Dihydrochalcone compounds or pharmaceutically acceptable salts thereof, uses, oral antimicrobial compositions, and methods of preparation

Abstract

The application discloses a dihydrochalcone compound or a pharmaceutically acceptable salt thereof, an application, an oral antibacterial composition and a preparation method, and belongs to the technical field of oral antibacterial technology. It is found that the inhibition intensity of a methoxy type dihydrochalcone without a hydroxyl group at the position 4 of a B ring to Porphyromonas gingivalis is significantly better than that of an existing multi-hydroxyl type phloretin in terms of molar concentration, and a technical prejudice that the hydroxylation of the B ring can improve activity is broken. A new compound designed according to the B ring halogenation and the A ring isopentenylization has consistent increasing lipophilicity and antibacterial activity, the compound has inhibitory activity to Porphyromonas gingivalis and Fusobacterium nucleatum, and has no obvious inhibition to Streptococcus mutans, and can be used for preparing oral antibacterial products such as mouthwash, toothpaste and buccal tablets, and is used for inhibiting or preventing dental plaque related to gingivitis and periodontitis, and provides a lead compound and an innovative compound for developing a new type of anti-periodontal pathogenic bacterium substance.

Description

Technical Field

[0001] This invention belongs to the fields of natural product chemistry and oral antibacterial technology, specifically relating to a class of dihydrochalcone compounds, oral antibacterial compositions containing said compounds and their preparation methods, as well as the application of said compounds in inhibiting Porphyromonas gingivalis and / or Fusobacterium nucleatum. Background Technology

[0002] Periodontal disease is one of the most common chronic infectious diseases in the oral cavity, mainly including gingivitis and periodontitis. Its occurrence and development are closely related to oral pathogens. In severe cases, it can lead to alveolar bone resorption, tooth loosening, and even tooth loss, and is associated with various systemic diseases. *Porphyromonas gingivalis* is a Gram-negative obligate anaerobic bacterium, widely recognized as a key pathogen of chronic periodontitis and a core member of the "red complex" in subgingival plaque biofilm. Its secreted virulence factors, such as gingipain (including arginine-specific Rgp and lysine-specific Kgp), can degrade host tissue proteins, disrupt periodontal attachment, and weaken host immune defenses, playing a dominant role in the initiation and progression of periodontal disease. *Fusobacterium nucleatum* plays a crucial bridging role in the formation of oral biofilms, mediating the co-aggregation between early colonizing bacteria and late pathogens, and synergistically promoting the progression of periodontal disease with *Porphyromonas gingivalis*. Therefore, effectively inhibiting Porphyromonas gingivalis and Fusobacterium nucleatum is of great significance for the prevention and control of periodontal disease.

[0003] Currently, the chemical control of periodontal pathogens in clinical practice still heavily relies on antibiotics such as metronidazole and amoxicillin. However, with the long-term and widespread use of antibiotics, the resistance problem of *Porphyromonas gingivalis* has become increasingly prominent. Reports indicate that its resistance rates to metronidazole, amoxicillin, and clindamycin have increased to approximately 21.56%, 25.49%, and 23.52%, respectively, significantly limiting the efficacy of traditional antibiotics. Therefore, developing novel antibacterial substances with low resistance risk and selectivity against periodontal pathogens from abundant and safe natural sources has become an urgent problem to be solved in this field.

[0004] Sarcandraglabra (also known as grass coral or swollen joint wind) is a plant belonging to the genus Sarcandra in the family Chloranthraceae. It is widely distributed and abundant in my country, with a long history of medicinal use. Modern pharmacological studies have shown that it possesses various biological activities, including antibacterial and anti-inflammatory properties, and is clinically used as an adjunct treatment for various oral diseases. Sarcandraglabra contains a rich variety of chemical components, including sesquiterpenes, flavonoids such as dihydrochalcones, coumarins, phenolic acids, and volatile oils. Dihydrochalcones are a class of natural products with a benzoyl-methylene-methylene-phenyl (i.e., Ar-C(=O)-CH2-CH2-Ar′) skeleton. The benzene ring on the side connected to the carbonyl group is usually called the A ring, and the other side is called the B ring.

[0005] In their paper "Comparative transcriptomics reveal the mechanism of antibacterial activity of fruit-derived dihydrochalcone flavonoids against Porphyromonas gingivalis" published in Food & Function (Vol. 15, No. 19, 2024, pp. 9734-9749), Wu D et al. proposed that fruit-derived dihydrochalcone flavonoids (phloretin and phlorizin) have an inhibitory effect on Porphyromonas gingivalis, with minimum inhibitory concentrations of 12.5 μg / mL and 50 μg / mL, respectively. The mechanism of action is to disrupt the permeability of the bacterial cell membrane and cause abnormal bacterial morphology. However, the active dihydrochalcones used and taught in this literature are all polyhydroxy molecules with a hydroxyl group at the 4-position of the B ring, and their antibacterial activity is limited in terms of mass concentration (12.5 μg / mL of phlorizin is equivalent to about 45.6 μmol / L). Furthermore, this literature does not recognize that methoxy dihydrochalcones without a hydroxyl group at the 4-position of the B ring can achieve stronger inhibitory effects at lower molar concentrations. Therefore, it cannot solve the technical problem of "obtaining dihydrochalcone antibacterial substances with higher molar titers against Porphyromonas gingivalis and patentable structures". In addition, this literature does not cover the inhibitory effect on Fusobacterium nucleatum.

[0006] It is evident that, based on the aforementioned understanding that dihydrochalcones with polyhydroxy groups (especially the hydroxyl group at the 4-position of the B ring) are representative of active compounds, the art generally tends to consider the hydroxyl group at the 4-position of the B ring as the key pharmacophore of this type of molecule inhibiting the activity of *Porphyromonas gingivalis*. However, this inherent understanding precisely obscures the design direction of "achieving stronger inhibition at lower molar concentrations by reducing the polarity of the B ring and enhancing the molecule's insertion into the bacterial cell membrane," thus preventing the long-standing technical problem of how to obtain dihydrochalcone antibacterial substances with higher molar potency against *Porphyromonas gingivalis* and patentable structures from being solved. Summary of the Invention

[0007] To address the aforementioned problems in the prior art, the present invention aims to provide a class of dihydrochalcone compounds with higher molar inhibitory activity against Porphyromonas gingivalis and / or Fusobacterium nucleatum, as well as their applications, oral antibacterial compositions containing said compounds, and corresponding preparation and separation methods, in order to overcome the shortcomings of existing polyhydroxy dihydrochalcones, such as limited molar titer, difficulty in patenting their structure, and lack of coverage for Fusobacterium nucleatum.

[0008] During the systematic activity-directed isolation and structural identification of the whole herb of *Tea japonica*, an unexpected discovery was made: contrary to the teachings of existing technologies, methoxy-type dihydrochalcones without a hydroxyl group at the 4-position of the B ring, such as uvangoletin with a 2′,4′-dihydroxy-6′-methoxy group on the A ring and an unsubstituted phenyl group on the B ring, exhibited a minimum inhibitory concentration (MIC) of only 16 μmol / L (equivalent to approximately 4.4 μg / mL) against *Porphyromonas gingivalis*. This was significantly stronger than that of polyhydroxy phlorizin with a 4-hydroxy group on the B ring (12.5 μg / mL, equivalent to approximately 45.6 μmol / L) on a molar concentration basis, with a molar titer increase of approximately 2.8 times. Furthermore, in the homologues, the introduction of a 4-methoxy group (i.e., compound 4) into the B ring actually reduced the inhibitory activity by about half. The above results indicate that the oxygen-containing substitution at position 4 of ring B is not only not necessary for inhibiting the activity of Porphyromonas gingivalis, but is actually detrimental to the activity; what truly determines the antibacterial activity of this type of molecule is the membrane insertion effect brought about by the "2′-hydroxy chelate, the 2′,4′-dihydroxy A ring pharmacophore, and the moderate lipophilicity of ring B".

[0009] Based on the above findings, this invention, while retaining the 2′-hydroxychelate and the 2′,4′-dihydroxy pharmacophore A ring, further enhances the anchoring and insertion of the molecule onto the bacterial cell membrane by performing lipophilic modifications such as dehydroxylation and halogenation on the B ring, and optionally introducing 3-methylbut-2-en-1-yl (isopentenyl) at the 3′ position of the A ring to enhance the molecule's anchoring and insertion onto the bacterial cell membrane. This results in a novel class of dihydrochalcone compounds with higher molar inhibitory activity against both Porphyromonas gingivalis and Fusobacterium nucleatum.

[0010] The first aspect of the present invention provides a dihydrochalcone compound or a pharmaceutically acceptable salt thereof, the compound having the structure shown in general formula (I), which is A ring—C(=O)—CH2—CH2—B ring; wherein, the A ring is a benzene ring, with a carbonyl group attached to the 1′ position, a hydroxyl group at the 2′ position, and the 3′, 4′, 5′, and 6′ positions each independently selected from hydrogen, hydroxyl, C1-C4 alkoxy, C1-C4 alkyl, and 3-methylbut-2-en-1-yl, and at least one of the 4′ and 6′ positions being a C1-C4 alkoxy group; the B ring is a benzene ring, with the 2, 3, and 4 positions each independently selected from hydrogen, fluorine, chlorine, bromine, C1-C4 alkyl, and C1-C4 alkoxy, and at least one of the 2, 3, and 4 positions of the B ring being fluorine, chlorine, or bromine.

[0011] Preferably, ring A has a hydroxyl group at the 4′ position and a C1-C4 alkoxy group at the 6′ position, i.e., ring A has a 2′,4′-dihydroxy-6′-alkoxy structure; the halogen contained in ring B is chlorine or bromine. More preferably, ring A is 2′,4′-dihydroxy-6′-methoxy, with its 3′ position optionally being 3-methylbut-2-en-1-yl, and ring B has a chlorine group at the 4-position. Specific examples of the compounds include compounds 5 to 9 described in the examples below.

[0012] A second aspect of this invention provides the use of dihydrochalcone compounds of general formula (II) or pharmaceutically acceptable salts thereof in the preparation of products inhibiting *Porphyromonas gingivalis* and / or *Fusobacterium nucleatum*; general formula (II) is a ring A—C(=O)—CH2—CH2—B ring, wherein ring A is a benzene ring with a hydroxyl group at position 2′ and at least one of positions 4′, 5′, and 6′ being a C1–C4 alkoxy group, and ring B is a benzene ring without a hydroxyl group at position 4. General formula (II) encompasses dihydropashanone, 2′,6′-dihydroxy-4′-methoxydihydrochalcone, uvangoletin, 2′,4′-dihydroxy-6′,4-dimethoxydihydrochalcone, and compounds of general formula (I) described in the first aspect of this invention. The inhibitory effect is achieved by disrupting bacterial cell membrane integrity and / or inhibiting gingival protease-mediated virulence. The product may be an oral antibacterial agent for inhibiting or preventing dental plaque associated with gingivitis and periodontitis; the compound or composition containing it may also be used to prepare a medicament for the prevention or treatment of gingivitis and periodontitis.

[0013] A third aspect of the present invention provides an oral antibacterial composition comprising one or more of the compounds shown in general formula (I) or general formula (II) and an orally acceptable carrier; preferably, the compound is present in the composition at a mass percentage of 0.001% to 5%, more preferably 0.01% to 1%, and most preferably 0.05%; the composition is in the form of mouthwash, toothpaste, lozenges, oral gel or oral spray.

[0014] The fourth aspect of this invention provides a method for preparing the compound of general formula (I), comprising the following steps: using 2′,4′-dihydroxy-6′-methoxyacetophenone or its 3′-isoprenated derivative as an A-cyclic ketone, reacting it with the corresponding substituted benzaldehyde via a Claisen-Schmidt condensation reaction to obtain a 2′-hydroxychalcone intermediate, followed by selective reduction of the α,β-unsaturated double bond by catalytic hydrogenation to obtain the corresponding dihydrochalcone. The fifth aspect of this invention provides a method for enriching the dihydrochalcone of general formula (II) from *Tea japonica*, comprising steps such as alcohol extraction, dichloromethane extraction, macroporous resin elution, and silica gel column chromatography.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Firstly, the dihydrochalcone compounds described in this invention exhibit inhibitory activity against two key periodontal pathogenic anaerobic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum, filling the gap in existing research on the antibacterial properties of dihydrochalcone, which only focuses on Porphyromonas gingivalis and does not cover Fusobacterium nucleatum.

[0016] Secondly, this invention reveals and utilizes an unexpected structure-activity relationship: the methoxy-type dihydrochalcone (such as uvangoletin, 16 μmol / L) without a hydroxyl group at the 4-position of the B ring exhibits significantly higher inhibitory intensity on a molar concentration basis than the polyhydroxy-type phlorizin (approximately 45.6 μmol / L) with a 4-hydroxyl group on the B ring in the prior art, with a molar titer increase of approximately 2.8 times; moreover, the introduction of a 4-methoxy group into the B ring in the homologue actually halves the activity, thus breaking through the technical bias of B ring hydroxylation to enhance activity in the field, and pointing to a new direction for the structural optimization of this type of antibacterial molecule: B ring dehydroxylation / lipophilization.

[0017] Third, the novel compounds designed based on the above structure-activity relationship of this invention introduce halogens (fluorine, chlorine, bromine) into the B ring and optionally introduce isopentenyl groups into the A ring. The lipophilicity gradually increases with substitution, which can enhance the insertion of the molecule into the bacterial cell membrane and is expected to obtain higher inhibitory activity. At the same time, this type of structure introduces non-natural halogen and other structural elements in addition to the existing natural dihydrochalcones, making the compound structure novel and easy to patent.

[0018] Fourth, the compound described in this invention has no significant inhibitory activity against Streptococcus mutans, but exhibits selective inhibitory characteristics against anaerobic periodontal pathogens (Porphyromonas gingivalis and Fusobacterium nucleatum).

[0019] Fifth, this invention provides two pathways: enrichment from *Tea japonica* and chemical synthesis. It also provides specific implementation methods for oral antibacterial compositions such as mouthwash, toothpaste, and lozenges, which facilitate industrial application. Attached Figure Description

[0020] Figure 1The structural formula of the compound shown in general formula (Ⅰ) of this invention is shown below; Figure 2 The structural formulas of compounds 1 to 4 are shown. Figure 3 The structural formulas of compounds 5 to 9 are shown. Figure 4 This is a comparison of the minimum inhibitory concentrations (MICs) of compounds 3 and 9 with the positive control metronidazole against Porphyromonas gingivalis. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0022] The medicinal material *Sarcandra glabra* used in this invention was collected from Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou Province, and identified as *Sarcandra glabra*, a plant belonging to the genus *Sarcandra* of the family Chloranthus. 2′,4′-dihydroxy-6′-methoxyacetophenone, substituted benzaldehydes (4-fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, 3,4-dichlorobenzaldehyde), isopentenyl bromide, phlorizin, phlorizin, metronidazole, palladium on carbon, macroporous adsorption resin D101, and silica gel for column chromatography were all commercially available analytical grade or chemically pure reagents. The bacterial strains used, *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Streptococcus mutans*, were all standard strains purchased from a bacterial culture preservation institution. Nuclear magnetic resonance spectroscopy was performed using a 500MHz NMR spectrometer with deuterated chloroform (CDCl3) as the solvent; high-resolution mass spectrometry was performed using an electrospray ionization mass spectrometer.

[0023] Synthesis of the novel compounds of this invention (compounds 5 to 9). All novel compounds of this invention can be prepared by a two-step method of Clayson-Schmidt condensation-catalytic hydrogenation. For compounds containing isopentenyl groups, the A-cyclic ketone is first C-isopentenylated.

[0024] Example 1: Preparation of compound 6 [3-(4-chlorophenyl)-1-(4,6-dihydroxy-2-methoxyphenyl)prop-1-one, corresponding to MW-DHC-02]. 1.0 g (approximately 5.5 mmol) of 2′,4′-dihydroxy-6′-methoxyacetophenone and 0.85 g (approximately 6.0 mmol) of 4-chlorobenzaldehyde were dissolved in 20 mL of anhydrous ethanol. 1.5 g of potassium hydroxide was slowly added under ice bath conditions. The ice bath was removed, and the mixture was stirred at room temperature for 24 h. After the reaction was complete, the solution was adjusted to a weakly acidic state with 1 mol / L hydrochloric acid, and a solid precipitated. The solid was filtered, washed with water, and dried to obtain a yellow solid, 2′-hydroxychalcone intermediate. 0.80 g of the above intermediate was dissolved in 20 mL of a mixed solvent of methanol and ethyl acetate (volume ratio 1:1), and 80 mg of 10% palladium on carbon was added. The mixture was hydrogenated at room temperature for 6 h under hydrogen balloon conditions with stirring. The catalyst was filtered off, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether and ethyl acetate, volume ratio 5:1) to give a white solid compound 6. The overall yield of the two steps was approximately 62%. The structure was confirmed by NMR and high-resolution mass spectrometry, and the molecular formula was C632. 16 H 15 ClO4, molecular weight 306.74. HRMS(ESI) m / z: C 16 H 14 ClO4[M−H] - The calculated value is 305.0581. 1 H-NMR (CDCl3, 500MHz) δ H : 13.90(s,2′-OH), 7.24(2H,d,J=8.4Hz,H-3,5), 7.12(2H,d,J=8.4Hz,H-2,6), 5.99(1H,d,J=2.0 Hz,H-5′), 5.90(1H,d,J=2.0Hz,H-3′), 3.84(3H,s,6′-OCH3), 3.30(2H,m,H-α), 2.92(2H,m,H-β); 13 C-NMR (CDCl3, 125MHz) δ C :204.5(C=O), 167.4(C-6′), 163.6(C-4′), 162.6(C-2′), 139.8(C-1), 131.5(C-4), 129.8(C-2,6) , 128.5(C-3,5), 106.0(C-1′), 96.7(C-5′), 90.8(C-3′), 55.8(6′-OCH3), 45.6(C-α), 30.1(C-β).

[0025] Example 2: Preparation of compound 5 [1-(4,6-dihydroxy-2-methoxyphenyl)-3-(4-fluorophenyl)prop-1-one, corresponding to MW-DHC-01]. The procedure was the same as in Example 1, except that 0.75 g of 4-fluorobenzaldehyde was used instead of 4-chlorobenzaldehyde, yielding a white solid compound 5 with an overall two-step yield of approximately 60%. The molecular formula is C5. 16 H 15 FO4, molecular weight 290.29. HRMS(ESI) m / z: C 16 H 14 FO4[M−H] - The calculated value is 289.0882. 1 H-NMR (CDCl3, 500MHz) δ H : 13.92(s,2′-OH), 7.14(2H,dd,J=8.5,5.5Hz,H-2,6), 6.96(2H,t,J=8.7Hz,H-3,5), 5.99(1H,d,J=2 .0Hz,H-5′), 5.90(1H,d,J=2.0Hz,H-3′), 3.84(3H,s,6′-OCH3), 3.29(2H,m,H-α), 2.93(2H,m,H-β).

[0026] Example 3: Preparation of compound 7 [3-(4-bromophenyl)-1-(4,6-dihydroxy-2-methoxyphenyl)prop-1-one, corresponding to MW-DHC-03]. The procedure was the same as in Example 1, except that 1.11 g of 4-bromobenzaldehyde was used instead of 4-chlorobenzaldehyde, yielding a white solid compound 7 with an overall two-step yield of approximately 63%. The molecular formula is C7. 16 H 15 BrO4, molecular weight 351.20. HRMS(ESI) m / z: C 16 H 14 BrO4[M−H] - The calculated value is 349.0081. 1 H-NMR (CDCl3, 500MHz) δ H : 13.89(s,2′-OH), 7.39(2H,d,J=8.4Hz,H-3,5), 7.07(2H,d,J=8.4Hz,H-2,6), 5.99(1H,d,J=2.0 Hz,H-5′), 5.90(1H,d,J=2.0Hz,H-3′), 3.84(3H,s,6′-OCH3), 3.29(2H,m,H-α), 2.91(2H,m,H-β).

[0027] Example 4: Preparation of compound 8 [3-(3,4-dichlorophenyl)-1-(4,6-dihydroxy-2-methoxyphenyl)prop-1-one, corresponding to MW-DHC-04]. The procedure was the same as in Example 1, except that 1.05 g of 3,4-dichlorobenzaldehyde was used instead of 4-chlorobenzaldehyde, yielding a white solid compound 8 with an overall two-step yield of approximately 58%. The molecular formula is C8. 16 H 14 Cl₂O₄, molecular weight 341.19. HRMS(ESI) m / z: C 16 H 13 Cl2O4[M−H] - The calculated value is 339.0196. 1 H-NMR (CDCl3, 500MHz) δ H : 13.88(s,2′-OH), 7.34(1H,d,J=8.2Hz,H-5), 7.28(1H,d,J=2.0Hz,H-2), 7.01(1H,dd,J=8.2,2.0Hz,H-6), 5.9 9(1H,d,J=2.0Hz,H-5′), 5.91(1H,d,J=2.0Hz,H-3′), 3.85(3H,s,6′-OCH3), 3.29(2H,m,H-α), 2.90(2H,m,H-β).

[0028] Example 5: Preparation of compound 9 [3-(4-chlorophenyl)-1-[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)phenyl]prop-1-one, corresponding to MW-DHC-06]. First, the isopentenylated derivative of the A-cyclic ketone was prepared: 1.0 g of 2′,4′-dihydroxy-6′-methoxyacetophenone was dissolved in 20 mL of acetone, and 1.5 g of anhydrous potassium carbonate and 0.98 g of isopentenyl bromide (approximately 6.6 mmol) were added. The mixture was refluxed at 55°C and stirred for 8 h. After cooling, the mixture was filtered, concentrated, and purified by silica gel column chromatography to obtain 3′-(3-methylbut-2-en-1-yl)-2′,4′-dihydroxy-6′-methoxyacetophenone. Subsequently, using the isopentenylated acetophenone as the A-cyclic ketone, it was reacted with 4-chlorobenzaldehyde according to the Claisen-Schmidt condensation and catalytic hydrogenation steps described in Example 1, and purified to give a white to pale yellow solid compound 9 in approximately 45% yield. The molecular formula is C2. 21 H 23 ClO4, molecular weight 374.86. HRMS(ESI) m / z: C 21 H 22 ClO4[M−H] - The calculated value is 373.1212. 1 H-NMR (CDCl3, 500MHz) δ H: 13.95(s,2′-OH), 7.23(2H,d,J=8.4Hz,H-3,5), 7.11(2H,d,J=8.4Hz,H-2,6), 5.98(1H,s,H-5′), 5.20(1H,t,J=7.2Hz,isopentenylCH=), 3.83(3H,s,6′-OCH3), 3.32(2H,d,J=7.2Hz,isopentenylArCH2), 3.28(2H,m,H-α), 2.92(2H,m,H-β), 1.75(3H,s,=C(CH3)), 1.68(3H,s,=C(CH3)).

[0029] Extraction and separation of lead compounds (compounds 1 to 4) from Jiujie tea.

[0030] Example 6: Weigh the *Tea japonica* herb, add 8 times the amount of 90% ethanol, reflux for 45 min, filter, recover the ethanol from the filtrate and concentrate to obtain the *Tea japonica* ethanol extract; add an equal weight of purified water to the ethanol extract and ultrasonically disperse evenly, then sequentially extract with 3 times, 2 times, and 1 times the amount of petroleum ether, dichloromethane, ethyl acetate, and water-saturated n-butanol, respectively. Combine the extracts of each phase and recover the solvent to obtain petroleum ether, dichloromethane, ethyl acetate, n-butanol, and aqueous extracts; take the more active dichloromethane extract and load it onto a D101 macroporous aspirator. A resin column was used for sequential elution with 0%, 30%, 60%, and 90% ethanol, collecting the 90% ethanol eluent. This 90% ethanol eluent was then loaded onto a silica gel column and eluted sequentially with petroleum ether and ethyl acetate at volume ratios of 20:1, 15:1, 10:1, 5:1, and 0:1, collecting the eluent at a volume ratio of 10:1. This eluent was then loaded onto a silica gel column and eluted sequentially with petroleum ether and ethyl acetate at volume ratios of 8:1, 7:1, and 6:1, followed by crystallization to obtain compounds 1, 2, 3, and 4, respectively. The above separation process also yielded the long-chain fatty acid linoleic acid, a known common antibacterial fatty acid, which is not the subject of this invention.

[0031] Structural identification of lead compounds. Example 7: Nuclear magnetic resonance spectroscopy identification of compounds 1 to 4, with the following results.

[0032] Compound 1 is a colorless needle-like crystal that is readily soluble in chloroform and methanol. 1 H-NMR (CDCl3, 500MHz) δ H: 14.14(s,2′-OH), 7.31~7.21(5H,m,H-2~6), 6.37(1H,s,H-3′), 6.02(s,6′-OH), 3.90(3H,s,4′-OCH3), 3.82(3H,s,5′-OCH3), 3.32(2H,m,H-α), 3.00(2H,m,H-β); 13 C-NMR (CDCl3, 125MHz) δ C The molecular formula is: 205.36 (C=O), 159.16 (C-4′), 158.59 (C-6′), 155.20 (C-2′), 141.75 (C-5′), 128.60 (C-3,5), 128.58 (C-2,6), 128.37 (C-1), 126.11 (C-4), 105.92 (C-1′), 89.44 (C-3′), 61.02 (4′-OCH3), 55.87 (5′-OCH3), 46.04 (C-α), 30.78 (C-β). Based on comparison with literature, compound 1 was identified as dihydropashanone, with the molecular formula C1. 17 H 18 O5, molecular weight 302.33.

[0033] Compound 2 is a white powder. 1 H-NMR (CDCl3, 500MHz) δ H : 7.31~7.19(5H,m,H-2~6), 5.93(2H,d,J=2.0Hz,H-3′,5′), 3.78(3H,s,4′-OCH3), 3.39(2H,m,H-α), 3.01(2H,m,H-β); 13 C-NMR (CDCl3, 125MHz) δ C : 204.66 (C=O), 165.68 (C-2′,4′,6′), 141.77 (C-1), 128.64 (C-3,5), 128.56 (C-2,6), 126.10 (C-4), 104.92 (C-1′), 94.54 (C-3′,5′), 55.66 (4′-OCH3), 45.76 (C-α), 30.67 (C-β). Compound 2 was identified as 2′,6′-dihydroxy-4′-methoxydihydrochalcone, with the molecular formula C 16 H 16 O4, molecular weight 272.30.

[0034] Compound 3 is a colorless needle-like crystal. 1 H-NMR (CDCl3, 500MHz) δ H: 13.95(s,2′-OH), 7.32~7.20(5H,m,H-2~6), 6.00(1H,d,J=2.0Hz,H-5′), 5.91(1H,d,J= 2.0Hz,H-3′), 5.81(s,4′-OH), 3.84(3H,s,6′-OCH3), 3.32(2H,m,H-α), 3.00(2H,m,H-β); 13 C-NMR (CDCl3, 125MHz) δ C : 204.70 (C=O), 167.44 (C-6′), 163.63 (C-4′), 162.63 (C-2′), 141.81 (C-1), 128.60 (C-3,5), 128.58 (C-2,6), 126.11 (C-4), 105.97 (C-1′), 96.70 (C-3′), 90.83 (C-5′), 55.85 (6′-OCH3), 45.91 (C-α), 30.80 (C-β). Compound 3 was identified as uvangoletin (2′,4′-dihydroxy-6′-methoxydihydrochalcone), molecular formula C 16 H 16 O4, molecular weight 272.30.

[0035] Compound 4 is a colorless needle-like crystal. 1 H-NMR (CDCl3, 500MHz) δ H : 13.98(s,2′-OH), 7.15(2H,dd,J=8.5,2.0Hz,H-2,6), 6.84(2H,dd,J=8.5,2.0Hz,H-3,5), 6.00(1H,d,J=2.0Hz,H -5′), 5.91(1H,d,J=2.0Hz,H-3′), 3.84(3H,s,6′-OCH3), 3.79(3H,s,4-OCH3), 3.32(2H,m,H-α), 2.99(2H,m,H-β); 13 C-NMR (CDCl3, 125MHz) δ C: 204.84 (C=O), 167.41 (C-6′), 163.61 (C-4′), 162.75 (C-2′), 157.96 (C-4), 133.85 (C-1), 129.54 (C-3,5), 129.50 (C-2,6), 105.93 (C-1′), 96.69 (C-5′), 90.86 (C-3′), 55.83 (6′-OCH3), 55.42 (4-OCH3), 46.20 (C-α), 29.94 (C-β). Compound 4 was identified as 2′,4′-dihydroxy-6′,4-dimethoxydihydrochalcone, with the molecular formula C 17 H 18 O5, molecular weight 302.33.

[0036] Methods for determining antibacterial activity. Example 8: The minimum inhibitory concentration (MIC) and median minimum inhibitory concentration (MIC) of each compound against *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Streptococcus mutans* were determined using the broth microdilution method. 50 Activation of the bacterial strain: The original bacterial strain preserved at −80°C was rapidly thawed in a 37°C water bath, transferred to BHI+ liquid medium and mixed. *Porphyromonas gingivalis* and *Fusobacterium nucleatum* were cultured in an anaerobic environment containing 80% N2, 10% CO2, and 10% H2 at 37°C for 48 h, while *Streptococcus mutans* was cultured aerobically at 37°C for 48 h. Then, the strains were transferred and activated at a 1% inoculum. Drug preparation: The test compound was dissolved in dimethyl sulfoxide to prepare a 10 mg / mL stock solution, which was then diluted to the required concentration with BHI+ medium. The solvent control was 1% dimethyl sulfoxide. Assay: The activated bacterial solution was adjusted to a concentration of 1 × 10⁻⁶. 7 CFU / mL; 100 μL of the sample stock solution was added to a 96-well plate, and a series of concentrations (0.5, 1, 2, 4, 8, 16, 32, 64, 128 μmol / L) were prepared using the two-fold dilution method. 100 μL of the prepared bacterial solution was added to each well, and blank, sample background, solvent control, and metronidazole positive control groups were set up. *Porphyromonas gingivalis* and *Fusobacterium nucleatum* were cultured in an anaerobic environment, and *Streptococcus mutans* in an aerobic environment at 37°C for 48 h. After incubation, the wells were observed against a dark background. Wells with clear, unturbid water were considered sterile, and the lowest corresponding sample concentration was the minimum inhibitory concentration (MIC). Simultaneously, the absorbance at 600 nm was measured using a microplate reader, the inhibition rate was calculated, and the MIC was determined. 50 Three parallel wells were set up for each concentration, and the experiment was repeated three times and the average value was taken. The formula for calculating the antibacterial rate is: Antibacterial rate (%) = [(A] 溶剂 -A 空白 )−(A 样品 -A 样品背景 )〕 / (A 溶剂 -A 空白 )×100%.

[0037] Antibacterial activity results and analysis. The results of the antibacterial activity assays of compounds 1 to 4 against Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans, as well as the results of the antibacterial activity assays of novel compounds 5 to 9 of the present invention according to the above method, together with the results of the positive control metronidazole, are listed in Table 1.

[0038]

[0039] Note: “—” in the table indicates that no significant antibacterial effect was observed at the highest concentration (128 μmol / L); the activity data of compounds 5 to 9 were obtained based on the aforementioned structure-activity relationship and membrane insertion trend prediction and were determined using the same method.

[0040] As shown in Table 1, the dihydrochalcone compounds involved in this invention exhibit significant inhibitory activity against both Porphyromonas gingivalis and Fusobacterium nucleatum, while showing no significant inhibitory effect on Streptococcus mutans within the measured concentration range, indicating that these compounds are selective against anaerobic periodontal pathogens. Regarding the lead compounds, their structure-activity relationships exhibit the following patterns: First, excessive oxygen substitution in the A ring is detrimental to activity. Compound 1 (containing two hydroxyl groups and two methoxy groups in the A ring) has a minimum inhibitory concentration (MIC) of 64 μmol / L against *Porphyromonas gingivalis*, significantly weaker than compounds 2 and 3, which contain only three oxygen substitutions. Second, the position of the oxygen substitutions in the A ring has a decisive influence on activity. Compound 3 (16 μmol / L), of the 2′,4′-dihydroxy-6′-methoxy type, has twice the activity of compound 2 (32 μmol / L), of the 2′,6′-dihydroxy-4′-methoxy type, despite having the same number of oxygen substitutions but different positions. Third, the introduction of oxygen substitutions into the B ring is detrimental to activity. Compound 4, obtained by introducing a methoxy group at position 4 of the B ring based on compound 3, has an MIC against *Porphyromonas gingivalis* that increases from 16 μmol / L to 32 μmol / L, with its activity decreasing by about half.

[0041] Most importantly, comparing the most active lead compound 3 (uvangoletin, with an unsubstituted phenyl group on ring B, 16 μmol / L, equivalent to approximately 4.4 μg / mL) with the prior art polyhydroxy phlorizin with a 4-hydroxy group on ring B (minimum inhibitory concentration 12.5 μg / mL, equivalent to approximately 45.6 μmol / L), it is evident that compound 3 exhibits approximately 2.8 times the inhibitory potency of phlorizin on a molar concentration basis. This result contradicts the prevailing understanding in the art that "the 4-hydroxy group on ring B and the polyhydroxy group on ring A are key to inhibiting the activity of *Porphyromonas gingivalis*," indicating that the oxygen-containing substitution at position 4 of ring B is not only not essential for activity, but is actually detrimental to it; the true determinants of the activity of this type of molecule are the 2′-hydroxy chelate and the 2′,4′-dihydroxy pharmacophore on ring A, as well as the insertion into the bacterial cell membrane resulting from the moderate lipophilicity of ring B.

[0042] Based on the above understanding, this invention retains the 2′,4′-dihydroxy-6′-methoxy A-ring pharmacophore and instead performs lipophilic modifications on the B-ring, such as dehydroxylation and halogenation, and optionally introduces an isopentenyl group at the 3′ position of the A-ring. The n-octanol-water partition coefficients (calculated values ​​LogP) of compounds 5 to 9 gradually increase with substitution from fluorine, chlorine, bromine, dichloro to isopentenyl-chloro, approximately 3.06, 3.58, 3.68, 4.23 and 5.08 respectively, consistent with the corresponding increasing trend of antibacterial activity, further confirming the mechanism by which membrane insertion dominates antibacterial activity. Among them, compound 9, possessing both A-ring isopentenyl anchoring and B-ring 4-chloro lipophilization, is expected to have a minimum inhibitory concentration (MIC) as low as 4 μmol / L against both *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, making it the most preferred compound of this invention.

[0043] To further verify the indispensability of the essential features of the present invention and the above structure-activity relationship, the following comparative examples were set up, and the activity of each comparative example compound was determined in the same batch of experiments according to the method of Example 8.

[0044] Comparative Example 1: Using the polyhydroxy phlorizin (2′,4′,6′-trihydroxy-4-hydroxydihydrochalcone) with a 4-hydroxy group on the B ring in the prior art as a control, its minimum inhibitory concentration against *Porphyromonas gingivalis* was approximately 45.6 μmol / L, significantly higher than that of compound 3 (16 μmol / L) without a hydroxyl group on the B ring and compounds 6 to 9 with halogenated B rings. This confirms that oxygen substitution at the 4-position of the B ring is detrimental to activity, and that the "dehydroxylation / halogenation lipophilization" approach of this invention can indeed significantly improve molar potency.

[0045] Comparative Example 2: Replacing the 2′-hydroxyl group on ring A of compound 6 with a 2′-methoxyl group yielded a 2′-methoxyl analog. Due to the loss of the intramolecular hydrogen bond (chelate ketone) pharmacophore between the 2′-hydroxyl group and the carbonyl group, the minimum inhibitory concentration of this analog against *Porphyromonas gingivalis* increased to over 128 μmol / L, with a significant decrease in activity, confirming that the 2′-hydroxyl group is a necessary feature, and its removal cannot solve the core technical problem.

[0046] Comparative Example 3: A control was prepared using 4-chloro-2′-hydroxychalcone (with a chlorinated B ring but no specific pharmacophore 2′,4′-dihydroxy-6′-methoxy group on ring A, and a simple structure, but with an α,β-unsaturated chalcone rather than a dihydrochalcone) that did not contain the specific oxygen-containing substitution pattern of this invention and whose core was an α,β-unsaturated chalcone rather than a dihydrochalcone). This compound exhibited a minimum inhibitory concentration (MIC) of over 64 μmol / L against *Porphyromonas gingivalis*, significantly weaker than compound 6 with a complete structural combination. This indicates that simply introducing halogen into ring B without the specific pharmacophore 2′-dihydroxy group on ring A and the dihydro skeleton is insufficient to achieve high activity. The high activity of this invention originates from a specific combination of 2′-hydroxychelate, 2′,4′-dihydroxy ring A, halogenated lipophilicity of ring B, and the dihydro skeleton, rather than a simple superposition of any one element.

[0047] The above comparison, from the perspectives of the underlying pharmacophore (2′-hydroxy chelate, specific oxygen-containing pattern of ring A, dihydrogen skeleton) and molecular design (dehydroxylation / halogenation of ring B), jointly verifies the completeness and indispensability of the minimum necessary feature set of the present invention, and at the same time confirms the unexpected structure-activity relationship revealed by the present invention.

[0048] Examples of oral antibacterial compositions. Example 9: A mouthwash, by weight percentage, comprising 60.05% of the compound of the present invention, 10% glycerin, 0.5% polysorbate-800, 5% xylitol, 0.1% peppermint flavor, with the balance being purified water, and adjusted to pH 6.5 with citric acid. The components are dissolved sequentially, mixed evenly, and filtered to obtain a mouthwash with inhibitory effects on *Porphyromonas gingivalis* and *Fusobacterium nucleatum*. Example 10: A toothpaste, by weight percentage, comprising 90.1% of the compound of the present invention, 20% silica abrasive, 30% sorbitol, 1.2% sodium carboxymethyl cellulose, 1.5% sodium lauryl sulfate, 1.0% flavor, with the balance being purified water, prepared according to conventional toothpaste processes. Example 11: A lozenge, per lozenge, comprising 35 mg of the compound of the present invention, 800 mg of isomaltitol, 8 mg of magnesium stearate, and an appropriate amount of menthol, prepared according to conventional tableting processes. The content of the compound of the present invention in the above composition can be adjusted within the range of 0.001% to 5% (composition) or the corresponding dosage, preferably 0.01% to 1%, and most preferably about 0.05%.

[0049] Based on the above embodiments and comparative examples, it can be seen that: this invention uses *Tea japonica* as a natural source and obtains four lead compounds—dihydropashanone, 2′,6′-dihydroxy-4′-methoxydihydrochalcone, uvangoletin, and 2′,4′-dihydroxy-6′,4-dimethoxydihydrochalcone—through activity-directed separation. Furthermore, it reveals for the first time the inhibitory activity of this type of methoxydihydrochalcone against two key periodontal pathogenic anaerobic bacteria: *Porphyromonas gingivalis* and *Fusobacterium nucleatum*. On this basis, this invention achieves a breakthrough in… The existing technology, which holds that "the 4-hydroxyl group of the B ring and the polyhydroxyl group of the A ring are key to activity," has revealed an unexpected structure-activity relationship: oxygen substitution at the 4-position of the B ring is detrimental to activity, while moderate lipophilicity of the B ring enhances activity through membrane insertion. Based on this, a series of novel dihydrochalcone compounds with halogenated B rings and isopentenylated A rings were designed and synthesized. Their lipophilicity and antibacterial activity show a consistent increasing trend, with the most preferred compound, compound 9, showing a minimum inhibitory concentration (MIC) as low as 4 μmol / L against both pathogens. This invention also provides oral antibacterial compositions including mouthwash, toothpaste, and lozenges containing these compounds, along with their preparation methods and a separation method from *Tea japonica* (a type of herb), which can be used to inhibit or prevent dental plaque associated with gingivitis and periodontitis, and have significant application value.

[0050] The inhibitory effects of the compounds of this invention on *Porphyromonas gingivalis* and *Fusobacterium nucleatum* are mainly achieved through two pathways: disrupting the integrity of the bacterial cell membrane and inhibiting the virulence mediated by gingival protease. Regarding the disruption of cell membrane integrity, the dihydrochalcone molecule of this invention has a polar head at one end with a 2′-hydroxychelate and an A-ring phenolic hydroxyl group, and a lipophilic tail at the other end consisting of a B-ring and optionally an isopentenyl group, exhibiting amphiphilic structural characteristics similar to surfactant molecules. This type of molecule can insert its lipophilic tail into the phospholipid bilayer of the cell membrane of the Gram-negative anaerobic bacterium *Porphyromonas gingivalis*, disrupting the orderly arrangement of membrane lipids, increasing cell membrane permeability, and consequently causing the leakage of intracellular contents such as potassium ions, nucleic acids, and proteins. This leads to cell shrinkage and damage, ultimately resulting in bacterial death. Under a scanning electron microscope, obvious depressions and damage were observed on the surface of the bacteria treated with the compound of this invention. The release of lactate dehydrogenase increased with increasing drug concentration. Under a confocal laser scanning microscope, the proportion of dead bacteria increased. All of the above phenomena indicate that the bacterial cell membrane has been substantially damaged.

[0051] The antibacterial activity of the compounds in this invention shows a clear positive correlation with their lipophilicity, further confirming the dominant role of membrane insertion at the mechanistic level. The partitioning of molecules between the phospholipid membrane and the aqueous phase is mainly determined by their n-octanol-water partition coefficient (LogP). The larger the LogP, the more likely the molecules are to partition into and remain in the phospholipid membrane, thus facilitating insertion into and disruption of the membrane structure. The calculated LogP value of the lead compound 3 (uvangoletin) in this invention is approximately 2.92. Through the sequential introduction of fluorine, chlorine, bromine, and dichloro into the B ring, and the introduction of an isopentenyl group at the 3′ position of the A ring, the calculated LogP values ​​of compounds 5 to 9 increase sequentially to approximately 3.06, 3.58, 3.68, 4.23, and 5.08, respectively. Correspondingly, their minimum inhibitory concentration against *Porphyromonas gingivalis* decreases from 16 μmol / L to 4 μmol / L, showing a highly consistent trend. It is worth noting that since the compounds of the present invention are used in topical oral antibacterial preparations such as mouthwash, toothpaste, and lozenges, their effects do not require oral absorption through the gastrointestinal tract. Therefore, their high lipophilicity does not constitute a drug-forming obstacle, but rather facilitates the retention and accumulation of the compounds on the oral mucosa and in dental plaque biofilm, thereby enhancing the local antibacterial effect.

[0052] Regarding the virulence inhibition mediated by gingival protease, gingival protease, including arginine-specific Rgp and lysine-specific Kgp, is a key virulence factor for *Porphyromonas gingivalis* in acquiring iron and nutrients (especially in the degradation and utilization of hemoglobin) and in damaging host tissues and weakening host immunity. The dihydrochalcone skeleton can interfere with *Porphyromonas gingivalis*' utilization of hemoglobin and inhibit gingival protease activity, thereby weakening its pathogenicity while inhibiting bacterial growth. Notably, when the C-ring of dihydrochalcone closes and transforms into a flavonoid structure, the aforementioned interference with gingival protease and hemoglobin utilization is significantly reduced. Therefore, this invention retains the open-chain dihydro skeleton with —C(=O)—CH2—CH2— as the linker arm, without reducing or cyclizing it, which is crucial for maintaining activity.

[0053] Furthermore, periodontal pathogens often exist subgingivally in the form of biofilms. These biofilms hinder the penetration of conventional antibacterial drugs, leading to a significant increase in the concentration required for their antibacterial effect. The membrane-inserted antibacterial molecule of this invention, due to its strong lipophilicity, facilitates penetration of the biofilm matrix and its action on the bacteria within it. Therefore, the oral antibacterial composition of this invention can also be used to inhibit the formation of oral biofilms. Regarding selectivity, *Streptococcus mutans* is a facultative anaerobic Gram-positive bacterium. Its cell wall is rich in thick peptidoglycan, and its membrane lipid composition differs significantly from that of Gram-negative obligate anaerobic bacteria such as *Porphyromonas gingivalis* and *Fusobacterium nucleatum*. The compound of this invention showed no significant inhibitory effect on *Streptococcus mutans* within the measured concentration range, indicating its selectivity against Gram-negative anaerobic periodontal pathogens. This suggests that it may reduce non-specific effects on other oral flora while inhibiting periodontal pathogens.

[0054] Based on the results of the foregoing examples and comparative examples, the structure-activity relationship of this type of dihydrochalcone inhibiting *Porphyromonas gingivalis* is further explained below. First, the 2′-hydroxyl group on ring A is an indispensable pharmacophore. This 2′-hydroxyl group can form an intramolecular hydrogen bond with the adjacent carbonyl group to constitute a chelate structure, which appears as a strong deshielded single peak signal at approximately 14 in the 1H NMR spectrum. This is crucial for maintaining the active conformation and polar head of the molecule; in Comparative Example 2, replacing the 2′-hydroxyl group with a 2′-methoxy group disrupted the intramolecular hydrogen bond, resulting in a significant decrease in activity, fully confirming this point.

[0055] Second, the number and position of oxygen-containing substitutions in ring A significantly affect activity. In terms of number, excessive oxygen-containing substitutions in ring A are detrimental to activity. Compound 1, with four oxygen-containing substitutions (two hydroxyl groups and two methoxy groups) in ring A, exhibits significantly weaker activity than compounds 2 and 3, which contain only three oxygen-containing substitutions. In terms of position, compound 3, with its 2′,4′-dihydroxy-6′-methoxy form, is twice as active as compound 2, which has the same number of oxygen-containing substitutions but different positions, indicating that 2′,4′-dihydroxy-6′-methoxy is the preferred ring A substitution mode. Therefore, this invention preferably fixes the pharmacophore in ring A as 2′,4′-dihydroxy-6′-methoxy.

[0056] Third, the substitution of the B ring has a decisive and contrary-to-current effect on activity. In the prior art, active dihydrochalcones generally have a 4-hydroxyl group on the B ring, leading the art to believe that the 4-hydroxyl group on the B ring is key to activity. However, this invention found that compound 3, with an unsubstituted phenyl group on the B ring, exhibits significantly stronger activity, on a molar concentration basis, than polyhydroxy phlorizin with a 4-hydroxyl group on the B ring. Furthermore, compound 4, obtained by introducing a methoxy group at the 4-position of the B ring into compound 3, shows a halved activity, indicating that oxygen substitution at the 4-position of the B ring is not only not necessary for activity but also detrimental to it. Therefore, this invention focuses on dehydroxylation and halogenation of the B ring: introducing halogens into the B ring can improve the lipophilicity of the molecule without increasing polarity. The type and number of halogens affect the degree of lipophilicity. Generally, as the halogen changes from fluorine, chlorine, and bromine, and from monohalogenation to dihalogenation, the lipophilicity increases, and the corresponding antibacterial activity also increases. Therefore, this invention preferably uses chlorine or bromine as the halogen on the B ring, and further enhances activity through 3,4-dihalogenation and other polyhalogenation methods.

[0057] Fourth, the introduction of an isopentenyl group (3-methylbut-2-en-1-yl) at the 3′ position of ring A can serve as a strongly lipophilic membrane anchoring group, further enhancing the insertion and anchoring of the molecule to the bacterial cell membrane. Therefore, compound 9, obtained by introducing an isopentenyl group at the 3′ position of ring A based on the halogenation of ring B, possesses the dual enhancement of membrane anchoring of ring A and lipophilicity of ring B, making it the compound with the best activity in this invention. In summary, the structure-activity relationship revealed in this invention can be summarized as follows: 2′-hydroxy chelate and 2′,4′-dihydroxy ring A are the essential polar pharmacophores; the lipophilicity provided by the dehydroxylation / halogenation of ring B and the isopentenylation of ring A is the key to enhancing membrane insertion and improving molar efficacy. The specific combination of these two factors jointly determines the highly efficient inhibitory activity of the compound of this invention against *Porphyromonas gingivalis* and *Fusobacterium nucleatum*.

[0058] The dihydrochalcone compounds described in this invention are readily soluble in organic solvents such as chloroform, dichloromethane, methanol, ethanol, and dimethyl sulfoxide, but have low solubility in water. When preparing oral compositions, the compounds can be first dissolved in a small amount of ethanol or a suitable oral-acceptable solvent before being mixed with the remaining components, or their dispersibility in an aqueous matrix can be improved using solubilizing agents such as surfactants or cyclodextrins. These compounds are stable under normal temperature and light-protected conditions, facilitating storage and formulation processing.

[0059] In terms of applications, the compounds and compositions of this invention can be used to inhibit or prevent dental plaque associated with gingivitis and periodontitis, showing broad application prospects. Firstly, this invention uses the abundant and safe *Tea japonica* as a natural source, aligning with the development trend of developing oral antibacterial active substances from natural products. Secondly, the compounds of this invention exert their antibacterial effects through physicochemical mechanisms such as disrupting cell membrane integrity, which differs from the target of traditional antibiotics, potentially reducing the risk of bacterial resistance and providing a new option for overcoming the increasingly serious resistance of *Porphyromonas gingivalis* to antibiotics such as metronidazole and amoxicillin. Thirdly, the compounds of this invention can be combined with commonly used oral care ingredients such as fluoride, chlorhexidine, and plant essential oils to formulate various dosage forms such as mouthwash, toothpaste, lozenges, oral gels, and oral sprays, possessing functions such as anti-periodontal pathogens, anti-caries, and breath freshening, facilitating industrial-scale promotion and application.

[0060] Those skilled in the art should understand that, based on the technical solutions of this invention, equivalent selections and combinations of substituents in general formulas (I) and (II) within the defined range, such as selecting C1-C4 alkoxy groups as methoxy, ethoxy, n-propoxy, or isopropoxy, selecting C1-C4 alkyl groups as methyl, ethyl, or propyl, selecting halogens as fluorine, chlorine, or bromine, and introducing halogens into the 2, 3, or 4 positions of the B ring, all constitute equivalent variations of this invention. Furthermore, the compounds of this invention can be formulated as pharmaceutically acceptable salts, solvates, or hydrates as needed, and also include possible tautomers; all these forms fall within the protection scope of this invention.

[0061] To further verify the completeness of the present invention, the following two comparative examples are provided. Comparative Example 4: Using the α,β-unsaturated chalcone corresponding to compound 6 (i.e., the 2′-hydroxychalcone intermediate obtained in Example 1 without catalytic hydrogenation, whose core is chalcone rather than dihydrochalcone) as a control, the activity was determined according to the method of Example 8. The results showed that the minimum inhibitory concentration of this chalcone against Porphyromonas gingivalis was significantly higher than that of the dihydrochalcone compound 6 obtained by hydrogenation reduction, indicating that the open-chain dihydrogen skeleton with —C(=O)—CH2—CH2— as the linker arm is a necessary structure to maintain activity. Changing it to a chalcone skeleton containing α,β-unsaturated double bonds would weaken the activity, which is consistent with the aforementioned mechanism by which the dihydrogen skeleton is beneficial for inhibiting gingival protease and hemoglobin utilization. Comparative Example 5: 2′,4′,6′-trihydroxy-3-(4-chlorophenyl)prop-1-one (i.e., a dihydrochalcone with a trihydroxy ring and a 4-chloro ring on ring B) containing no alkoxy groups in ring A was used as a control. The results showed that this compound was less active than compound 6, which has a 6′-methoxy group in ring A. This indicates that the presence of at least one C1-C4 alkoxy group in ring A is a necessary feature of this invention. A purely polyhydroxy ring A cannot achieve the antibacterial effect of this invention. This also explains why the general formula (II) of this invention specifies that at least one of the 4′, 5′, and 6′ positions of ring A must be a C1-C4 alkoxy group, thus distinguishing it from existing polyhydroxy dihydrochalcones.

[0062] To further characterize the antibacterial properties and mechanism of action of the compounds of this invention, the most preferred compound 9 was used as a representative for supplementary verification. Minimum bactericidal concentration (MBC) determination: 10 μL of the culture from each well corresponding to the minimum inhibitory concentration (MIC) and above in Example 8 was evenly spread onto BHI+ solid medium plates and incubated for 72 h under the corresponding conditions. Colonies were then counted, and the lowest drug concentration corresponding to the elimination of more than 99.9% of the initial bacterial load was defined as the minimum bactericidal concentration. The results showed that the minimum bactericidal concentration of compound 9 against *Porphyromonas gingivalis* was approximately 8 μmol / L, close to its minimum inhibitory concentration, indicating that this compound has a bactericidal rather than merely inhibitory effect on *Porphyromonas gingivalis*.

[0063] Time-bacterial curve determination: Porphyromonas gingivalis in the logarithmic growth phase was adjusted to 1×10⁻⁶. 6 Compound 9 was added at concentrations of CFU / mL to achieve final concentrations of 1, 2, and 4 times the minimum inhibitory concentration (MIC). The mixture was incubated anaerobically at 37°C, and samples were taken at 0, 2, 4, 8, 12, and 24 hours. After serial dilution, viable bacteria were plated and counted, and time-kill curves were plotted. The results showed that the viable bacterial count gradually decreased with increasing drug concentration and treatment time. At 4 times the MIC, the viable bacterial count decreased by more than three logarithmic orders after 24 hours, exhibiting a clear concentration- and time-dependent bactericidal characteristic.

[0064] Cell membrane permeability assay: *Porphyromonas gingivalis* bacterial cultures treated with different concentrations of compound 9 were centrifuged, and the supernatant was collected. The absorbance at 260 nm was measured to reflect the leakage of intracellular nucleic acid substances. Simultaneously, the activity of lactate dehydrogenase in the supernatant was measured. The results showed that with increasing drug concentration, both the absorbance at 260 nm and the lactate dehydrogenase activity of the supernatant significantly increased, indicating that compound 9 treatment led to increased cell membrane permeability and leakage of intracellular substances in *Porphyromonas gingivalis*, directly confirming the mechanism by which the compound of this invention exerts its antibacterial effect by disrupting bacterial cell membrane integrity. Furthermore, in a hemoglobin-containing culture system, melanin formation in *Porphyromonas gingivalis* colonies treated with compound 9 was inhibited, suggesting that it interferes with the hemoglobin utilization and gingival protease-mediated virulence of *Porphyromonas gingivalis*, consistent with the aforementioned mechanism.

[0065] Compounds 5 to 9, prepared in Examples 1 to 5 of this invention, all had a purity of over 98% as determined by high-performance liquid chromatography (HPLC), meeting the requirements for bioactivity assays and formulation applications. Each compound was a white to slightly yellow solid, readily soluble in chloroform, methanol, ethanol, and dimethyl sulfoxide; its structure was confirmed by 1H NMR, 1C NMR, and high-resolution mass spectrometry, and the spectral data were consistent with the target structure. Lead compounds 1 to 4, after comparison with spectral data reported in the literature, were confirmed as dihydropashanone, 2′,6′-dihydroxy-4′-methoxydihydrochalcone, uvangoletin, and 2′,4′-dihydroxy-6′,4-dimethoxydihydrochalcone, respectively. Their purity, determined by HPLC, was also over 95%, sufficient to support the reliability of the antibacterial activity assay results of this invention.

[0066] The oral antibacterial composition of this invention can be used according to conventional oral care methods. Taking the mouthwash described in Example 9 as an example, an appropriate amount (approximately 10-15 mL) can be taken after brushing teeth in the morning and evening, rinsed for 30 seconds to 1 minute, and then spat out, used 1-2 times daily. Taking the toothpaste described in Example 10 as an example, an appropriate amount can be applied to a toothbrush and brushed 2-3 times daily, with each brushing session lasting at least 2 minutes. Taking the lozenges described in Example 11 as an example, they can be taken after meals, allowing them to slowly dissolve in the mouth, 2-4 tablets daily. During use, the composition should be allowed to fully contact the gums and plaque to maximize its inhibitory effect on *Porphyromonas gingivalis* and *Fusobacterium nucleatum*. The dosage of the compound in the composition can be adjusted appropriately according to the dosage form, frequency of use, and the user's oral health condition, generally aiming for an effective concentration that is not lower than its minimum inhibitory concentration in the oral cavity. For users at higher risk of gingivitis or periodontitis, the frequency of use or the content of the active ingredient can be appropriately increased under the guidance of a physician or oral care professional.

[0067] The medicinal material used in this invention, *Sarcandra glabra*, should be accurately identified as the dried whole herb of *Sarcandra glabra*, a plant belonging to the genus *Sarcandra* of the family Chloranthus, to avoid confusion with closely related species or adulterants, thus ensuring the stability and consistency of the active ingredients. Before use, the medicinal material should be selected, impurities removed, dried, and then pulverized and sieved to obtain a powder of suitable particle size, increasing the contact area between the medicinal material and the solvent and facilitating the full extraction of active ingredients. To improve extraction efficiency and protect heat-sensitive active ingredients, the extraction process should be carried out under light-protected conditions and at a suitable temperature. The concentration of ethanol, extraction time, and solvent volume used in reflux extraction can be appropriately optimized according to the batch of medicinal material within the range described in this invention. The above extraction and separation processes should minimize the oxidation and degradation of active ingredients, and attention should be paid to the precise control of the elution solvent ratio in each separation step to ensure the purity and antibacterial activity of the final dihydrochalcone compound.

[0068] To ensure the accuracy and reproducibility of the antibacterial activity assay results, strict quality control should be implemented for the bacterial strains and assay system during the experiment. The *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Streptococcus mutans* used should all be identified standard strains, with a limited number of passages. Purity and activity confirmation should be performed before use to avoid the influence of strain contamination or degeneration on the assay results. Each assay should include a blank control group, a solvent control group, a sample background control group, and a control group with metronidazole as a positive control. The assay result is considered valid only if the blank control group shows no bacterial growth, the solvent control group shows normal bacterial growth, and the positive control group exhibits the expected antibacterial effect. Three parallel wells should be set for each drug concentration, and each experiment should be independently repeated three times, with the average value taken as the final result to reduce random error. When determining the minimum inhibitory concentration (MIC), the turbidity of each well should be visually observed against a dark background, and confirmed by combining the absorbance value measured at 600 nm using an ELISA reader, thus avoiding interference from the color of the sample itself on visual interpretation. Through the above quality control measures, it can be ensured that the minimum inhibitory concentration and half-maximal inhibitory concentration data reported in this invention are true and reliable.

[0069] After preparation, the content of dihydrochalcone compounds in the oral antibacterial composition of this invention can be determined by high-performance liquid chromatography (HPLC) to ensure that the content of the active ingredient meets the design requirements. Simultaneously, routine tests are performed on the composition's pH value, appearance, homogeneity, and stability. For mouthwash, the pH value should be controlled within a suitable range of 5.0–7.5 to balance compound stability and oral comfort. For toothpaste and lozenges, uniformity of properties, even distribution of active ingredients, and absence of stratification or clumping should be ensured. Stability studies show that the content and antibacterial activity of the dihydrochalcone compounds in the composition remain stable during storage at room temperature and away from light, meeting the product shelf-life requirements. In summary, this invention not only provides lead compounds of dihydrochalcones with higher molar potency against periodontal pathogens and innovative compounds with novel structures, but also provides corresponding preparation methods, separation methods, oral antibacterial compositions, and their quality control and usage methods, constituting a complete technical solution from compound and composition to application.

Claims

1. A dihydrochalcone compound or a pharmaceutically acceptable salt thereof, characterized in that, The dihydrochalcone compounds have the structure shown in general formula (I), which is A ring—C(=O)—CH2—CH2—B ring; wherein, A ring is a benzene ring, with a carbonyl group attached to the 1′ position, a hydroxyl group at the 2′ position, and the 3′, 4′, 5′, and 6′ positions each independently selected from hydrogen, hydroxyl, C1-C4 alkoxy, C1-C4 alkyl, and 3-methylbut-2-en-1-yl, and at least one of the 4′ and 6′ positions is a C1-C4 alkoxy group; B ring is a benzene ring, with the 2, 3, and 4 positions each independently selected from hydrogen, fluorine, chlorine, bromine, C1-C4 alkyl, and C1-C4 alkoxy groups, and at least one of the 2, 3, and 4 positions of the B ring is fluorine, chlorine, or bromine.

2. The dihydrochalcone compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, Ring A has a hydroxyl group at position 4′ and an alkoxy group at position 6′; Ring B contains chlorine or bromine halogens.

3. The dihydrochalcone compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, Ring A is 2′,4′-dihydroxy-6′-methoxy, with its 3′ position optionally being 3-methylbut-2-en-1-yl; the 4 position of ring B is chlorine.

4. The dihydrochalcone compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The compounds are selected from: 1-(4,6-dihydroxy-2-methoxyphenyl)-3-(4-fluorophenyl)prop-1-one; 3-(4-chlorophenyl)-1-(4,6-dihydroxy-2-methoxyphenyl)prop-1-one; 3-(4-bromophenyl)-1-(4,6-dihydroxy-2-methoxyphenyl)prop-1-one; 3-(3,4-dichlorophenyl)-1-(4,6-dihydroxy-2-methoxyphenyl)prop-1-one; 3-(4-chlorophenyl)-1-[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)phenyl]prop-1-one.

5. The dihydrochalcone compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, The compound is 3-(4-chlorophenyl)-1-[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)phenyl]prop-1-one.

6. The dihydrochalcone compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The alkoxy group at the 6′ position of ring A, from C1 to C4, is either methoxy or ethoxy.

7. The dihydrochalcone compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The fluorine, chlorine, or bromine contained in ring B is located at position 4 of ring B.

8. The use of dihydrochalcone compounds of general formula (II) or pharmaceutically acceptable salts thereof in the preparation of products inhibiting Porphyromonas gingivalis and / or Fusobacterium nucleatum; characterized in that, General formula (II) is A ring—C(=O)—CH2—CH2—B ring, where ring A is a benzene ring with a hydroxyl group at position 2′ and at least one of positions 4′, 5′, and 6′ being a C1 to C4 alkoxy group, and ring B is a benzene ring without a hydroxyl group at position 4.

9. The application according to claim 8, characterized in that, The dihydrochalcone compounds are selected from one or more of dihydropashanone, 2′,6′-dihydroxy-4′-methoxydihydrochalcone, uvangoletin, 2′,4′-dihydroxy-6′,4-dimethoxydihydrochalcone, and the compound of general formula (I) according to claim 1.

10. The application according to claim 8, characterized in that, The minimum inhibitory concentration of the dihydrochalcone compounds against Porphyromonas gingivalis is 16–64 μmol / L.

11. The application according to claim 8, characterized in that, The half-maximal inhibitory concentration (MCIC) of the dihydrochalcone compounds against Porphyromonas gingivalis is not higher than 16 μmol / L.

12. The application according to claim 8, characterized in that, The inhibition is achieved by disrupting bacterial cell membrane integrity and / or inhibiting gingival protease-mediated virulence.

13. The application according to claim 8, characterized in that, The product is also used to inhibit Fusobacterium nucleatum, and the minimum inhibitory concentration of the compound against Fusobacterium nucleatum is 16–128 μmol / L.

14. The application according to claim 8, characterized in that, The product is an oral antibacterial agent that inhibits or prevents dental plaque associated with gingivitis and periodontitis.

15. The application according to claim 8, characterized in that, The product is a medication for the prevention or treatment of gingivitis and periodontitis.

16. An oral antibacterial composition, characterized in that, It comprises one or more of the compound of general formula (I) of claim 1 or the compound of general formula (II) of claim 8, and an oral-acceptable carrier.

17. The oral antibacterial composition according to claim 16, characterized in that, The compound is present in the composition at a mass percentage of 0.001% to 5%, preferably 0.01% to 1%.

18. The oral antibacterial composition according to claim 16, characterized in that, The composition is available in the form of mouthwash, toothpaste, lozenges, oral gel, or oral spray.

19. The oral antibacterial composition according to claim 16, characterized in that, It also contains one or more of fluorides, chlorhexidine, and plant essential oils.

20. The oral antibacterial composition according to claim 16, characterized in that, The composition is used to inhibit the formation of oral biofilms.

21. The oral antibacterial composition according to claim 16, characterized in that, The pH value of the composition is 5.0 to 7.

5.

22. The method for preparing the compound of general formula (I) according to claim 1, characterized in that, The process includes the following steps: using 2′,4′-dihydroxy-6′-methoxyacetophenone or its 3′-isoprenated derivative as an A-cyclic ketone, and reacting it with the corresponding substituted benzaldehyde via a Claisen-Schmidt condensation reaction to obtain a 2′-hydroxychalcone intermediate, followed by selective reduction of the α,β-unsaturated double bond via catalytic hydrogenation to obtain the corresponding dihydrochalcone.

23. The preparation method according to claim 22, characterized in that, The Clayson-Schmidt condensation reaction uses potassium hydroxide or piperidine as a base and alcohol as a solvent; the catalytic hydrogenation uses palladium on carbon as a catalyst and hydrogen as a hydrogen source.

24. The preparation method according to claim 22, characterized in that, The isopentenylation of the 3′ position of the A-ring ketone is carried out in acetone or N,N-dimethylformamide using isopentenyl bromide as the alkylating agent and potassium carbonate as the base.

25. A method for enriching dihydrochalcone of general formula (II) according to claim 8 from Jiujie tea, characterized in that, include: Nine-section tea was extracted by reflux with 90% ethanol, and the solvent was recovered to obtain an alcohol extract; after being dispersed in water, it was extracted with dichloromethane to obtain a dichloromethane extract; the dichloromethane extract was eluted with 90% ethanol through a D101 macroporous adsorption resin, and then eluted with petroleum ether-ethyl acetate at a volume ratio of 10:1 through a silica gel column.

26. The method according to claim 25, characterized in that, The D101 macroporous adsorption resin was eluted sequentially with 0%, 30%, 60%, and 90% ethanol, and the 90% ethanol eluent was collected; the silica gel column was eluted sequentially with petroleum ether-ethyl acetate gradients at volume ratios of 20:1, 15:1, 10:1, and 5:1, and the 10:1 volume eluent was collected.

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