L, arranged in a checkerboard pattern, and the MIC values ​​of each combination were determined. The FICI calculation formula is: FICI = (MIC(A combined) / MIC(A alone)) + (MIC(B combined) / MIC(B alone)), where FICI <= 0.5 indicates a synergistic effect, 0.5 < FICI <= 1 indicates an additive effect, 1 < FICI <= 2 indicates an unrelated effect, and FICI > 2 indicates an antagonistic effect.

[0034] Experimental results showed that when the concentrations of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone were 4 μmol / L and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone were 16 μmol / L, the combined administration completely inhibited the growth of *Porphyromonas gingivalis*, with a FICI of 4 / 16 + 16 / 64 = 0.25 + 0.25 = 0.5, indicating a synergistic effect. When the concentrations of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone were 8 μmol / L and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone were 8 μmol / L, the FICI of 8 / 16 + 8 / 64 = 0.5 + 0.125 = 0.625, indicating an additive effect. Based on multiple sets of data, the FICI range of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone against *Porphyromonas gingivalis* was 0.375-0.625, with most combinations exhibiting synergistic to additive effects, confirming a good combined antibacterial effect of the two compounds. This synergistic effect may be related to the different molecular targets through which the two compounds exert their antibacterial activity: the 2',6'-dihydroxy substitution mode of ring A in 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone facilitates interaction with the hydrophobic core of the bacterial cell membrane lipid bilayer, disrupting membrane integrity; while the 4',5'-dimethoxy substitution of ring B in 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone may exert its antibacterial effect by inhibiting the activity of bacterial metabolic enzymes.

[0035] To verify the hypothesized synergistic mechanism, the effect of combined drug administration on the cell membrane permeability of *Porphyromonas gingivalis* was evaluated using propidium iodide (PI) fluorescence staining. *Porphyromonas gingivalis* was exposed to media containing 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone alone (16 μmol / L), 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone alone (64 μmol / L), and a combination of both (4 μmol / L + 16 μmol / L) for 2 h. After treatment, the cells were stained with PI (final concentration 10 μg / mL) for 15 min and observed under a fluorescence microscope. The proportion of PI-positive bacteria in the combined treatment group (78.3 + / - 6.2%) was significantly higher than that in the 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone monotherapy group (45.6 + / - 5.8%) and the 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone monotherapy group (21.2 + / - 4.3%), confirming that the combined use of the two compounds can significantly enhance the disruptive effect on bacterial cell membranes. This result supports the hypothesis that the two compounds exert a synergistic effect through different targets.

[0036] The FICI (Fluid-Induced Cognition) of the combined drug therapy against *Fusobacterium nucleatum* was further determined. The results showed that when 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone (8 μmol / L) was used in combination with 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone (32 μmol / L), the FICI = 8 / 16 + 32 / 128 = 0.5 + 0.25 = 0.75, indicating an additive effect. Overall, the combined effect of the two compounds was more pronounced against *Porphyromonas gingivalis* (FICI reached a synergistic level), while an additive effect was observed against *Fusobacterium nucleatum*, both superior to either compound used alone. This provides sufficient scientific evidence for the formulation containing two active ingredients simultaneously.

[0037] IV. Formulation design and optimization of self-microemulsifying oral sprays.

[0038] 1-(2,4-Dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone are both lipid-soluble compounds, practically insoluble in purified water (solubility < 0.01 mg / mL), with a solubility of approximately 2-5 mg / mL in propylene glycol and 15-30 mg / mL in medium-chain triglycerides (MCTs). Traditional surfactant solubilization strategies are insufficient to meet the clarity and stability requirements of oral sprays. Therefore, this invention employs a self-microemulsifying drug delivery system (SMEDDS) to pre-dissolve the active ingredient in the oil phase. A high-HLB nonionic surfactant spontaneously forms nanoscale microemulsion droplets (particle size 20-80 nm) under oral saliva dilution conditions, thereby achieving rapid solubilization and release of the active ingredient.

[0039] The selection criteria for each component in the formulation are as follows: Medium-chain triglycerides (MCT, C8-C10) were chosen for the oil phase due to their superior solubility for dihydrochalcone compounds and their status as a commonly used excipient in oral preparations, demonstrating good safety. Polyoxyethylene hydrogenated castor oil (HLB value 12-16) was selected as the surfactant due to its strong emulsifying ability, widely verified oral safety, and lack of noticeable irritating taste. Propylene glycol was chosen as the co-surfactant, acting both as a solubilizer to increase the solubility of the active ingredient in the mixture and as a co-surfactant to reduce the oil-water interfacial tension, promoting self-microemulsification. Hydroxypropyl methylcellulose (HPMC) was chosen as the mucosal adhesion polymer, which forms a gel-like film on the oral mucosa, prolonging the contact time between the active ingredient and the oral mucosa. The buffer system uses a citrate-sodium citrate buffer pair to control the pH of the spray within the range of 5.5-6.5. This pH range is beneficial for maintaining the chemical stability of dihydrochalcone compounds and is close to the physiological pH environment of the oral cavity (6.2-7.0), so it will not irritate the oral mucosa.

[0040] The optimal formulation ratio was determined using a pseudo-ternary phase diagram method. Using MCT as the oil phase, polyoxyethylene hydrogenated castor oil as the surfactant, and propylene glycol as the co-surfactant, pseudo-ternary phase diagrams were constructed with fixed surfactant-to-co-surfactant mass ratios (Km) of 1:3, 1:4, and 1:5. Under these fixed Km values, mixtures were prepared with different oil phase proportions (10%-90%), purified water was added dropwise, and the changes in the system's appearance were observed. The composition range of the spontaneously formed transparent or translucent microemulsion regions was recorded. The results showed that the microemulsion region area was largest when Km = 1:4, indicating the highest self-microemulsification efficiency. Under the condition of Km = 1:4, when the oil phase accounted for 5%-15% of the mixture (oil phase + surfactant + co-surfactant), a clear and transparent nanoemulsion spontaneously formed after 100-fold dilution with purified water.

[0041] The droplet size of the microemulsion formed after diluting the optimized formulation 100-fold with purified water was determined using dynamic light scattering (DLS, Malvern Zetasizer Nano ZS). The results showed an average droplet size of 38.6 ± 4.2 nm (n = 3) and a polydispersity index (PDI) of 0.186 ± 0.023, indicating a uniform droplet size distribution within the nanometer range. Transmission electron microscopy (TEM, JEM-2100) revealed that the droplets were regularly spherical, uniform in size, and exhibited no aggregation.

[0042] To verify the dilution stability of the self-microemulsifying system, the optimized formulation was diluted 50-fold, 100-fold, and 200-fold with purified water, pH 6.8 artificial saliva, and pH 1.2 hydrochloric acid solution, respectively. The solutions were then allowed to stand at room temperature for 24 hours to observe changes in appearance and measure particle size. The results showed that the microemulsions diluted with purified water and artificial saliva remained clear and transparent within 24 hours, without precipitation or stratification, and the particle size change was within 10%. The microemulsion diluted with pH 1.2 hydrochloric acid solution remained stable for 4 hours, but slight turbidity appeared after 24 hours, with a particle size increase of approximately 30%. This was attributed to a conformational change in the polyoxyethylene segments of the polyoxyethylene hydrogenated castor oil under strong acid conditions. Considering that oral sprays are used in an oral environment (pH 6.2-7.0), these results confirm that the self-microemulsifying system of this invention exhibits excellent dilution stability under oral use conditions.

[0043] Solubility Enhancement Evaluation: The apparent solubility of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone in different media was measured. The results are as follows: 0.008 + / - 0.002 mg / mL in purified water; 0.42 + / - 0.05 mg / mL in an aqueous solution containing 1% polysorbate 80; and 3.85 + / - 0.31 mg / mL in a self-microemulsifying system (formulation of this invention) containing 2% MCT + 3.5% polyoxyethylene hydrogenated castor oil + 14% propylene glycol. The self-microemulsifying system of this invention increases the apparent solubility of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone by approximately 481 times compared to pure water, and by approximately 9.2 times compared to the traditional polysorbate 80 solubilization scheme, fully demonstrating the significant advantages of the self-microemulsifying delivery system in improving the water solubility of dihydrochalcone compounds.

[0044] V. Examples of Spray Formulations

[0045] Example 1: Basic prescription oral spray. The components were weighed according to the following percentages by weight: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone 0.3%, 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone 0.15%, medium-chain triglycerides (MCT) 2%, polyoxyethylene hydrogenated castor oil 3.5%, propylene glycol 14%, hydroxypropyl methylcellulose (HPMCK4M) 0.3%, citric acid 0.15%, sodium citrate 0.35%, menthol 0.1%, steviol glycosides 0.05%, methylparaben 0.12%, and purified water to 100%. The preparation method is as follows: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone were added to MCT and stirred in a 45 °C water bath until completely dissolved. Polyoxyethylene hydrogenated castor oil was added, and stirring continued for 15 min until homogeneous and transparent, forming a self-microemulsifying concentrate. Separately, an appropriate amount of purified water was added to HPMC, and the mixture was stirred until swollen and completely dissolved for 2 h. Citric acid, sodium citrate, steviol glycosides, and methylparaben were added sequentially, and stirred until completely dissolved. Propylene glycol was added to the self-microemulsifying concentrate and mixed well. This mixture was then slowly added to the aqueous phase, and stirring was continued for 30 min under magnetic stirring at 300 rpm. Menthol was dissolved in a small amount of ethanol (approximately 0.5 mL) and added dropwise to the mixture. Purified water was added to the total volume. The pH was adjusted to 6.0 with 0.1 mol / L NaOH or HCl. After being filtered and sterilized through a 0.22 μm microporous membrane (PVDF material), the solution is filled into a 20 mL metering spray pump bottle (approximately 0.1 mL per puff), sealed with a pressure valve, and the product is ready. The finished product is a pale yellow to slightly yellow clear to slightly milky liquid with a refreshing minty aroma.

[0046] Example 2: High-concentration prescription oral spray. The following components were weighed according to their mass percentages: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone 0.5%, 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone 0.3%, 2',6'-dihydroxy-4'-methoxydihydrochalcone (compound 2) 0.1%, medium-chain triglycerides (MCT) 3%, polyoxyethylene hydrogenated castor oil 5%, propylene glycol 20%, hydroxypropyl methylcellulose (HPMC K4M) 0.5%, citric acid 0.2%, sodium citrate 0.4%, menthol 0.15%, steviol glycosides 0.08%, methylparaben 0.15%, and purified water to 100%. The preparation method was the same as in Example 1, with the final pH adjusted to 5.8. The finished product was a pale yellow, slightly opalescent liquid.

[0047] Example 3: Low-concentration prescription oral spray. The following components were weighed according to their mass percentages: 0.1% 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, 0.05% 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, 1% medium-chain triglycerides (MCT), 2% polyoxyethylene hydrogenated castor oil, 10% propylene glycol, 0.1% hydroxypropyl methylcellulose (HPMC K4M), 0.1% citric acid, 0.25% sodium citrate, 0.05% menthol, 0.02% steviol glycosides, 0.1% methylparaben, and purified water to 100%. The preparation method was the same as in Example 1, with the final pH adjusted to 6.5. The finished product was a nearly colorless to slightly yellow clear liquid.

[0048] Example 4: A four-component compound active oral spray. The following components were weighed according to their mass percentages: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone 0.2%, 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone 0.1%, 2',6'-dihydroxy-4'-methoxydihydrochalcone (compound 2) 0.05%, 2',4'-dihydroxy-6',4-dimethoxydihydrochalcone (compound 4) 0.05%, medium-chain triglycerides (MCT) 2%, polyoxyethylene hydrogenated castor oil 4%, propylene glycol 16%, hydroxypropyl methylcellulose (HPMC K4M) 0.3%, citric acid 0.15%, sodium citrate 0.35%, menthol 0.1%, steviol glycosides 0.05%, methylparaben 0.12%, and purified water added to 100%. The preparation method was the same as in Example 1, with the final pH adjusted to 6.0. The finished product is a pale yellow, slightly opalescent liquid.

[0049] Example 5: Controlled oral spray (without self-microemulsification system). The following components were weighed according to their mass percentages: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone 0.3%, 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone 0.15%, polysorbate 80 1%, propylene glycol 15%, hydroxypropyl methylcellulose (HPMC K4M) 0.3%, citric acid 0.15%, sodium citrate 0.35%, menthol 0.1%, steviol glycosides 0.05%, methylparaben 0.12%, and purified water to 100%. Preparation method: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone were dissolved in propylene glycol, and polysorbate 80 was added for solubilization and stirring. Then, the mixture was added to the aqueous phase containing the remaining components, stirred until homogeneous, and the pH was adjusted to 6.0. The solution was then filtered and filled into containers. This formulation does not contain a self-microemulsifying system and serves as a control to evaluate the synergistic effect of the self-microemulsifying delivery system.

[0050] VI. Evaluation of the physicochemical properties of the formulation.

[0051] The physicochemical properties of the sprays prepared in Examples 1-5 were systematically evaluated, including appearance, pH value, particle size, polydispersity index (PDI), content determination, and stability study.

[0052] Appearance and pH Measurement: The sprays in Examples 1 to 4 were all pale yellow to slightly yellow clear to slightly opalescent liquids, without precipitation or stratification, and could evenly cover the oral mucosa surface after spraying. The spray in Example 5 was also a pale yellow liquid, but its clarity was slightly worse than that of Examples 1-4. The pH measurement results for each example are as follows: the pH of Example 1 was 6.02 + / - 0.05, the pH of Example 2 was 5.81 + / - 0.03, the pH of Example 3 was 6.48 + / - 0.06, the pH of Example 4 was 5.98 + / - 0.04, and the pH of Example 5 was 6.05 + / - 0.05, all within the target range of 5.5-6.5.

[0053] Particle size and PDI determination: The sprays from each example were diluted 100 times with purified water, and the average particle size and PDI of the microemulsion droplets were determined using dynamic light scattering. The results showed that the average particle size of Example 1 was 38.6 + / - 4.2 nm, and the PDI was 0.186 + / - 0.023; the average particle size of Example 2 was 52.3 + / - 5.1 nm, and the PDI was 0.212 + / - 0.031; the average particle size of Example 3 was 25.8 + / - 2.9 nm, and the PDI was 0.154 + / - 0.018; and the average particle size of Example 4 was 42.1 + / - 3.8 nm, and the PDI was 0.195 + / - 0.025. Example 5 (control formulation) has a droplet size of 286.5 + / - 42.7 nm and a PDI of 0.478 + / - 0.068, which are significantly larger than those of Examples 1-4, since it does not contain a self-microemulsification system. This shows that the self-microemulsification delivery system can significantly reduce the droplet size to the nanoscale.

[0054] Content determination: The contents of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone in each example were determined by high performance liquid chromatography (HPLC). Chromatographic conditions: An Agilent ZORBAX Eclipse XDB-C18 column (250 mm x 4.6 mm, 5 μm) was used; the mobile phase was methanol-0.1% phosphoric acid aqueous solution (70:30, v / v); the flow rate was 1.0 mL / min; the detection wavelength was 290 nm; the column temperature was 30 °C; and the injection volume was 10 μL. Under the above conditions, the retention times of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone were 8.2 min and 10.5 min, respectively, with symmetrical peak shapes and a resolution greater than 1.5. The content was calculated using the external standard method. In each example, the ratio of the measured content of the active ingredient to the labeled amount (labeled percentage content) was within the range of 95%-105%, which meets the requirements of the pharmacopoeia.

[0055] Accelerated stability study: The spray from Example 1 was placed in a constant temperature and humidity chamber at 40 °C and 75% relative humidity for accelerated stability testing. Samples were taken at 0, 1, 2, 3, and 6 months to test appearance, pH value, content, and microbial limits. The results showed that during the 6-month accelerated test, the appearance of the spray did not change significantly, the pH value changed within 0.1, and the content decrease rates of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone were 3.2% and 4.1%, respectively, both within 5%. The microbial limit test was passed. These results indicate that the spray of the present invention has good physical and chemical stability.

[0056] Viscosity Measurement: The viscosity of the sprays in each example was measured at 25 °C using a rotational viscometer (Brookfield DV-II+Pro). The viscosity of Example 1 was 8.6 + / - 0.4 mPa·s, Example 2 was 12.3 + / - 0.7 mPa·s, Example 3 was 5.2 + / - 0.3 mPa·s, Example 4 was 9.1 + / - 0.5 mPa·s, and Example 5 was 7.8 + / - 0.4 mPa·s. The viscosity of each example was within the applicable range (1-50 mPa·s) for metered spray pump bottles, indicating good atomization and uniform droplet coverage. The viscosity changes of Example 1 at different temperatures (4 °C, 25 °C and 37 °C) were measured, and the results were 12.8 + / - 0.6 mPa.s, 8.6 + / - 0.4 mPa.s and 6.1 + / - 0.3 mPa.s, respectively. This indicates that the viscosity decreases moderately with increasing temperature, which is beneficial to the flow and uniform distribution of the spray in the oral temperature environment of 37 °C.

[0057] Long-term stability study: The spray from Example 1 was stored at 25 °C / 60% RH (long-term test) and 4 °C (refrigeration conditions), and samples were taken and tested at 0, 3, 6, 9, and 12 months. The results of the 25 °C long-term test showed no significant change in the appearance of the spray after 12 months. The pH decreased from the initial 6.02 to 5.91, the content of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone decreased by 2.8%, the content of 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone decreased by 3.5%, and the particle size increased from 38.6 nm to 43.2 nm, all within acceptable ranges. Under 4 °C refrigeration conditions, the changes in various indicators were even smaller after 12 months; the content of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone decreased by only 1.2%, and the particle size remained essentially unchanged. The above data supports the fact that the shelf life of the spray of the present invention is not less than 24 months under 25 °C conditions, and the storage period can be further extended under 4 °C refrigeration conditions.

[0058] VII. Evaluation of the in vitro antibacterial effect of the spray.

[0059] The in vitro antibacterial effects of the sprays from Examples 1 and 5 (control formulations) were evaluated to verify the synergistic effect of the self-microemulsification delivery system on antibacterial activity.

[0060] Experimental Methods: The microdilution method was used. The sprays from Examples 1 and 5 were serially diluted 8-1024 times using BHI+ medium. The final concentration of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone in the diluted mixture was expressed as the final concentration. *Porphyromonas gingivalis* and *Fusobacterium nucleatum* were used as test strains, and the MIC values ​​were determined according to the aforementioned method. Simultaneously, pure 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone (dissolved in DMSO) was used as a drug control.

[0061] Experimental results showed that the MIC (microemulsification concentration) of Example 1 (self-microemulsifying formulation) against *Porphyromonas gingivalis* (based on the concentration of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone) was 8 μmol / L, while the MIC of Example 5 (control formulation) was 32 μmol / L, and the MIC of pure 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone was 16 μmol / L. The MIC (microemulsifying formulation) of Example 1 (self-microemulsifying formulation) against *Fusobacterium nucleatum* (based on the concentration of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone) was 8 μmol / L, while the MIC of Example 5 (control formulation) was 64 μmol / L, and the MIC of pure 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone was 16 μmol / L. The above results demonstrate that the self-microemulsifying delivery system can reduce the MIC of the spray against *Porphyromonas gingivalis* to half that of the pure drug, and similarly reduce the MIC against *Fusobacterium nucleatum* to half that of the pure drug. This is because the nanoscale microemulsion droplets increase the contact area between the active ingredient and the bacterial cell surface, promoting drug penetration into the bacterial cell membrane. In contrast, the control formulation (Example 5) without the self-microemulsifying system, due to its larger droplet size (approximately 286 nm) and slower release rate of the active ingredient, had a higher MIC than the pure drug, further confirming the importance of the self-microemulsifying delivery system in improving antibacterial efficacy.

[0062] Antibacterial activity was also evaluated for Examples 2 (high-concentration formulation) and 4 (four-component compound formulation). Example 2, due to its higher concentration of active ingredients, had a MIC (based on total dihydrochalcone concentration) of 4 μmol / L against *Porphyromonas gingivalis* and 8 μmol / L against *Fusobacterium nucleatum*, both superior to Example 1. Although the concentration of a single component in Example 4 was lower than in Example 1, the simultaneous presence of four dihydrochalcone compounds resulted in a synergistic effect across multiple targets, leading to an MIC of 8 μmol / L against *Porphyromonas gingivalis*, comparable to Example 1. Example 3 (low-concentration formulation) had a higher MIC value, 16 μmol / L against *Porphyromonas gingivalis* and 32 μmol / L against *Fusobacterium nucleatum*, but these values ​​remained within the effective inhibitory concentration range, making it suitable for daily oral health maintenance.

[0063] To evaluate the effect of the spray on *Porphyromonas gingivalis* biofilm, the inhibitory effect of Example 1 on *Porphyromonas gingivalis* biofilm formation was determined using the crystal violet staining method. *Porphyromonas gingivalis* (1 x 10⁻⁶) was inoculated into 96-well microplates. 7 The spray from Example 1 (CFU / mL) was added at different dilutions, anaerobically cultured at 37 °C for 72 h, and then the culture medium was discarded. The sample was gently washed three times with phosphate buffer, stained with 0.1% crystal violet for 20 min, dissolved in 33% glacial acetic acid, and the absorbance at 590 nm was measured. The results showed that Example 1 reduced the formation of *Porphyromonas gingivalis* biofilm by 42.6 ± 5.8% at a concentration of 1 / 4 MIC (2 μmol / L) and by 68.3 ± 4.5% at a concentration of 1 / 2 MIC (4 μmol / L). This indicates that the spray of the present invention is not only effective against planktonic bacteria, but also significantly inhibits the formation of periodontal pathogenic bacteria biofilms at sub-inhibitory concentrations. This characteristic has important clinical significance for the prevention and control of periodontal disease.

[0064] 8. Evaluation of oral mucosal adhesion.

[0065] The retention performance of the spray from Example 1 on the oral mucosa was evaluated using an in vitro mucosal flushing method. Fresh porcine buccal mucosa (purchased from a local slaughterhouse and used within 2 hours of collection) was cut into 2 cm x 2 cm pieces, cleaned with physiological saline, and fixed on a glass slide tilted at 30°. The spray from Example 1 was applied to the mucosal surface (0.1 mL each time), allowed to stand for 1 min, and then continuously flushed the mucosal surface with artificial saliva (pH 6.8) at 37 °C at a flow rate of 0.5 mL / min. The flushing solution was collected at 5, 10, 15, 20, 30, and 60 min, respectively. The content of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone in the flushing solution was determined by HPLC, and the residual rate of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone on the mucosal surface was calculated. Meanwhile, Example 5 (a control formulation without HPMC) and pure 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone aqueous suspension (0.3%) were used as controls.

[0066] The results showed that, after 30 min of application of the spray containing HPMC, the residual rate of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone on the mucosal surface was 62.8 ± 5.3%, and after 60 min, the residual rate was still 38.5 ± 4.1%. In contrast, the residual rate of the spray without HPMC was only 28.4 ± 3.7% at 30 min, decreasing to 12.6 ± 2.8% at 60 min. The pure 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone aqueous suspension had the lowest residual rate at 30 min, only 15.2 ± 3.1%, decreasing to 6.3 ± 1.9% at 60 min. These results indicate that the introduction of HPMC significantly increased the oral mucosal retention time of the spray, with the residual rate at 30 min being approximately 2.2 times higher than the control without HPMC, which is beneficial for the sustained release of the active ingredient on the oral mucosal surface to exert its antibacterial effect.

[0067] To further quantify and evaluate mucosal adhesion, an adhesion test mode was used on a texture analyzer (TA.XT Plus, Stable MicroSystems) with fresh porcine buccal mucosa as the matrix. The maximum desorption force (Fmax) and adhesion work (AUC) of each formulation were measured at 37 °C and 100% relative humidity. The results showed that the Fmax of Example 1 was 0.326 + / - 0.042 N, and the AUC was 0.187 + / - 0.028 N·mm; the Fmax of the control formulation (without HPMC) was 0.089 + / - 0.015 N, and the AUC was 0.038 + / - 0.009 N·mm. The maximum desorption force of Example 1 was 3.66 times that of the control formulation, and the adhesion work was 4.92 times that of the control formulation. This confirms that HPMC imparts excellent mucosal adhesion properties to the spray, effectively prolonging the drug's residence time on the oral mucosa and creating favorable conditions for the sustained release and absorption of the active ingredient. Furthermore, experiments on the effect of different concentrations of HPMC (0.1%, 0.3%, and 0.5%) on adhesion showed that adhesion increased significantly when the HPMC concentration increased from 0.1% to 0.3%, and the increase slowed down when it increased from 0.3% to 0.5%. Considering both adhesion performance and the flowability of the spray (excessive HPMC concentration will increase viscosity and affect spraying effect), the preferred HPMC concentration is 0.2%-0.4%.

[0068] IX. Safety evaluation of oral mucosa.

[0069] The oral mucosal irritation and cytotoxicity of the spray from Example 1 were evaluated. The oral mucosal irritation test employed a modified hamster cheek pouch method. Eight healthy Syrian golden hamsters (half male and half female, weighing 100-120 g) were randomly divided into an experimental group and a control group, with four hamsters in each group. The experimental group received the spray from Example 1 (0.1 mL / time) twice daily on the cheek pouch mucosa, while the control group received physiological saline instead. The administration continued for 14 days. During the administration period, the cheek pouch mucosa was observed for congestion, edema, and ulceration daily. Twenty-four hours after the last administration, the cheek pouch mucosa was harvested for histopathological examination (HE staining). The results showed that no significant congestion, edema, or ulceration was observed in the cheek pouch mucosa of the experimental group throughout the administration period. Histopathological examination revealed intact mucosal epithelium and no significant inflammatory cell infiltration in the lamina propria, showing no significant difference compared to the control group. This indicates that the spray of the present invention is non-irritating to the oral mucosa.

[0070] The cytotoxicity assay was performed using the MTT assay, with human oral keratinocytes (HOK) as the target cells. HOK cells were seeded in 96-well plates (5 x 10^3 cells / well) and cultured adherently for 24 h. The spray from Example 1 was diluted 10-fold, 50-fold, 100-fold, 200-fold, and 500-fold with complete culture medium and added to each well. After 24 h of incubation, MTT solution (5 mg / mL) was added, and the cells were cultured for another 4 h. The supernatant was discarded, and formazan crystals were dissolved in DMSO. The absorbance was measured at 490 nm, and cell viability was calculated. The results showed that when the spray was diluted 50-fold or higher, the HOK cell viability was greater than 90%. At a 100-fold dilution, the cell viability was 96.3% + / - 2.1%, and at a 200-fold dilution, it was 98.7% + / - 1.5%. Considering that the oral spray will be rapidly diluted by saliva after application (the flow rate of oral saliva is about 0.3-0.5 mL / min), the actual drug concentration that comes into contact with oral mucosal cells is far lower than the 50-fold dilution concentration. Therefore, the spray of the present invention has no obvious toxicity to oral mucosal cells within the concentration range of use.

[0071] Hemolysis test: Fresh anticoagulated blood from healthy individuals was collected by centrifugation, and red blood cells were washed three times with physiological saline to prepare a 2% red blood cell suspension. The spray from Example 1 was diluted to 5, 10, 20, 50, and 100 times, respectively. 0.5 mL of each solution was added to 4.5 mL of the red blood cell suspension, incubated at 37 °C for 3 h, and the supernatant was collected by centrifugation. The absorbance was measured at 540 nm. Distilled water was used as a positive control (complete hemolysis), and physiological saline as a negative control. The results showed that the hemolysis rate was less than 5% (actual measured values ​​were 1.8%-3.6%) when the spray was diluted 10 times or more, and less than 2% when diluted 20 times, indicating that the spray of this invention has no risk of hemolysis.

[0072] Taste and User Experience Evaluation: Twenty healthy volunteers (aged 22-45, half male and half female) were invited to evaluate the taste of the spray from Example 1. Each volunteer sprayed two puffs (approximately 0.2 mL) into their mouth as usual and rated it on five dimensions: taste, smell, irritation, cooling sensation, and overall acceptability, using a scale of 1-5 (1 being the worst and 5 being the best). The results showed that the taste score was 3.8 + / - 0.6, the smell score was 4.2 + / - 0.5, the irritation score was 4.5 + / - 0.4 (higher values ​​indicate less irritation), the cooling sensation score was 4.1 + / - 0.5, and the overall acceptability score was 4.0 + / - 0.5. None of the volunteers reported any adverse experiences such as oral mucosal discomfort, taste abnormalities, or tooth discoloration, and all felt their mouths were fresh within 30 minutes after use. The above results indicate that the spray of the present invention has a good taste and user compliance, and does not produce the common side effects of chlorhexidine antibacterial agents such as tooth discoloration and taste changes.

[0073] The technical effect of this oral spray stems from the synergistic effect of the active ingredient, delivery system, and mucosal adhesion polymer. At the level of the active ingredient, 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, through its A-ring 2',6'-dihydroxy structure, forms hydrogen bonds and hydrophobic interactions with anionic phospholipids such as phosphatidylethanolamine and phosphatidylglycerol in the bacterial cell membrane, leading to increased membrane permeability and leakage of cell contents, thereby exerting a bactericidal effect. Meanwhile, 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, through its B-ring 4',5'-dimethoxy structure, interferes with bacterial fatty acid metabolism pathways. The two compounds act on different targets to produce a synergistic antibacterial effect (FICI = 0.375-0.5). At the delivery system level, the self-microemulsification system spontaneously forms nanoemulsion droplets with a particle size of 20-80 nm in the oral saliva environment, significantly increasing the contact surface area between the drug and bacterial cells. Simultaneously, the nanodroplets can promote the penetration of the active ingredient into the bacterial cell through membrane fusion, reducing the formulation's MIC to half that of the pure drug. At the mucosal adhesion level, HPMC forms a gel layer on the oral mucosa surface, prolonging the drug retention time to over 30 minutes, ensuring the continuous release of the active ingredient. Furthermore, the active ingredient of this invention exhibits selective inhibitory effects on periodontal pathogens (Porphyromonas gingivalis and Fusobacterium nucleatum) while having no significant effect on Streptococcus mutans in the normal oral flora. This characteristic is beneficial for maintaining the oral microecological balance and overcomes the deficiency of traditional chemical antibacterial agents that cause dysbiosis due to broad-spectrum bactericidal activity.

[0074] From a structure-activity relationship perspective, the four dihydrochalcone compounds selected in this invention (1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, compound 2, and compound 4) all possess a 1,3-diphenyl-1-propanone core structure, but the substituent patterns of ring A and ring B differ. The ring A of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone is substituted with 2',4'-dihydroxy-6'-methoxy, compound 2 is substituted with 2',6'-dihydroxy-4'-methoxy, compound 4 is substituted with 2',4'-dihydroxy-6'-methoxy-4-methoxy, while 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone is substituted with 2'-hydroxy-4',5'-dimethoxy. Comparison of the antibacterial activities of various compounds revealed that compounds containing a 2'-hydroxyl group and an adjacent phenolic hydroxyl group (such as the 2',4'-dihydroxyl group in 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone) on ring A exhibited stronger antibacterial activity. This may be related to the ability of the phenolic hydroxyl group to form chelates with metal ions on the bacterial cell membrane, thereby disrupting the membrane structure. While the increase in methoxyl substitution on ring B (such as the 4',5'-dimethoxyl group in 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone) reduced the direct membrane-damaging ability, it may have promoted transmembrane permeation by enhancing the lipophilicity of the molecule, thus interfering with intracellular bacterial metabolism. This complementary structure-activity relationship provides a reasonable molecular explanation for the synergistic antibacterial effect of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, and also provides guidance for further improving antibacterial activity through structural optimization.

[0075] The embodiments of the present invention are not limited to the specific embodiments described above. Those skilled in the art can make various equivalent changes or substitutions based on the technical solutions of the present invention, and all such changes or substitutions should be included within the protection scope of the present invention.

Claims

1. A dihydrochalcone oral spray characterized in that, The spray comprises the following components in weight percentages: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone 0.1%-0.5%, 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone 0.05%-0.3%, medium-chain triglycerides 1%-3%, polyoxyethylene hydrogenated castor oil 2%-5%, propylene glycol 10%-20%, hydroxypropyl methylcellulose 0.1%-0.5%, and citric acid-sodium citrate buffer 0.2%- 0.8%, menthol 0.05%-0.2%, steviol 0.02%-0.1%, methylparaben 0.1%-0.15%, purified water balance to 100%, the pH of the spray is 5.5-6.5; the 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone are both monomeric compounds obtained by systematic separation and purification from the ethanol extract of the whole plant of Coral Gynostemma pentaphyllum.

2. The oral spray according to claim 1, characterized in that, The mass percentage of 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone is 0.2%-0.4%, and the mass percentage of 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone is 0.1%-0.2%.

3. The oral spray according to claim 1, characterized in that, The spray also contains 0.02%-0.1% of 2',6'-dihydroxy-4'-methoxydihydrochalcone and / or 0.02%-0.1% of 2',4'-dihydroxy-6',4-dimethoxydihydrochalcone.

4. The oral spray according to claim 1, characterized in that, said medium-chain triglycerides have a carbon chain length of C8-C 10 said polyoxyethylene hydrogenated castor oil has an HLB value of 12-16.

5. The oral spray according to claim 1, characterized in that, The mass ratio of the polyoxyethylene hydrogenated castor oil to propylene glycol is 1:3 to 1:

5.

6. The method for preparing a dihydrochalcone-based oral spray according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1, Preparation of active ingredients: Take the dried and pulverized whole herb of *Sargassum fusiforme*, add 8 times the amount of 90% ethanol for reflux extraction, filter and collect the filtrate to recover the ethanol and obtain the alcohol extract. Extract the alcohol extract sequentially with petroleum ether, dichloromethane, ethyl acetate and n-butanol, collect the dichloromethane extract, load the dichloromethane extract onto a D101 macroporous resin column and elute with a gradient of ethanol at different concentrations, collect the 90% ethanol eluent, load the 90% ethanol eluent onto a silica gel column and elute with a petroleum ether-ethyl acetate gradient. The mixture was then crystallized and purified to obtain monomeric compounds 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone, respectively. Step two, preparation of the self-microemulsion concentrate: 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone and 2',6'-dihydroxy-3',4'-dimethoxydihydrochalcone were dissolved in medium-chain triglycerides and heated at 40-50 °C. Stir at °C until completely dissolved, add polyoxyethylene hydrogenated castor oil and continue stirring until uniform and transparent; Step 3, preparation of oral spray: Add propylene glycol to the microemulsion concentrate and mix evenly. Separately, dissolve hydroxypropyl methylcellulose, citric acid, sodium citrate, steviol glycosides and methylparaben in purified water. Slowly add the propylene glycol-microemulsion mixture to the aqueous phase and stir to mix. Add menthol, add purified water to the total volume, adjust the pH to 5.5-6.5, filter through a 0.22um microporous membrane, and fill into a metered spray pump bottle to obtain the final product.

7. The preparation method according to claim 6, characterized in that, The reflux extraction temperature in step one is 80 °C, the extraction time is 45 min, and the extraction is performed twice.

8. The preparation method according to claim 6, characterized in that, The conditions for silica gel column elution in step one are as follows: first, perform preliminary gradient elution with a petroleum ether-ethyl acetate volume ratio of 20:1 to 5:1, collect the eluent fraction with a petroleum ether-ethyl acetate volume ratio of 10:1, and then perform fine gradient elution with a petroleum ether-ethyl acetate volume ratio of 8:1 to 6:1 and crystallize for purification.

9. The preparation method according to claim 6, characterized in that, The stirring temperature in step two is 45 °C, and the stirring time is 15-30 min.

10. The preparation method according to claim 6, characterized in that, The microporous filter membrane mentioned in step three is made of polyvinylidene fluoride, and the release volume per press of the metering spray pump bottle is 0.08-0.12 mL.