Use of uvangoletin in the preparation of an anti-periodontitis medicament

Abstract

This invention belongs to the interdisciplinary field of natural product medicinal chemistry and oral medicine, specifically disclosing the application of uvangoletin in the preparation of anti-periodontalgia drugs. Uvangoletin is 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, with the molecular formula C. 16 H 16 O4, experiments confirmed that its MIC for Porphyromonas gingivalis was 16 μmol / L, MIC 50 The concentration is 10.28 μmol / L, the MIC for Fusobacterium nucleatum is 16 μmol / L and the MIC50 is 6.86 μmol / L. It has no inhibitory activity against Streptococcus mutans and has selective anti-periodontal pathogenic bacteria characteristics. It can be made into oral preparations such as gels, mouthwashes or toothpaste.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of natural product medicinal chemistry and oral medicine, and more specifically relates to the application of uvangoletin, a dihydrochalcone compound isolated from *Sarcandra glabra*, in the preparation of oral medications for the treatment and prevention of periodontitis, which inhibit the growth and reproduction of *Porphyromonas gingivalis* and *Fusobacterium nucleatum*. Background Technology

[0002] Periodontitis is a chronic infectious disease caused by periodontal pathogens, characterized by progressive destruction of the periodontal supporting tissues, and is the leading cause of tooth loss in adults. Epidemiological surveys show that more than one billion people worldwide suffer from periodontitis to varying degrees. This disease not only seriously affects oral health and quality of life, but is also closely related to the occurrence and development of various systemic diseases such as cardiovascular disease, diabetes, rheumatoid arthritis, Alzheimer's disease, and adverse pregnancy outcomes, placing a heavy economic burden and posing a significant health threat to public health systems.

[0003] Porphyromonas gingivalis (Porphyromonas gingivalis ) and Fusobacterium nucleatum ( Fusobacterium nucleatum *Porphyromonas gingivalis* is a recognized key pathogen of periodontitis. As a Gram-negative anaerobic bacterium, it is considered a major pathogen of chronic periodontitis, directly damaging periodontal tissues by secreting virulence factors such as gingival protease and lipopolysaccharide, while simultaneously inducing an excessive immune response in the host, leading to alveolar bone resorption and loss of periodontal attachment. *Fusobacterium nucleatum* plays a crucial bridging role in the formation of subgingival plaque biofilm, promoting the adhesion and aggregation of late-term colonizing bacteria such as *Porphyromonas gingivalis*, thus accelerating the pathological progression of periodontitis. Effectively inhibiting the growth and reproduction of these two key pathogens is an important strategy for controlling and treating periodontitis.

[0004] Currently, the main drugs used in clinical treatment of periodontitis include metronidazole, minocycline, and chlorhexidine. Although these drugs have strong antibacterial activity, long-term use can easily lead to drug resistance and often causes adverse reactions such as gastrointestinal discomfort, oral flora imbalance, and tooth staining. Therefore, developing novel anti-periodontalgia drugs with high safety, low drug resistance, and wide availability has significant clinical and social value.

[0005] Natural products have attracted widespread attention in the field of antibacterial drug development due to their structural diversity and broad-spectrum biological activities. *Sarcandra glabra* (a type of herb) Sarcandra glabra *Sarcandra glabra*, belonging to the genus *Sarcandra* of the family Chloranthaceae, is widely distributed and abundant in Guizhou, Sichuan, Yunnan, Anhui, Fujian, and Jiangxi provinces of my country. It has a long history of medicinal use, possessing various biological activities such as anti-tumor, antibacterial, anti-inflammatory, fracture-healing-promoting, and analgesic effects. The chemical components of *Sarcandra glabra* mainly include sesquiterpenes, flavonoids, coumarins, triterpenoids, phenolic acids, and volatile oils; however, research on the antibacterial properties of its dihydrochalcone components in oral cavity is still lacking.

[0006] Dihydrochalcones are an important subclass of flavonoids, with a molecular skeleton consisting of A and B rings linked by a saturated three-carbon chain. They are widely distributed in natural products. Previous studies have reported that some dihydrochalcones possess anti-inflammatory, antioxidant, and anti-leishmaniatic activities; however, their inhibitory activity against key periodontitis pathogens, namely *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, has not been previously reported.

[0007] Uvangoletin, chemically named 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, has the molecular formula C1. 16 H 16 O4, with a molecular weight of 272.30, is a natural dihydrochalcone compound isolated from various plants. While uvangoletin is known to possess anti-inflammatory activity, its inhibitory activity against periodontal pathogens and its potential application in the treatment of oral diseases have never been previously discovered or reported.

[0008] In the prior art, the published invention patents related to *Sarcandra glabra* mainly focus on the following aspects: using crude extracts of *Sarcandra glabra* or its polysaccharide components in combination with other traditional Chinese and Western medicines for the treatment of oral diseases. For example, Chinese invention patent application CN1596924A discloses a compound traditional Chinese and Western medicine formula for oral and pharyngeal use containing *Sarcandra glabra* extract, and Chinese invention patent CN116492369B discloses the combination of *Sarcandra glabra* polysaccharide and ginger-processed *Elsholtzia ciliata* polysaccharide for oral care products. However, the above-mentioned prior art all use crude extracts or polysaccharide components of *Sarcandra glabra* as the basis for active substances, without delving into the specific monomer compound level. There is a lack of systematic research on the oral antibacterial activity of various monomer compounds in *Sarcandra glabra*, especially the specific inhibitory effect on key pathogenic bacteria of periodontitis. In addition, although international patent application US20110201503 involves uvangoletin compounds, its application field is herbicides, which is completely different from oral antibacterial agents and cannot provide technical inspiration for this invention. Therefore, the application of uvangoletin monomer compounds isolated from *Sarcandra glabra* in the preparation of anti-periodontalgia drugs has not been reported in the prior art, nor can any inspiration be obtained from known technical solutions.

[0009] From the perspective of antimicrobial drug development, naturally derived monomeric compounds have clear advantages over crude extracts: their chemical structures are well-defined, their quality is highly controllable, they facilitate systematic pharmacological and toxicological evaluations, they are conducive to establishing standardized quality control systems and production process specifications, and they are more likely to obtain market approval through drug review and approval procedures. Therefore, isolating and screening monomeric compounds with specific anti-periodontal pathogenic bacteria activity from traditional medicinal plants such as *Sarcandra glabra*, and systematically evaluating their application potential in oral preparations, is of great significance for promoting the research and development of natural product antimicrobial drugs.

[0010] In conclusion, there is an urgent need to discover natural compounds with good activity against periodontal pathogens, providing new options for the development of novel oral antibacterial drugs or health products. Summary of the Invention

[0011] The purpose of this invention is to provide the application of uvangoletin in the preparation of anti-periodontic drugs, fill the gap in the application of uvangoletin in the field of oral antibacterial, and provide a new source of natural active compounds for the treatment and prevention of periodontitis.

[0012] Another object of the present invention is to provide an oral pharmaceutical composition containing uvangoletin and its application in the treatment of oral infectious diseases such as periodontitis and gingivitis.

[0013] To achieve the above objectives, the present invention adopts the following technical solution: The application of uvangoletin in the preparation of anti-periodontalgia drugs. The chemical name of uvangoletin is 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, with the molecular formula C1. 16 H 16 O4 has a molecular weight of 272.30.

[0014] Furthermore, the anti-periodontalgia drug is a drug that inhibits Porphyromonas gingivalis and / or Fusobacterium nucleatum.

[0015] Furthermore, the dosage form of the anti-periodontalgia drug is selected from one of the following: gel, mouthwash, toothpaste, lozenge, oral spray, oral patch, or oral sustained-release film.

[0016] Furthermore, the mass concentration of uvangoletin in the anti-periodontalgia drug is 0.001%~5%.

[0017] Furthermore, the uvangoletin is derived from the isolated and purified product or chemically synthesized product of Sarcandra glabra plant extract.

[0018] The present invention also provides an oral pharmaceutical composition containing uvangoletin as an active ingredient and pharmaceutically acceptable excipients, the oral pharmaceutical composition being used to treat periodontitis.

[0019] Furthermore, the oral pharmaceutical composition also contains anti-inflammatory agents and / or active ingredients that promote periodontal tissue repair.

[0020] The beneficial effects of this invention are as follows: First, this invention is the first to discover and verify that uvangoletin has significant inhibitory activity against *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, key pathogens of periodontitis. In vitro antibacterial experiments showed that uvangoletin had a minimum inhibitory concentration (MIC) of 16 μmol / L and a median MIC50 of 10.28 μmol / L against *Porphyromonas gingivalis*; and a MIC of 16 μmol / L and a MIC50 of 6.86 μmol / L against *Fusobacterium nucleatum*. This discovery lays a solid experimental foundation for the development of uvangoletin in the field of periodontitis treatment.

[0021] Secondly, this invention discovers that uvangoletin has selective inhibitory characteristics against periodontal pathogens, specifically, within the concentration range that effectively inhibits Porphyromonas gingivalis and Fusobacterium nucleatum, it also inhibits Streptococcus mutans, a resident oral bacterium. Streptococcus mutans It does not produce significant inhibitory effects (MIC>128 μmol / L). This selective inhibitory characteristic helps maintain the balance of the oral microecology and avoid oral flora imbalance, which is superior to the adverse effects of traditional broad-spectrum antibacterial drugs in oral applications.

[0022] Third, uvangoletin is derived from natural plants such as *Sarcandra glabra*, with abundant and widely distributed raw material resources. In particular, the *Sarcandra glabra* resources in Guizhou, Yunnan, and Sichuan provinces of my country are enormous, providing ample raw material guarantees for industrial applications. Compared with chemically synthesized antibacterial drugs, naturally derived uvangoletin offers better safety prospects and a lower environmental burden.

[0023] Fourth, the oral pharmaceutical composition containing uvangoletin provided by this invention can be prepared into various oral preparations such as gels, mouthwashes, and toothpastes. It is convenient to use, has good patient compliance, and has good prospects for clinical translation and market application value.

[0024] Fifth, this invention systematically traced and ultimately located uvangoletin, the key active ingredient against *Porphyromonas gingivalis*, from the ethanol extract of *Sarcandra glabra* using an activity-directed separation strategy, establishing a complete activity tracking and quality control pathway from crude extract to monomeric compound. This pathway not only provides technical support for the large-scale preparation of uvangoletin but also provides methodological reference for further exploration of other natural compounds with oral antibacterial activity from *Sarcandra glabra*. Furthermore, in vitro comparative experiments determined that uvangoletin exhibited the best antibacterial activity against *Porphyromonas gingivalis* and *Fusobacterium nucleatum* among the five isolated compounds, providing a scientific basis for its use as a lead compound for subsequent structural optimization and in-depth pharmacodynamic studies. Detailed Implementation

[0025] The present invention will be further described in detail below through specific embodiments. These embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0026] Example 1: Extraction, separation and purification of uvangoletin from Coral Gynostemma pentaphyllum.

[0027] Weigh 1000 g of dried, pulverized whole *Sarcandra glabra* herb and place it in a round-bottom flask. Add 8 times the volume (8000 mL) of 90% ethanol solution and extract under reflux for 45 min. After extraction, filter while hot and collect the filtrate. Repeat the extraction twice more with the same conditions on the residue. Combine the filtrates from the three extractions and recover ethanol and water under reduced pressure at 50°C using a rotary evaporator to obtain the ethanol extract of *Sarcandra glabra*.

[0028] Take the extract of *Sargassum fusiforme* and add an equal mass of purified water. After ultrasonic dispersion, systematic solvent extraction is performed sequentially using petroleum ether (3 times, 2 times, and 1 times each), dichloromethane (3 times, 2 times, and 1 times each), ethyl acetate (3 times, 2 times, and 1 times each), and saturated water with n-butanol (3 times, 2 times, and 1 times each). The solvent extracts are combined and the solvent is recovered under reduced pressure to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract, n-butanol extract, and aqueous extract.

[0029] Take the dichloromethane extract and load it onto a D101 macroporous adsorption resin column (column diameter 5 cm, column height 60 cm). Elute sequentially with 0% ethanol (pure water), 30% ethanol, 60% ethanol and 90% ethanol. Collect each eluent and concentrate under reduced pressure to recover the solvent, to obtain 0% ethanol eluent, 30% ethanol eluent, 60% ethanol eluent and 90% ethanol eluent, respectively.

[0030] Combine the 60% and 90% ethanol eluates and load them onto a silica gel column (200-300 mesh, 3 cm diameter, 40 cm height). Perform gradient elution with a petroleum ether-ethyl acetate mixture, using eluents at volume ratios of 20:1, 15:1, 10:1, 5:1, and 0:1 (pure ethyl acetate). Analyze the combined fractions by thin-layer chromatography (TLC). Collect the fraction obtained from the petroleum ether-ethyl acetate (10:1 volume ratio) elution.

[0031] The petroleum ether-ethyl acetate (volume ratio 10:1) eluent was again loaded onto a silica gel column (200-300 mesh, column diameter 2 cm, column height 30 cm) for fine separation. Gradient elution was performed with petroleum ether-ethyl acetate (volume ratios) of 8:1, 7:1, and 6:1, respectively. TLC analysis was performed to combine identical components. After concentration under reduced pressure, the mixture was purified by crystallization to obtain the compound uvangoletin.

[0032] The obtained uvangoletin was a colorless needle-like crystal, readily soluble in chloroform and methanol. Structural identification was performed using nuclear magnetic resonance spectroscopy (NMR). The 1H NMR spectrum (CDCl3, 500 MHz) showed: δH 13.95 (1H, s, 2'-OH), 7.32–7.19 (5H, m, H-2–H-6), 6.00 (1H, d, J=2.0 Hz, H-5'), 5.91 (1H, d, J=2.0 Hz, H-3'), 5.81 (1H, s, 6'-OH), 3.84 (3H, s, OCH3-6'), 3.32 (2H, m, H-α), 3.00 (2H, m, H-β). Carbon NMR (13C-NMR, CDCl3, 125 MHz) showed the following δC values: 204.70 (C=O), 167.44 (C-6'), 163.63 (C-4'), 162.63 (C-2'), 141.81 (C-1), 128.60 (C-3, C-5), 128.58 (C-2, C-6), 126.11 (C-4), 105.97 (C-1'), 96.70 (C-3'), 90.83 (C-5'), 55.85 (OCH3-6'), 45.91 (C-α), and 30.80 (C-β). Based on the comparison with literature, the above NMR data confirmed that the compound is uvangoletin, namely 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, with the molecular formula C16H16O4 and a molecular weight of 272.30.

[0033] Example 2: Determination of the in vitro antibacterial activity of uvangoletin against Porphyromonas gingivalis.

[0034] Strains and Culture Conditions: The experimental strain was the standard strain of *Porphyromonas gingivalis*. The original bacterial culture, stored at -80°C, was rapidly thawed in a 37°C water bath. The contents were transferred to a test tube containing 10 mL of BHI+ liquid medium, mixed thoroughly, and then placed in an anaerobic incubator. The culture was kept at 37°C for 48 h in an anaerobic atmosphere with 80% N2, 10% CO2, and 10% H2 to complete the resuscitation process. The resuscitated strain was then inoculated into BHI+ liquid medium at a 1% inoculum and incubated anaerobicly at 37°C for 48 h to complete the activation process.

[0035] Minimum inhibitory concentration (MIC) determination: The microbroth dilution method was used. A 10 mg / mL stock solution of uvangoletin was prepared by dissolving uvangoletin in dimethyl sulfoxide (DMSO). This stock solution was then serially diluted twofold with BHI+ medium to achieve final uvangoletin concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μmol / L in each well of a 96-well plate. Activated *Porphyromonas gingivalis* was then added to adjust the concentration to 1 × 10⁻⁶. 7 CFU / mL, add 100 μL of bacterial culture to each well. Set up a blank control group, a sample background control group, a 1% DMSO solvent control group, and a metronidazole positive control group. After incubating the 96-well plate in an anaerobic environment at 37°C for 48 h, observe the growth of each well against a dark background. A well with clear, bright images and no turbidity is considered sterile, and the corresponding sample concentration is the minimum inhibitory concentration (MIC). Simultaneously, measure the absorbance (A600) of each well at 600 nm using a microplate reader. Calculate the inhibition rate using the formula: Inhibition rate (%) = [(A solvent - A blank) - (A sample - A sample background)] / (A solvent - A blank) × 100%. Three replicate wells are set up for each sample concentration, and the entire experiment is independently repeated three times. The average value is taken, and the MIC50 value is calculated using nonlinear regression analysis.

[0036] Experimental results: The MIC of uvangoletin against *Porphyromonas gingivalis* was 16 μmol / L, and the MIC50 was 10.28 μmol / L. The MIC of metronidazole (positive control) was 0.16 μmol / L, and the MIC50 was 0.0415 μmol / L. These results indicate that uvangoletin has clear inhibitory activity against *Porphyromonas gingivalis*.

[0037] Further analysis of the experimental data revealed that uvangoletin achieved an inhibition rate of approximately 40% against *Porphyromonas gingivalis* at a concentration of 8 μmol / L, over 90% at 16 μmol / L, and nearly 100% at 32 μmol / L and higher concentrations, exhibiting a clear dose-response relationship. The steepness of this dose-response curve suggests that uvangoletin significantly affects the metabolic activity and proliferative capacity of *Porphyromonas gingivalis* within the sub-MIC concentration range. From a pharmacological perspective, the MIC50 (10.28 μmol / L) of uvangoletin against *Porphyromonas gingivalis* corresponds to a mass concentration of approximately 2.80 μg / mL. This concentration level is easily achievable and maintained in topical oral formulations, providing a feasible concentration basis for its clinical application in dentistry, indicating that this compound is suitable for development as a topical oral anti-periodontalgia agent.

[0038] Example 3: Determination of the in vitro antibacterial activity of uvangoletin against Fusobacterium nucleatum.

[0039] Strains and culture conditions: The experimental strain was the standard strain of *Fusobacterium nucleatum*. The strain resuscitation and activation methods were the same as those for *Porphyromonas gingivalis* in Example 2, i.e., static incubation at 37°C in BHI+ liquid medium under anaerobic gas conditions of 80% N2, 10% CO2, and 10% H2.

[0040] MIC determination method: Same as in Example 2, using the micro-broth dilution method, with the final concentration gradient of uvangoletin set at 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μmol / L. The bacterial concentration was adjusted to 1×10⁻⁶ μmol / L. 7 The results were observed and the A600 value was measured after anaerobic culture at 37°C for 48 h with CFU / mL.

[0041] Experimental results: The MIC of uvangoletin against *Fusobacterium nucleatum* was 16 μmol / L, and the MIC50 was 6.86 μmol / L. The MIC of metronidazole (positive control) was 0.16 μmol / L, and the MIC50 was 0.0396 μmol / L. These results indicate that uvangoletin also exhibits significant inhibitory activity against *Fusobacterium nucleatum*, and the MIC50 value (6.86 μmol / L) is lower than that against *Porphyromonas gingivalis* (10.28 μmol / L), suggesting that uvangoletin demonstrates stronger inhibitory efficacy against *Fusobacterium nucleatum*.

[0042] Example 4: Determination of the in vitro antibacterial activity of uvangoletin against Streptococcus mutans.

[0043] Strains and culture conditions: The experimental strain was Streptococcus mutans (Streptococcus mutans). Streptococcus mutans Standard strain. The original bacterial culture, stored at -80°C, was rapidly thawed in a 37°C water bath. The contents were transferred entirely to a test tube containing 10 mL of BHI+ liquid medium, mixed thoroughly, and then placed in an incubator for static incubation at 37°C with aerobic conditions for 48 h to complete the recovery process. The recovered strain was then inoculated into BHI+ liquid medium at a 1% inoculum and activated by static incubation at 37°C with aerobic conditions for 48 h.

[0044] MIC determination method: Same as in Example 2, using the micro-broth dilution method, but the culture conditions were changed to an aerobic environment at 37°C. The final concentration gradient of uvangoletin was set to 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μmol / L. The bacterial concentration was adjusted to 1×10⁻⁶. 7 The results were observed after CFU / mL aerobic incubation at 37°C for 48 h.

[0045] Experimental results: Within the concentration range of 0.5–128 μmol / L, uvangoletin did not produce significant inhibitory activity against *Streptococcus mutans*, and turbidity was observed in all wells at all concentrations. The MIC was >128 μmol / L. The MIC for the metronidazole positive control was 1.6 μmol / L. These results indicate that uvangoletin lacks inhibitory activity against *Streptococcus mutans*, further confirming its selective inhibitory characteristics against periodontitis-specific pathogens (*Porphyromonas gingivalis* and *Fusobacterium nucleatum*).

[0046] The aforementioned selective inhibition results have significant clinical translational implications. While *Streptococcus mutans* is a major pathogen of dental caries, it is also an important component of the normal oral flora, playing a crucial role in maintaining oral microecological balance. Traditional broad-spectrum antibiotics such as metronidazole and chlorhexidine, while treating periodontitis, often cause non-selective killing of normal oral flora, leading to oral microecological imbalance and even secondary infections such as fungal infections. uvangoletin exhibits selective inhibition of anaerobic pathogens (*Porphyromonas gingivalis* and *Fusobacterium nucleatum*) but does not significantly inhibit the facultative anaerobic *Streptococcus mutans*. This differential antimicrobial spectrum suggests that uvangoletin may exert its antimicrobial effect by specifically interfering with the energy metabolism or membrane structure of anaerobic bacteria, rather than broadly destroying all oral microorganisms. Therefore, in clinical applications, it holds promise for better protecting the oral microecological balance and reducing the risk of treatment-related adverse reactions. This discovery provides new drug candidates for developing precise antibacterial strategies targeting periodontitis pathogens and lays the foundation for in-depth research on the antibacterial mechanism of uvangoletin.

[0047] Example 5: Comparison of antibacterial activity of uvangoletin with other dihydrochalcone compounds of *Sarcandra glabra*.

[0048] During the separation process in Example 1, five monomeric compounds were simultaneously obtained: dihydropashanone (compound 1), 2',6'-dihydroxy-4'-methoxydihydrochalcone (compound 2), uvangoletin (compound 3), 2',4'-dihydroxy-6',4-dimethoxydihydrochalcone (compound 4), and linoleic acid (compound 5). Following the methods in Examples 2-4, the antibacterial activities of the five compounds against *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Streptococcus mutans* were determined, and the experimental results are summarized below.

[0049] Activity against *Porphyromonas gingivalis* (MIC / MIC50, μmol / L): Compound 1 64 / 29.73, Compound 2 32 / 16.10, Compound 3 (uvangoletin) 16 / 10.28, Compound 4 32 / 18.72, Compound 5 32 / 18.56. Activity against *Fusobacterium nucleatum* (MIC / MIC50, μmol / L): Compound 1 128 / 56.71, Compound 2 64 / 28.69, Compound 3 (uvangoletin) 16 / 6.86, Compound 4 32 / 14.35, Compound 5 16 / 3.27. Activity against *Streptococcus mutans*: None of the five compounds showed significant inhibitory activity in the concentration range of 0.5–128 μmol / L, and all MICs were >128 μmol / L.

[0050] The comparative experimental results showed that among the five compounds isolated from *Sargassum fusiforme*, uvangoletin (compound 3) exhibited the strongest inhibitory activity against *Porphyromonas gingivalis* (MIC = 16 μmol / L, which is 1 / 2 to 1 / 4 of the activity of other compounds), and also showed excellent inhibitory efficacy against *Fusobacterium nucleatum* (MIC = 16 μmol / L, MIC50 = 6.86 μmol / L). The bisphenol A hydroxyl substitution pattern of 2'-OH and 6'-OH on the A ring of uvangoletin, along with the specific combination of the methoxy group at the 4'-position, endows it with unique spatial configuration and electronic distribution characteristics. This structural feature may be the structural basis for its superior antibacterial activity compared to other dihydrochalcone compounds. Compared to compound 1 (dihydropashanone), the MIC value of uvangoletin decreased by 4-fold, indicating that the type and position of the substituents on the A ring have a significant impact on antibacterial activity.

[0051] Example 6: Preparation of an oral gel containing uvangoletin.

[0052] Dissolve 0.5 g of uvangoletin in an appropriate amount of anhydrous ethanol until completely dissolved. Separately, dissolve 1.0 g of carbomer 940 in an appropriate amount of purified water, stir until fully swollen, and allow to stand to degas. Add 5.0 g of glycerin (humectant), 0.05 g of benzalkonium chloride (preservative), and 0.1 g of menthol (cooling agent) to the swollen carbomer matrix in sequence, and stir thoroughly. Slowly add the uvangoletin ethanol solution to the above matrix, and continue stirring until uniformly dispersed. Adjust the pH to 6.5-7.0 with triethanolamine, add purified water to 100 g, and stir well to obtain an oral gel containing 0.5% (w / w) uvangoletin. This gel is a pale yellow, uniform, semi-transparent gel with a fine texture and good spreadability, suitable for topical application to the gums.

[0053] Example 7: Preparation of mouthwash containing uvangoletin.

[0054] Take 0.2 g of uvangoletin and add 2.0 g of polyoxyethylene castor oil (Cremophor EL) as a solubilizer, stirring until the uvangoletin is completely dissolved and dispersed. Separately, take 3.0 g of xylitol, 0.1 g of citric acid, 0.3 g of sodium citrate, 0.5 g of peppermint oil, and 0.2 g of sodium benzoate, and dissolve them in an appropriate amount of purified water, stirring until completely dissolved. Add the uvangoletin solubilizer to the above aqueous solution, mix thoroughly, and bring the volume to 1000 mL with purified water. Filter to remove insoluble particles and fill into brown glass bottles. This yields a mouthwash containing 0.02% (w / v) uvangoletin. This mouthwash is a pale yellow to colorless clear liquid with a refreshing minty aroma. Use 10-15 mL each time, gargle for 1 minute, and spit it out. Use 2-3 times daily.

[0055] Example 8: Preparation of oral toothpaste containing uvangoletin.

[0056] Dissolve 1.0 g of uvangoletin in an appropriate amount of propylene glycol. Separately, take 45.0 g of sorbitol, 10.0 g of glycerin, 18.0 g of hydrated silica (abrasive), 1.2 g of sodium carboxymethyl cellulose (thickener), 1.5 g of sodium dodecyl sulfate (foaming agent), 0.76 g of sodium monofluorophosphate (fluoride active ingredient), 0.15 g of sodium saccharin, 1.0 g of peppermint flavor, and 0.1 g of methylparaben. Following standard toothpaste manufacturing processes, sequentially mix and grind the humectant, thickener, and abrasive until homogeneous. Then add the uvangoletin propylene glycol solution and other excipients, vacuum stir to degas, and adjust the total amount to 100 g with purified water. This yields an oral toothpaste containing 1.0% (w / w) uvangoletin. This toothpaste has a white to pale yellow uniform paste appearance, a fine texture, and a refreshing peppermint aroma, suitable for daily brushing.

[0057] Example 9: Preparation of an oral spray containing uvangoletin.

[0058] Dissolve 0.1 g of uvangoletin in an appropriate amount of anhydrous ethanol. Separately, take 5.0 g of polyethylene glycol 400, 3.0 g of glycerin, 0.05 g of menthol, and an appropriate amount of sodium citrate-citrate buffer (pH 6.0-6.5), add purified water to 100 mL, mix thoroughly, filter through a 0.22 μm microporous membrane for sterilization, and then fill into a high-density polyethylene (HDPE) bottle with a micro-spray pump head. This yields an oral spray containing 0.1% (w / v) uvangoletin. When using, aim the spray nozzle at the affected gum area and spray 2-3 times each time, 3-4 times daily.

[0059] Example 10: Preparation of an oral sustained-release patch containing uvangoletin.

[0060] Dissolve 0.3 g of uvangoletin in an appropriate amount of ethanol. Separately, dissolve 2.0 g of hydroxypropyl methylcellulose (HPMC) in an appropriate amount of purified water to prepare a matrix solution. Add the uvangoletin ethanol solution to the HPMC matrix solution, mix thoroughly, and then add 0.5 g of glycerol (plasticizer) and 0.2 g of carbomer 934P (bioadhesive). Pour the mixture onto a glass plate and dry it in a 40°C oven for 8–12 h until a film forms. Cut the film into circular patches with a diameter of 10 mm. Each patch contains approximately 1.5 mg of uvangoletin. When using, apply the patch to the surface of the gingival lesion area. The slow release of uvangoletin maintains an effective antibacterial concentration locally for 4–6 h.

[0061] Example 11: The entire process of uvangoletin's activity-guided separation and its activity tracking and verification against periodontal pathogens.

[0062] This embodiment describes the complete process and activity tracking data at each stage of the systematic isolation of uvangoletin from the whole plant of *Porphyromonas gingivalis*, guided by the inhibition of *Porphyromonas gingivalis* activity.

[0063] The first stage involved pre-screening the antibacterial activity of the *Sargassum fusiforme* ethanol extract. The ethanol extract was prepared according to the method in Example 1, and its inhibitory effect on *Porphyromonas gingivalis* was determined using the inhibition loop method. The ethanol extract was dissolved in DMSO and then serially diluted with physiological saline to 0.25, 0.5, 1, 2, and 4 mg / mL. *Porphyromonas gingivalis* (1×10⁻⁶ mg / mL) was then... 8 The solution (CFU / mL) was spread onto blood agar plates, and 100 μL of the drug solution was added using the punching method. After anaerobic incubation at 37°C for 72 h, the diameter of the inhibition zone was measured. The results showed that only a concentration of 4 mg / mL produced an inhibition zone with a diameter of 11.28 ± 0.64 mm, while concentrations below 4 mg / mL did not produce any visible inhibition zones.

[0064] The second stage involved systematic solvent extraction and screening of active components. The alcohol extract was subjected to systematic extraction with petroleum ether, dichloromethane, ethyl acetate, n-butanol, and aqueous layers. The MICs of the five extracts were determined using the micro-broth dilution method. The results showed that only the dichloromethane extract exhibited significant inhibitory activity against *Porphyromonas gingivalis*, with an MIC of 25 μg / mL and an MIC50 of 8.00 μg / mL. The MICs of the petroleum ether extract were 50 μg / mL, the ethyl acetate extract was 12.5 μg / mL, and the n-butanol extract was 100 μg / mL. The aqueous extract showed no activity.

[0065] In the third stage, the dichloromethane extract was separated into four eluents (0%, 30%, 60%, and 90% ethanol eluents) by D101 macroporous resin column chromatography. Activity assays showed that the 90% ethanol eluent exhibited the strongest antibacterial activity.

[0066] In the fourth stage, the 90% ethanol eluent was eluted by silica gel column chromatography with a gradient of petroleum ether-ethyl acetate in different ratios to obtain five fractions: A (20:1), B (15:1), C (10:1), D (5:1), and E (0:1). Activity assays showed that fraction C (10:1 eluent) exhibited the strongest antibacterial activity, with a MIC of 0.5 μg / mL and a MIC50 of 0.37 μg / mL, representing a significant increase in activity compared to the previous stage, thus confirming the effectiveness of the activity-directed separation strategy.

[0067] In the fifth stage, fraction C (10:1 eluent) was purified by fine silica gel column chromatography and crystallization to obtain five monomeric compounds, including uvangoletin. Antibacterial activity data for each compound are shown in Example 5. Throughout the activity-directed separation process, the antibacterial activity gradually increased from the crude extract level (MIC = 4 mg / mL, inhibition loop method) to the monomeric compound level (uvangoletin MIC = 16 μmol / L, equivalent to 4.35 μg / mL). The activity tracking clues were clear, fully demonstrating that uvangoletin is one of the key active ingredients of *Porphyromonas gingivalis* in inhibiting *Sarcandra glabra*.

[0068] Example 12: Stability study of uvangoletin in different oral preparations.

[0069] Stability studies were conducted on the oral gel prepared in Example 6, the mouthwash prepared in Example 7, and the toothpaste prepared in Example 8 at 25°C / 60% relative humidity (long-term test conditions) and 40°C / 75% relative humidity (accelerated test conditions). Samples were taken at 0, 1, 2, 3, and 6 months, and the uvangoletin content in each formulation was determined by high-performance liquid chromatography (HPLC). Changes in appearance and pH were also observed. HPLC conditions were as follows: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (volume ratio 40:60), flow rate: 1.0 mL / min, detection wavelength: 285 nm, column temperature: 30°C, injection volume: 10 μL.

[0070] Under conditions of 25°C / 60% relative humidity, the uvangoletin content in the oral gel remained at 97.2% after 6 months, with no significant changes in appearance and pH fluctuations within ±0.2. In mouthwash, the uvangoletin content remained at 95.8% after 6 months, with the solution remaining clear and transparent without precipitation. In oral toothpaste, the uvangoletin content remained at 98.1% after 6 months, with no significant changes in texture or color. Under accelerated conditions of 40°C / 75% relative humidity, the uvangoletin content retention rate in all three formulations was above 90% after 6 months, indicating that uvangoletin possesses good chemical stability in the three oral formulation matrices and meets the basic shelf-life requirements of oral formulation products. This stability data further supports the feasibility of uvangoletin as an antibacterial active ingredient in oral medications for industrial application.

[0071] Example 13: Evaluation of the inhibitory effect of uvangoletin on Porphyromonas gingivalis biofilm.

[0072] Periodontal pathogens typically exist in the oral environment as biofilms, and biofilm formation significantly enhances bacterial resistance to antimicrobial agents. Therefore, evaluating the effect of uvangoletin on *Porphyromonas gingivalis* biofilms is of significant practical importance. *Porphyromonas gingivalis* (1×10⁻⁶) biofilms were tested. 7 CFU / mL was inoculated into 96-well plates and anaerobically cultured in BHI+ medium at 37°C for 72 h to form a mature biofilm. After gently washing three times with sterile phosphate-buffered saline (PBS) to remove airborne bacteria, BHI+ medium containing different concentrations of uvangoletin (8, 16, 32, and 64 μmol / L) was added, and anaerobic culture was continued at 37°C for 24 h. 1% DMSO was used as the solvent control, and 0.2% chlorhexidine was used as the positive control. After treatment, the culture medium was discarded, the plates were washed twice with PBS, fixed with 99% methanol for 15 min, stained with 0.1% crystal violet for 15 min, and excess dye was washed away. Bound crystal violet was dissolved in 33% glacial acetic acid, and the absorbance at 570 nm was measured using a microplate reader to calculate the biofilm inhibition rate.

[0073] Experimental results showed that uvangoletin inhibited the mature biofilm of *Porphyromonas gingivalis* at a concentration of 32 μmol / L by 61.3%, and at a concentration of 64 μmol / L by 78.5%, indicating that uvangoletin can not only inhibit the growth of planktonic *Porphyromonas gingivalis*, but also disrupt the established bacterial biofilm structure to a certain extent. This result has significant clinical implications, as pathogenic bacteria in periodontal pockets mainly exist in biofilm form, and drugs capable of disrupting biofilms have greater application value in the treatment of periodontitis.

[0074] Example 14: Pharmacodynamic evaluation scheme of uvangoletin oral gel in a rat model of experimental periodontitis.

[0075] Forty male SPF-grade SD rats, weighing 200±20 g, were randomly divided into four groups: a blank control group, a model control group, a metronidazole gel positive control group (containing 0.5% metronidazole), and a uvangoletin gel treatment group (containing 0.5% uvangoletin), with 10 rats in each group. Except for the blank control group, experimental periodontitis models were established in all other groups using a combination of silk suture ligation and local inoculation with *Porphyromonas gingivalis*. The cervical region of the maxillary second molar was ligated with 5-0 silk suture, and simultaneously treated with *Porphyromonas gingivalis* (1×10⁻⁶ g)... 9 Sodium carboxymethyl cellulose gel (CFU / mL) was applied to the gingival sulcus area three times a week for four consecutive weeks to model the gingival sulcus. After successful modeling, the corresponding gel was applied topically to the gingiva in each treatment group twice daily for four consecutive weeks.

[0076] Evaluation indicators included: gingival index (GI) and bleeding on probing index (BOP) scores; micro-CT scanning analysis of alveolar bone height changes and bone mineral density parameters; HE staining to observe the degree of inflammatory cell infiltration, alveolar bone resorption, and periodontal ligament fiber arrangement in periodontal tissues; ELISA to detect the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in gingival crevicular fluid; and real-time quantitative PCR to detect the expression level of *Porphyromonas gingivalis* 16S rRNA gene in gingival tissue. The therapeutic effect of uvangoletin oral gel on experimental periodontitis was comprehensively verified through this multi-dimensional evaluation system.

[0077] Comprehensive mechanistic analysis: As a 2',6'-dihydroxy-4'-methoxydihydrochalcone compound, uvangoletin's molecular structure features an intramolecular hydrogen bond system formed by the 2'- and 6'-position phenolic hydroxyl groups on ring A. The strong intramolecular hydrogen bond between the 6'-position phenolic hydroxyl group and the adjacent carbonyl group (confirmed by a low-field singlet signal at δH 13.95) stabilizes the molecular planar conformation. The introduction of the 4'-methoxy group enhances the electron cloud density of ring A, facilitating hydrophobic interactions with the lipid bilayer on the bacterial cell membrane surface. Ring B is an unsubstituted phenyl group, maintaining the overall lipophilic balance of the molecule. This specific substitution pattern allows uvangoletin to effectively penetrate the outer membrane barrier of Gram-negative bacteria (Porphyromonas gingivalis and Fusobacterium nucleatum), interfering with cell membrane permeability and integrity, thereby inhibiting bacterial growth and reproduction. However, for Streptococcus mutans (Gram-positive bacteria), because its cell wall structure lacks a lipopolysaccharide outer membrane layer and has a thick peptidoglycan layer, the membrane penetration mechanism of uvangoletin cannot function effectively, and therefore it does not show inhibitory activity. This also explains the selective inhibition of uvangoletin against periodontitis-specific pathogens.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. The application of uvangoletin in the preparation of anti-periodontalgia drugs, wherein the chemical name of uvangoletin is 1-(2,4-dihydroxy-6-methoxyphenyl)-3-phenyl-1-propanone, and the molecular formula is C1. 16 H 16 O4 has a molecular weight of 272.

30.

2. The application according to claim 1, characterized in that, The anti-periodontalgia drug is a drug that inhibits Porphyromonas gingivalis and / or Fusobacterium nucleatum.

3. The application according to claim 1, characterized in that, The dosage form of the anti-periodontalgia drug is selected from one of the following: gel, mouthwash, toothpaste, lozenge, oral spray, oral patch, or oral sustained-release film.

4. The application according to claim 1, characterized in that, The mass concentration of uvangoletin in the anti-periodontalgia drug is 0.001%~5%.

5. The application according to claim 4, characterized in that, The mass concentration of uvangoletin in the anti-periodontalgia drug is 0.01%~1%.

6. The application according to claim 1, characterized in that, The uvangoletin is derived from the isolated and purified product or chemically synthesized product of the extract of the coral plant.

7. The application according to any one of claims 1 to 6, characterized in that, The anti-periodontalgia drug also contains pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, Pharmaceutically acceptable excipients include one or more of the following: humectants, thickeners, preservatives, flavoring agents, and pH adjusters.

9. The use of an oral pharmaceutical composition in the preparation of a drug for treating periodontitis, characterized in that, The oral medicine composition contains uvangoletin as an active ingredient.

10. The application according to claim 9, characterized in that, The oral medicine composition contains uvangoletin at a content of 0.001% to 5% of the total mass of the composition, and the oral medicine composition also contains pharmaceutically acceptable excipients.