Sarcandra glabra compound antibacterial gel as well as preparation method and application thereof

Abstract

The invention discloses glabrous sarcandra herb compound antibacterial gel as well as a preparation method and application thereof, and belongs to the technical field of oral medicine preparations, the gel is prepared from the following components in parts by weight: 2-8 parts of 90% ethanol eluate of glabrous sarcandra herb, 0.5-2 parts of eugenol, 0.3-1 part of gallic acid, 1-2 parts of carbomer 940, triethanolamine for adjusting the pH value to 6.5-7.0, 15-25 parts of glycerol and the balance of purified water. The 90% ethanol eluate of sarcandra glabra is rich in dihydrochalcone compounds and has remarkable inhibitory activity on porphyromonas gingivalis and fusobacterium nucleatum, the three components are compounded to generate a synergistic antibacterial effect, the 90% ethanol eluate of sarcandra glabra has no obvious inhibition on streptococcus mutans, and the selective effect on periodontal pathogenic bacteria is achieved.

Description

Technical Field

[0001] This invention belongs to the field of oral pharmaceutical preparation technology, specifically relating to Jiujiecha compound antibacterial gel, its preparation method and its application. Background Technology

[0002] Periodontitis is a chronic infectious disease caused by periodontal pathogens, primarily *Porphyromonas gingivalis* and *Fusobacterium nucleatum*. *Porphyromonas gingivalis* is one of the most critical pathogens in periodontitis, producing gingival proteases and lipopolysaccharides that can damage periodontal tissues and activate the host's inflammatory response. *Fusobacterium nucleatum* plays a crucial bridging role in the formation of the periodontal biofilm, connecting early colonizing bacteria with late-stage pathogens. Currently, clinical treatment for periodontitis mainly relies on nitroimidazole antibiotics such as metronidazole and tinidazole, as well as β-lactam antibiotics such as amoxicillin. However, long-term antibiotic use can easily lead to the development of drug-resistant strains, and systemic administration can cause gastrointestinal adverse reactions.

[0003] Chinese patent CN101978961B discloses a composition for treating gingivitis or periodontitis, mainly composed of amoxicillin, metronidazole, trimethoprim, and vitamin B6, belonging to a multi-antibiotic combination therapy. This therapy has drawbacks such as a high risk of antibiotic resistance and significant gastrointestinal side effects, and it does not optimize the dosage form for local administration into periodontal pockets. Chinese patent CN116492369B discloses a traditional Chinese medicine polysaccharide composition with anti-inflammatory and hemostatic functions. Its active ingredients are a combination of *Elsholtzia ciliata* polysaccharide and ginger-processed *Elsholtzia ciliata* polysaccharide, mainly used in oral care products such as toothpaste. This technical solution utilizes the anti-inflammatory activity of plant polysaccharides, rather than the direct antibacterial effect of small molecule compounds, and the toothpaste formulation makes it difficult to achieve effective drug retention within the periodontal pockets.

[0004] Sarcandra glabra, a plant belonging to the genus Sarcandra in the family Chloranthaceae, also known as "grass coral," has a long history of medicinal use, widely used in traditional Chinese medicine for treating oral inflammation. Modern pharmacological studies have shown that Sarcandra glabra contains abundant flavonoids, terpenes, and organic acids, among which dihydrochalcone compounds exhibit significant antibacterial activity. However, there are currently no reports on the formulation of periodontal pocket topical gels by combining the dihydrochalcone-enriched fraction of Sarcandra glabra with eugenol and gallic acid. Therefore, developing a natural product-based topical gel formulation targeting periodontal anaerobic pathogens has significant clinical value in reducing antibiotic use and lowering the risk of drug resistance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a compound antibacterial gel containing *Jiujiecha* (a type of herb) and its preparation method. This gel uses 90% ethanol eluent of *Jiujiecha* as the main ingredient, combined with eugenol and gallic acid, and employs carbomer 940 as the gel matrix to form a gel formulation suitable for local administration into periodontal pockets. The gel of this invention exhibits significant inhibitory activity against *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, the main pathogens of periodontitis. The three active components demonstrate a synergistic antibacterial effect, while showing no significant inhibitory effect on *Streptococcus mutans*, a normal commensal bacterium in the oral cavity, thus achieving selective inhibition of periodontal pathogens.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Firstly, the present invention provides a compound antibacterial gel containing, by weight, 2-8 parts of 90% ethanol eluent from *Jiujiecha*, 0.5-2 parts of eugenol, 0.3-1 part of gallic acid, 1-2 parts of carbomer 940, triethanolamine to adjust the pH to 6.5-7.0, 15-25 parts of glycerol, and purified water to a total of 100 parts. Secondly, the present invention provides a method for preparing the above-mentioned gel, including gel matrix preparation, active component solution preparation, mixing and blending, and degassing and filling steps.

[0007] Compared with the prior art, the present invention has the following advantages: It uses 90% ethanol eluent from *Tea japonica* as the main drug, which is rich in dihydrochalcone compounds. Uvanogleatin has a minimum inhibitory concentration (MIC) of 16 μmol / L against *Porphyromonas gingivalis*, and linoleic acid has a MIC of 16 μmol / L against *Fusobacterium nucleatum*. The three components produce a synergistic antibacterial effect, and the diameter of the inhibition zone of the compound gel is significantly larger than that of each individual component gel. The gel has no significant inhibition against *Streptococcus mutans*, achieving selective action. The carbomer 940 gel matrix has good bioadhesion, enabling continuous drug release within the periodontal pocket. Detailed Implementation

[0008] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, all reagents and instruments used in the embodiments are commercially available conventional products, and all experimental methods are conventional methods unless otherwise specified.

[0009] Example 1: Preparation of 90% ethanol eluent from Jiujie tea Take 5.0 kg of dried whole herb of *Tea japonica*, pulverize it into coarse powder with a particle size of about 2 mm using a pulverizer, pass it through a 20-mesh sieve, and place it in a 50 L round-bottom flask. First extraction: Add 10 times (50 L) of 90% (v / v) ethanol aqueous solution, heat and reflux at 80°C for 2 h, filter under reduced pressure while hot, and collect the filtrate. Second extraction: Add 8 times (40 L) of 90% (v / v) ethanol aqueous solution to the residue, reflux at 80°C for 1.5 h, filter under reduced pressure, and collect the filtrate. Combine the filtrates from both extractions, concentrate under reduced pressure at 50°C and -0.09 MPa using a rotary evaporator until there is no obvious alcohol odor in the distillate, yielding approximately 620 g of dark brown viscous extract.

[0010] The above extract was added to 2L of purified water and ultrasonically dispersed for 30 minutes to ensure uniform suspension. The mixture was then transferred to a 5L separatory funnel. Extraction was performed three times with an equal volume (2L) of petroleum ether (boiling range 60-90°C). After standing and separating the layers, the petroleum ether layer was discarded. Extraction was then performed three more times with an equal volume (2L) of dichloromethane. After standing and separating the layers, the dichloromethane layer was collected. The three dichloromethane extracts were combined, dried over anhydrous sodium sulfate, and then the solvent was recovered by vacuum distillation at 40°C using a rotary evaporator, yielding approximately 85g of a dark brown oily dichloromethane extract. The purpose of the petroleum ether extraction step is to remove lipid-soluble impurities such as chlorophyll and waxes beforehand, thereby improving the purity of the target active ingredient in the subsequent dichloromethane extract.

[0011] Take 85g of the above dichloromethane extract, use 200-300 mesh silica gel as the stationary phase (silica gel to sample mass ratio approximately 30:1), pack the column using a wet packing method, and perform gradient elution with a petroleum ether-ethyl acetate system. The elution gradients are successively: petroleum ether-ethyl acetate volume ratios of 20:1 (collecting 6 column volumes), 10:1 (collecting 8 column volumes), 5:1 (collecting 6 column volumes), 2:1 (collecting 4 column volumes), and 1:1 (collecting 4 column volumes). Each fraction is analyzed by thin-layer chromatography, and identical components are combined. After the above gradient elution is completed, elution is continued with a 90% (v / v) ethanol aqueous solution, and 6 column volumes of 90% ethanol eluent are collected. This fraction is concentrated to dryness under reduced pressure at 45°C to obtain approximately 12.6g of 90% ethanol eluent for *Tea japonica*, which is a brownish-yellow powder solid, with a yield of approximately 0.25% (based on dried medicinal material).

[0012] The 90% ethanol eluent of the obtained Jiujiecha tea was analyzed by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-0.1% formic acid aqueous solution gradient elution program (0–10 min methanol 40%–55%, 10–25 min methanol 55%–75%, 25–35 min methanol 75%–90%); flow rate: 1.0 mL / min; detection wavelength: 280 nm; column temperature: 30°C; injection volume: 10 μL. The chromatogram showed five major peaks in the fraction, with retention times of 8.2 min, 12.6 min, 15.8 min, 19.3 min, and 28.7 min, respectively.

[0013] The above fractions were further separated, purified, and structurally identified using preparative high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). A YMC-Pack ODS-A column (250 mm × 20 mm, 10 μm) was used, with a methanol-water gradient elution mobile phase at a flow rate of 8 mL / min. After multiple preparative separations, five pure compounds were obtained. Their structures were identified by 1H NMR, 1C NMR, and high-resolution mass spectrometry: Compound 1 was identified as dihydropashanone, with the molecular formula... Compound 2 is 2',6'-dihydroxy-4'-methoxydihydrochalcone, with the molecular formula 286.32. The molecular weight is 272.30; compound 3 is uvangoletin, with the molecular formula... The molecular weight is 302.32; compound 4 is 2',4'-dihydroxy-6',4-dimethoxydihydrochalcone, with the molecular formula... The molecular weight is 302.32; compound 5 is linoleic acid, with the molecular formula... The molecular weight is 280.45. Compounds 1-4 all belong to the dihydrochalcone class of compounds and have similar molecular weights. The skeleton structure has hydroxyl and methoxy substituents of varying numbers and positions on the A ring.

[0014] Example 2: Determination of antibacterial activity of monomeric compounds The minimum inhibitory concentrations (MICs) of the five compounds against periodontitis-associated pathogens were determined using the micro-broth dilution method. The tested bacterial strains included: *Porphyromonas gingivalis* ATCC 33277, *Fusobacterium nucleatum* ATCC 25586, and *Streptococcus mutans* ATCC 25175. Metronidazole (purity ≥99%, purchased from the National Institutes for Food and Drug Control) was used as the positive control. Stock solutions of the test compounds were prepared using dimethyl sulfoxide (DMSO) as the solvent (concentration 20 mmol / L), and the final concentration of DMSO in the experiment did not exceed 1%.

[0015] Cultures of *Porphyromonas gingivalis* and *Fusobacterium nucleatum* were prepared using a solution containing 5 μg / mL heme chloride and 1 μg / mL vitamin C. BHI broth medium was cultured at 37°C in an anaerobic workstation (80%). 10% 10% Incubate for 48 hours in a suitable atmosphere. *Streptococcus mutans* was cultured in BHI broth at 37°C with 5% [amount missing]. Incubate in an incubator for 24 hours. Dilute the activated bacterial culture with fresh culture medium to approximately [concentration missing]. CFU / mL available for use.

[0016] A two-fold dilution method was performed in a 96-well microplate: 100 μL of culture medium containing the test compound (final concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μmol / L) was added to each well, followed by 100 μL of the above bacterial suspension. Three parallel wells were set up for each concentration, along with blank control wells (sterile and drug-free), growth control wells (bacterial but drug-free), and solvent control wells (containing 1% dimethyl sulfoxide). The 96-well plate was placed in an anaerobic workstation or... After incubation at 37°C for 48 hours, the results were observed. The optical density of each well was measured at 600 nm using a microplate reader, and the lowest drug concentration with an inhibition rate greater than 90% compared to the control well was defined as the MIC. Simultaneously, calculations were performed... This refers to the minimum drug concentration that inhibits the growth of 50% of the strain. Each experiment was independently repeated three times, and the median was taken as the final result.

[0017] The experimental results are as follows: Compound 3 (urvanogleatin) showed the strongest inhibitory activity against Porphyromonas gingivalis, with a MIC of 16 μmol / L. The MIC for *Clostridium nucleatum* was 10.28 μmol / L; the MIC for *Clostridium nucleatum* was 32 μmol / L. Compound 5 (linoleic acid) showed the most significant inhibitory activity against *Clostridium nucleatum*, with an MIC of 16 μmol / L. The MIC against *Porphyromonas gingivalis* was 3.27 μmol / L; the MIC against *Porphyromonas gingivalis* was 64 μmol / L. Compound 1 (dihydropashanone) had an MIC of 32 μmol / L against *Porphyromonas gingivalis* and 64 μmol / L against *Fusobacterium nucleatum*. Compound 2 (2',6'-dihydroxy-4'-methoxydihydrochalcone) had an MIC of 64 μmol / L against *Porphyromonas gingivalis* and 32 μmol / L against *Fusobacterium nucleatum*. Compound 4 (2',4'-dihydroxy-6',4-dimethoxydihydrochalcone) had an MIC of 64 μmol / L against both pathogenic bacteria. Notably, the MICs of all five compounds against *Streptococcus mutans* were greater than 128 μmol / L, indicating that these compounds had no significant inhibitory effect on the normal oral commensal bacteria *Streptococcus mutans* and exhibited good species selectivity. The positive control, metronidazole, had a MIC of 0.16 μmol / L against *Porphyromonas gingivalis* and 0.5 μmol / L against *Fusobacterium nucleatum*. The solvent control wells containing 1% dimethyl sulfoxide had no effect on the growth of any of the three strains.

[0018] Example 3: Preparation of Nine-Section Tea Compound Antibacterial Gel Prepare the Jiujiecha compound antibacterial gel according to the following formula: 5g of Jiujiecha 90% ethanol eluent, 1g of eugenol, 0.5g of gallic acid, 1.5g of carbomer 940, appropriate amount of triethanolamine (about 0.8g), 20g of glycerin, and purified water to make up to 100g.

[0019] The specific preparation steps are as follows. Step (1) Gel matrix preparation: Accurately weigh 1.5g of Carbomer 940 and slowly and evenly sprinkle it into about 72g of purified water while continuously stirring. The stirring speed should be controlled at 100r / min to avoid generating too many bubbles, and the sprinkling time should be controlled at more than 10min to prevent clumping. After the sprinkling is completed, stop stirring, seal the container with plastic wrap, and place it in a constant temperature environment of 25°C for 12h to allow it to swell. After swelling, the gel matrix should be colorless and transparent, and should flow evenly with a glass rod without visible particles.

[0020] Step (2) Preparation of active component solution: Accurately weigh 5.00 g of 90% ethanol eluent from *Tea japonica*, 1.00 g of eugenol, and 0.50 g of gallic acid, and add them sequentially to 20.00 g of glycerol. Since gallic acid has low solubility in glycerol, it is first solubilized in a small amount of glycerol at 40°C for 10 min by ultrasonic treatment to promote dissolution. After complete dissolution, the *Tea japonica* eluent and eugenol are added. Continue stirring magnetically at 200 r / min for 30 min at 40°C until all components are completely dissolved, resulting in a uniform, clear, dark brown active component solution. Eugenol, as a liquid volatile component, should be added at the end of stirring to minimize loss.

[0021] Step (3) Mixing and Preparation: Start the mechanical stirrer and adjust the stirring speed to 300 r / min. Under continuous stirring, slowly add the above active component solution to the gel matrix in a thin stream, controlling the addition rate at about 5 mL / min, and observe the mixing uniformity while adding. After all the components are added, continue stirring for 20 min until the gel system is uniform and translucent. Then, add triethanolamine dropwise using a precision pipette, pausing stirring after each drop of about 0.1 mL, and measure the pH value of the gel using a precision pH meter. Stop adding triethanolamine when the pH reaches 6.8. The total amount of triethanolamine used is about 0.8 g. Continue stirring for 10 min to ensure that the pH in the gel system is fully uniform. If necessary, add purified water to a total of 100 g and stir well. The entire preparation process should be carried out under light-proof conditions to avoid photo-oxidative degradation of eugenol.

[0022] Step (4) Degassing and Filling: Transfer the gel to a vacuum degassing container, connect a vacuum pump, and degas under a vacuum of -0.08 MPa for 15 minutes, stirring gently every 5 minutes to help release air bubbles. After degassing, fill the gel into a 5 mL syringe-type applicator with a curved nozzle on a clean workbench, seal the package, and store in a cool, dark place.

[0023] The resulting gel was a light brown, semi-transparent, homogeneous gel with a fine texture and a slight clove odor. The gel had a pH of 6.8 and a viscosity of 35600±1800 mPa·s (measured at 25°C). This gel exhibited good thixotropy, maintaining its gel state when standing, flowing smoothly when squeezed with a syringe, and rapidly reverting to a gel state within the periodontal pocket, adhering to the periodontal tissue surface.

[0024] Example 4: Preparation of Nine-Section Tea Compound Antibacterial Gel The following formula was prepared: 2g of 90% ethanol eluent from *Tea japonica*, 0.5g of eugenol, 0.3g of gallic acid, 1g of carbomer 940, an appropriate amount of triethanolamine (approximately 0.5g), 15g of glycerol, and purified water to a final volume of 100g. The preparation method was the same as in Example 3, except that in step (1), the swelling time was 16h and the swelling temperature was 20°C, and in step (3), the stirring speed was 200r / min and the stirring time was 40min, and the pH was adjusted to 6.5. The resulting gel was a pale yellow, semi-transparent gel with a pH of 6.5 and a viscosity of 28200±1500mPa·s. This formula is suitable for maintenance treatment after the acute phase of periodontitis, with a lower concentration of active ingredients but less irritation.

[0025] Example 5: Preparation of Nine-Section Tea Compound Antibacterial Gel Prepared according to the following formula: 8g of 90% ethanol eluent from *Tea japonica*, 2g of eugenol, 1g of gallic acid, 2g of carbomer 940, appropriate amount of triethanolamine (approximately 1.2g), 25g of glycerol, and purified water to a final volume of 100g. The preparation method is the same as in Example 3, except that in step (1), the swelling time is 8h and the swelling temperature is 30°C; in step (3), the stirring speed is 500r / min and the stirring time is 20min; and the pH is adjusted to 7.0. After degassing, it is further pasteurized at 60°C for 30min. The resulting gel is a brown, semi-transparent gel with a pH of 7.0 and a viscosity of 42100±2200mPa·s. This formula has a high concentration of active ingredients and is suitable for intensive treatment during acute exacerbations of periodontitis.

[0026] Example 6: Preparation of Nine-Section Tea Compound Antibacterial Gel The following optimized formulation was prepared: 5g of 90% ethanol eluent from *Tea japonica*, 1.2g of eugenol, 0.6g of gallic acid, 1.5g of carbomer 940, an appropriate amount of triethanolamine (approximately 0.85g), 20g of glycerol, and purified water to a final volume of 100g. The preparation method was the same as in Example 3, with the pH adjusted to 6.8. The resulting gel was a light brown, semi-transparent gel with a pH of 6.8 and a viscosity of 36800±1600 mPa·s. This optimized formulation was determined through orthogonal experimental optimization, comprehensively balancing the performance in three dimensions: antibacterial activity, gel physical stability, and oral sensory acceptability. In subsequent antibacterial experiments, this optimized formulation exhibited the best overall antibacterial effect.

[0027] Example 7: Determination of the antibacterial activity of the compound gel The antibacterial effect of the compound gels prepared in Examples 3-6 against periodontal pathogens was determined using the agar diffusion method (drilling method). The test strains and culture conditions were the same as in Example 2. The activated bacterial solutions were adjusted to 0.5 McFarland turbidity standard (approximately 1.5 × 10⁻⁶). 8(CFU / mL) 200 μL of the test gel was evenly spread onto the surface of a BHI blood agar plate. Four wells were punched at equal intervals on the solidified agar plate using a 6 mm diameter sterile punch, and 50 μL of the test gel was added to each well. A blank gel matrix without active ingredient served as a negative control, and a gel containing 2% metronidazole served as a positive control. After incubation at 37°C under anaerobic conditions for 48 h, the diameter of the inhibition zone (including the well diameter) was measured using calipers. Each group had three replicates, and the experiment was independently repeated three times. Results are expressed as mean ± standard deviation.

[0028] Simultaneously, the following control gels were prepared to evaluate the individual contribution and synergistic effect of each component: Control gel A contained only 5g of 90% ethanol eluent of *Tea japonica* as the sole active ingredient; Control gel B contained only 1g of eugenol; Control gel C contained only 0.5g of gallic acid; Control gel D contained 5g of 90% ethanol eluent of *Tea japonica* and 1g of eugenol (excluding gallic acid); Control gel E contained 5g of 90% ethanol eluent of *Tea japonica* and 0.5g of gallic acid (excluding eugenol). The matrix composition of each control gel, namely 1.5g of carbomer 940, 20g of glycerol, appropriate amount of triethanolamine, and the balance of purified water, remained consistent with that in Example 3.

[0029] The experimental results are as follows. The diameter of the inhibition zone against *Porphyromonas gingivalis* was: 18.3±1.2 mm for Example 3 (Formula 1), 14.6±0.9 mm for Example 4 (low concentration formula), 20.1±1.3 mm for Example 5 (high concentration formula), and 19.5±0.9 mm for Example 6 (preferred formula). The results for each control gel were: 11.2±0.8 mm for control gel A (single *Tea japonica*), 12.5±1.0 mm for control gel B (single *eugenol*), 8.1±0.6 mm for control gel C (single *gallic acid*), 15.8±0.7 mm for control gel D (*Tea japonica* plus eugenol), and 14.3±0.9 mm for control gel E (*Tea japonica* plus gallic acid). The diameter of the inhibition zone of the positive control 2% metronidazole gel was 22.6±1.1 mm, and no inhibition zone was observed in the blank gel matrix.

[0030] The diameter of the inhibition zone against *Fusobacterium nucleatum* was as follows: Example 3: 16.8 ± 1.0 mm; Example 4: 13.2 ± 0.8 mm; Example 5: 18.5 ± 1.2 mm; Example 6: 17.9 ± 0.8 mm. The diameters of control gels were: A: 10.5 ± 0.7 mm; B: 9.8 ± 0.9 mm; C: 7.6 ± 0.5 mm; D: 14.2 ± 0.8 mm; E: 13.7 ± 0.6 mm. The diameter of the positive control was 20.1 ± 1.3 mm.

[0031] Inhibition zone diameter against Streptococcus mutans: The diameter of the inhibition zone of the compound gels in Examples 3-6 and all single-component and two-component control gels was less than 7 mm (judgment criterion: the diameter of the inhibition zone is less than 7 mm and is considered to have no antibacterial activity), further confirming that the gel of the present invention and its active components have no significant antibacterial effect on Streptococcus mutans in the normal oral flora.

[0032] The above results indicate that the antibacterial activity of the three-component compound gel is significantly superior to any single-component or two-component gel, exhibiting a clear synergistic effect. Taking Example 3 as an example, the degree of synergistic effect was analyzed: the inhibition zone diameter of the compound gel against *Porphyromonas gingivalis* was 18.3 mm, which was 63.4% larger than that of the single *Tea japonica* component (11.2 mm), 46.4% larger than that of eugenol alone (12.5 mm), and 125.9% larger than that of gallic acid alone (8.1 mm). Even compared to the two-component gel D (*Tea japonica* plus eugenol, 15.8 mm), which showed the highest activity, the three-component compound gel still increased by 15.8%, indicating that the addition of gallic acid made a crucial contribution to the overall synergistic effect. A similar synergistic effect was also verified in the antibacterial experiment against *Fusobacterium nucleatum*.

[0033] Example 8: Evaluation of the physicochemical properties and stability of the compound gel The physicochemical properties of the compound gels prepared in Examples 3-6 were systematically evaluated. Regarding appearance and uniformity, all four formulations yielded semi-transparent gel-like substances with a fine and uniform texture. Appropriate amounts of each gel were applied to glass slides and observed under a 10×40x optical microscope; no insoluble particles or phase separation were observed.

[0034] For pH measurement, a Mettler Toledo FiveEasy Plus precision pH meter was used. 1g of gel was diluted in 10mL of purified water and stirred before measurement. The pH values ​​of Examples 3-6 were 6.8, 6.5, 7.0, and 6.8, respectively, all within the range of 6.5-7.0 defined in the claims. This range is close to the physiological pH value of 6.5-6.9 within the periodontal pocket, which is beneficial for reducing irritation to periodontal tissues.

[0035] For viscosity measurement, an NDJ-5S rotational viscometer was used with a No. 4 rotor at a rotation speed of 12 r / min. Readings were taken after equilibration at 25 ± 0.5°C for 5 minutes. The gel viscosity of Example 3 was 35600 ± 1800 mPa·s, Example 4 was 28200 ± 1500 mPa·s, Example 5 was 42100 ± 2200 mPa·s, and Example 6 was 36800 ± 1600 mPa·s. Viscosity increased with increasing carbomer 940 dosage. Gels within the above viscosity ranges could be easily extruded using a syringe and maintained good retention within the periodontal pocket.

[0036] For the extensibility test, 1.0g of gel was placed in the center of a horizontal glass plate, and another glass plate of the same area was placed on top (total load 100g). After standing for 3 minutes, the diameter of the circular area formed by the gel expanding outwards was measured, and the average of three measurements was taken. The expansion diameter of Example 3 was 42.5±2.1mm, and that of Example 6 was 43.8±1.8mm, both indicating that the gel has moderate extensibility.

[0037] For stability testing, accelerated stability tests were conducted on the gels from Examples 3 and 6. Samples were placed under refrigerated conditions at 4°C, room temperature conditions at 25°C and 60% RH, and accelerated conditions at 40°C and 75% RH. Samples were taken at 0, 1, 2, 3, and 6 months to measure appearance, pH, viscosity, and the diameter of the inhibition zone against *Porphyromonas gingivalis*. Results showed that after 6 months of storage at 4°C and 25°C and 60% RH, the appearance of both gels showed no significant change, pH fluctuations did not exceed ±0.2 units, and viscosity changes were less than 8%. The diameter of the inhibition zone against *Porphyromonas gingivalis* in Example 3 decreased from the initial 18.3 ± 1.2 mm to 17.1 ± 1.4 mm, a decrease of only 6.6%. After 3 months of storage at 40°C and 75% RH, the gel color darkened slightly but remained homogeneous, and the pH and viscosity remained within acceptable ranges, with an antibacterial activity decrease of approximately 12%. The above results indicate that the gel of the present invention has good stability under normal storage conditions.

[0038] Example 9: In vitro drug release behavior study of compound gel The in vitro release behavior of the active ingredients in the compound gels of Examples 3 and 6 was studied using a modified Franz diffusion cell method. A 0.45 μm polyvinylidene fluoride microporous membrane was used as the diffusion membrane, with an effective diffusion area of ​​2.0 cm². The receiving medium was a 12 mL volume of pH 6.8 phosphate buffer containing 0.5% Tween-80, maintained at 37 ± 0.5°C, and magnetically stirred at 100 rpm to ensure thorough mixing in the recipient chamber. 0.50 g of gel was precisely measured and evenly coated onto the donor side of the diffusion membrane. Samples of 2 mL were taken from the recipient chamber at 0.5, 1, 2, 4, 6, 8, 12, and 24 h after drug administration (with an equal volume of isothermal fresh receiving medium added immediately). The samples were filtered through a 0.22 μm filter membrane and analyzed by high-performance liquid chromatography (HPLC). The detection indicators include the cumulative release of eugenol, gallic acid, and dihydrochalcone compounds (with uvangoletin as a representative indicator component), and the cumulative release percentage at each time point is calculated.

[0039] The drug release results of the compound gel in Example 3 are as follows. Eugenol showed a cumulative release rate of approximately 28% in the first 2 hours, exhibiting a certain burst release effect, which is beneficial for rapidly establishing an effective antibacterial concentration in the early stages of administration. Subsequently, the release rate tended to stabilize, with a cumulative release rate of approximately 45% at 4 hours, 60% at 8 hours, 70% at 12 hours, and approximately 78% at 24 hours. Gallic acid, due to its good water solubility (approximately 11.5 g / L at 25°C), was released relatively quickly, with a cumulative release rate of approximately 45% at 2 hours, 75% at 8 hours, and nearly complete release reaching approximately 95% at 24 hours. Uvangolide (representing dihydrochalcones) was released more slowly, with a cumulative release rate of approximately 15% at 2 hours, 30% at 8 hours, and approximately 52% at 24 hours, exhibiting a more pronounced sustained-release characteristic. The differentiated release kinetics of the three active ingredients create a multi-layered, time-sequential antibacterial pattern: gallic acid is released rapidly initially, weakening the nutrient supply to pathogens and disrupting the initial biofilm through iron chelation; eugenol experiences a peak release in the early to mid-stages, directly killing bacteria by disrupting bacterial cell membrane permeability; and dihydrochalcone compounds are released continuously and slowly, maintaining a long-lasting antibacterial concentration. This sequential release pattern facilitates once-daily clinical dosing regimens.

[0040] Example 10: Biocompatibility evaluation of the compound gel The cytotoxicity of the compound gel in Example 3 against human gingival fibroblasts (HGF) was evaluated using the CCK-8 assay. HGF cells were cultured at 5 × 10⁻⁶ cells / year. 3 Cells / well were seeded in 96-well cell culture plates and incubated at 37°C with 5% [unspecified concentration]. Cells were cultured in a saturated humidity incubator for 24 hours to allow complete cell adhesion. Cells were then treated with gel extraction buffer: 1g of gel was added to 10mL of serum-free DMEM medium and incubated at 37°C for 24 hours using a shaker. The supernatant was then sterilely filtered through a 0.22μm filter to obtain the stock extract. The stock extract was diluted with serum-free DMEM medium to 1:1, 1:2, 1:4, 1:8, and 1:16, respectively. The original medium in the 96-well plate was discarded, and 200μL of the different concentrations of extract was added, followed by incubation for 24 hours and 48 hours. Six parallel wells were set up for each concentration and time point. After incubation, 10μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance at 450nm was measured using a microplate reader. An untreated blank medium group served as a negative control (cell viability was 100%), and a medium containing 5% dimethyl sulfoxide (DMSO) served as a positive control. Cell viability is calculated using the formula: Cell viability = (Experimental group absorbance - Blank control absorbance) / (Negative control absorbance - Blank control absorbance) × 100%.

[0041] The results showed that, at dilution ratios of 1:2 and above, the HGF cell viability was greater than 85% after 24 and 48 hours of treatment with the gel extract, meeting the basic requirements for in vitro cytotoxicity evaluation of medical devices and pharmaceutical preparations in international standard ISO 10993-5 (cell viability greater than 70% is considered as no significant cytotoxicity). At a 1:4 dilution, cell viability was 91.3±3.8% after 24 hours and 88.7±4.2% after 48 hours. At a 1:1 concentration extract, cell viability was 72.3±4.5% after 24 hours and 68.1±5.2% after 48 hours, showing slight cytotoxicity. Considering that in actual clinical use, the gel is continuously diluted by gingival crevicular fluid and saliva within the periodontal pocket, and the actual drug contact concentration is much lower than that of the 1:1 extract, the gel of this invention has acceptable biocompatibility.

[0042] Example 11: Inhibitory effect of compound gel on periodontal pathogenic bacteria biofilm Periodontal biofilm is a key factor in the persistent development of periodontitis, and bacteria within the biofilm are far more resistant to antimicrobial drugs than bacteria in a planktonic state. This study investigates the inhibitory effect of the compound gels from Examples 3 and 6 on biofilm formation by *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, as well as their effect on clearing pre-formed biofilms.

[0043] Biofilm formation inhibition experiment: Porphyromonas gingivalis and Fusobacterium nucleatum were respectively used as... CFU / mL was initially inoculated into 24-well polystyrene cell culture plates, with 1 mL of bacterial culture added to each well. In the experimental group, a sterile filter paper disc (8 mm in diameter) loaded with 50 mg of the compound gel was placed in each well, while in the control group, a filter paper disc loaded with an equal volume of blank gel matrix was placed in each well. The plates were incubated anaerobically at 37°C for 72 h to establish a mature biofilm. After incubation, the filter paper discs were gently removed, and the plates were washed three times with sterile phosphate buffer to remove airborne bacteria. Biofilm biomass was quantitatively analyzed using crystal violet staining: 500 μL of 0.1% crystal violet solution was added, and the plates were stained at room temperature for 15 min. The plates were then washed three times with purified water, and 500 μL of 33% glacial acetic acid was added to dissolve the stained biofilm. The absorbance at 595 nm was measured using a microplate reader. The biofilm formation rate of each group was calculated with the absorbance of the blank gel matrix group as 100%.

[0044] Pre-biofilm removal experiment: *Porphyromonas gingivalis* biofilms were cultured in 24-well plates for 72 hours to allow them to mature. The culture medium was then aspirated, and fresh medium containing a compound gel extract (1 g gel extracted in 10 mL of BHI medium for 24 hours) was added, followed by anaerobic culture for another 24 hours. The control group received medium containing a blank gel matrix extract. After treatment, the amount of residual biofilm was quantitatively analyzed using crystal violet staining.

[0045] Experimental results showed that the compound gel of Example 3 inhibited the formation of *Porphyromonas gingivalis* biofilm by 68.5±4.3% and *Fusobacterium nucleatum* biofilm by 61.2±3.8%, significantly higher than the control gel A (containing only *Nine-section Tea* extract) which showed inhibition rates of 42.3% and 37.8% for the two bacteria, respectively. The preferred formulation of Example 6 further improved the inhibition rate, with inhibition rates of 72.1±3.6% for *Porphyromonas gingivalis* and 65.8±4.1% for *Fusobacterium nucleatum* biofilm formation. In the pre-biofilm removal experiment, after 24 hours of treatment with the extract of the compound gel of Example 3, the residual amount of pre-formed *Porphyromonas gingivalis* biofilm decreased by 53.7±5.1%, indicating that the gel of this invention not only effectively prevents the establishment of new biofilms but also has a certain ability to destroy existing biofilms. This anti-biofilm activity is consistent with the mechanism of action of gallic acid interfering with quorum sensing signals and inhibiting extracellular polysaccharide synthesis as described in the aforementioned comprehensive mechanism analysis. For the clinical treatment of periodontitis, drug formulations that possess both anti-plankton and anti-biofilm activities can more effectively eliminate pathogenic microorganisms in periodontal pockets and improve treatment outcomes.

[0046] Comparative Example 1: Nine-section tea extract gel A comparative study was conducted by preparing gels using a traditional water extract instead of the 90% ethanol eluent from *Tea japonica*. 100g of dried whole *Tea japonica* herb was added to 10 times (1L) of purified water and decocted twice, 1 hour each time. The decoctions were combined and concentrated under reduced pressure to 50mL to obtain a concentrated water extract of *Tea japonica*. 5g of this water extract was used to replace the 90% ethanol eluent from *Tea japonica* in Example 3, with the remaining component ratios and preparation methods identical to Example 3. The resulting gel showed an inhibition zone diameter of only 9.3±0.8mm against *Porphyromonas gingivalis* and 8.1±0.7mm against *Fusobacterium nucleatum*, both significantly lower than the compound gel in Example 3. This indicates that the traditional water extraction process cannot effectively enrich dihydrochalcone active ingredients, resulting in insufficient antibacterial activity. This comparative result confirms the necessity of the specific extraction and purification pathway employed in this invention—combining 90% ethanol reflux extraction with dichloromethane extraction and silica gel column chromatography with 90% ethanol elution—for obtaining highly active fractions.

[0047] Comparative Example 2: Monomeric gels without eugenol and gallic acid A gel was prepared using 5g of 90% ethanol eluate from *Tea japonica* as the sole active ingredient, without the addition of eugenol and gallic acid. The remaining matrix composition and preparation method were the same as in Example 3. The resulting single-component gel is the control gel A in Example 7. Its inhibition zone diameter against *Porphyromonas gingivalis* was 11.2±0.8mm, and against *Fusobacterium nucleatum* was 10.5±0.7mm, both significantly lower than the corresponding values ​​of 18.3mm and 16.8mm for the three-component compound gel in Example 3. It also showed a significant difference compared to control gel D (a two-component combination of *Tea japonica* and eugenol, 15.8mm against *Porphyromonas gingivalis*) and control gel E (a two-component combination of *Tea japonica* and gallic acid, 14.3mm). This result further confirms the irreplaceable role of eugenol and gallic acid in the compound system of this invention; the synergistic effect of the three is far greater than the antibacterial effect of using them alone or in combination.

[0048] Comparative Example 3: Carbomer 934 as a substitute for gel A gel was prepared using Carbomer 934 instead of Carbomer 940, with the same dosage of 1.5 g. The remaining component ratios and preparation methods were the same as in Example 3. The resulting gel had a viscosity of 18900 ± 1200 mPa·s, significantly lower than the gel obtained using Carbomer 940 in Example 3 (35600 ± 1800 mPa·s). In an in vitro adhesion experiment simulating a periodontal pocket environment (the gel was applied to the surface of the porcine oral mucosa and continuously rinsed with pH 6.8 phosphate buffer at 37°C at a flow rate of 0.5 mL / min), the Carbomer 934 gel completely detached within approximately 45 minutes, while the Carbomer 940 gel maintained effective adhesion for over 120 minutes. Carbomer 940 exhibits a higher crosslinking density and longer polymer chains than Carbomer 934, thus demonstrating superior bioadhesive properties and higher viscosity. For the specific application scenario of local drug delivery in periodontal pockets, the retention time of the drug in the periodontal pocket directly determines the efficacy. Therefore, Carbomer 940 is a more suitable gel matrix choice.

[0049] The multi-level synergistic antibacterial mechanism of the Jiujiecha compound antibacterial gel of this invention can be explained from three dimensions: molecular target, bacterial selectivity, and drug release kinetics.

[0050] At the molecular target level, the dihydrochalcone compounds, represented by uvangoletin, abundant in the 90% ethanol eluent of *Tea japonica*, possess excellent lipophilicity and hydrogen bond donor capabilities due to the 2',4'-dihydroxy substitution on its A ring. These compounds can interact hydrophobically with fatty acid chains in the phospholipid bilayer of bacterial cell membranes, and simultaneously form hydrogen bonds with phospholipid head groups through phenolic hydroxyl groups, altering membrane fluidity and permeability, leading to leakage of key intracellular substances such as nucleic acids and proteins. Eugenol, as a phenolic propene compound, also primarily acts on the bacterial cell membrane through its antibacterial mechanism. Previous studies have shown that eugenol can embed itself in the lipid bilayer of cell membranes, increasing membrane permeability, promoting the leakage of large intracellular molecules, and ultimately leading to cell death. More importantly, eugenol's enhancement of cell membrane permeability can significantly promote the penetration of other antibacterial components into the cell, thus producing a synergistic effect. This mechanism explains why the increase in antibacterial activity after combining *Tea japonica* eluent with eugenol is significantly greater than the simple arithmetic sum of their activities.

[0051] Gallic acid plays multiple roles in the compound system of this invention. First, as a polyphenolic antioxidant and iron ion chelator, gallic acid can form stable complexes with ferric ions, reducing the concentration of free iron in the microenvironment. The growth and virulence factor expression of *Porphyromonas gingivalis* are highly dependent on iron supply; gallic acid weakens the proliferation and pathogenicity of pathogenic bacteria from a nutrient competition perspective by limiting iron bioavailability. Second, gallic acid can interfere with bacterial quorum sensing signaling pathways, inhibiting the synthesis and secretion of extracellular polysaccharide matrix, thereby hindering the initial formation of biofilms and promoting the disintegration of established mature biofilms. Damaged or failed-to-form biofilms expose deep bacterial cells, making them more susceptible to direct attack from dihydrochalcone and eugenol. The three active components form a multi-target synergistic antibacterial system through three complementary pathways: enhanced cell membrane permeability, iron nutrient deprivation, and biofilm disruption. This is the core mechanism by which the compound gel of this invention has significant advantages over single-component formulations.

[0052] Regarding the reasons for the selective inhibition of bacterial species, the phenomenon that the gel of this invention has no inhibitory effect on *Streptococcus mutans* but exhibits selective inhibitory activity against *Porphyromonas gingivalis* and *Fusobacterium nucleatum* may be related to the fundamental differences in the lipid composition and metabolic characteristics of different bacterial species in their cell membranes. *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, as obligate anaerobic Gram-negative bacteria, have cell membranes with branched-chain fatty acids as the main lipid component, resulting in relatively high membrane fluidity. *Streptococcus mutans*, on the other hand, is a facultative anaerobic Gram-positive bacterium with a cell membrane mainly composed of straight-chain saturated fatty acids, resulting in a denser membrane structure and lower fluidity. Dihydrochalcone compounds may have a stronger ability to intercalate and perturb looser cell membranes containing branched-chain fatty acids, thus exhibiting a preferential inhibitory effect on anaerobic pathogens. Furthermore, obligate anaerobes have poor tolerance to oxidative stress, and the antioxidant activity and iron chelation of gallic acid may have a more significant interfering effect on their metabolism. This natural bacterial selectivity allows the gel of this invention to effectively inhibit periodontal pathogens without interfering with the normal oral microecological balance, avoiding the dysbiosis side effects commonly seen with broad-spectrum antibiotics.

[0053] Regarding the correlation between drug release kinetics and clinical application, the differentiated release behavior of the three active components in the gel of this invention has significant clinical implications. Gallic acid is rapidly released at the initial stage of administration, establishing an iron ion chelating field in the periodontal pocket microenvironment, reducing the local concentration of free iron, inhibiting the activity of iron-dependent gingival proteases, and slowing down the enzymatic destruction of host periodontal tissue by *Porphyromonas gingivalis*. Eugenol reaches its release peak 2–8 hours after administration, a period that coincides with the active window of bacterial re-proliferation in the periodontal pocket, achieving highly efficient killing of proliferating bacteria through membrane disruption. The sustained and slow release of dihydrochalcone compounds from *Tetracentron sinense* maintains a stable baseline of antibacterial concentration within 24 hours, preventing residual bacteria from rebounding and proliferating during periods of low drug concentration. This sequential, three-stage drug release strategy matches the growth kinetics of pathogenic bacteria in the periodontal pocket, theoretically enabling a once-daily dosing regimen and improving patient compliance. The effective adhesion time of the carbomer 940 gel matrix within the periodontal pocket for over 120 minutes provides a reliable dosage form guarantee for the implementation of the above sustained-release strategy.

[0054] In summary, the present invention, a compound antibacterial gel containing *Jiujiecha* (a type of herb) obtained through a specific extraction and purification pathway, yields a 90% ethanol eluent rich in dihydrochalcone compounds. This eluent, when scientifically combined with eugenol and gallic acid, forms an antibacterial system with a triple synergistic mechanism of enhanced membrane permeability, iron nutrient deprivation, and biofilm disruption. This system exhibits significant inhibitory activity and anti-biofilm activity against *Porphyromonas gingivalis* and *Fusobacterium nucleatum*, the main pathogens of periodontitis, while maintaining good selectivity and non-interference with *Streptococcus mutans*, a normal commensal bacterium in the oral cavity. The carbomer 940 gel matrix provides an excellent dosage form platform for sustained drug release and long-lasting retention of the active ingredients within the periodontal pocket. This entire technical solution provides a new strategy and product option for the non-antibiotic local treatment of periodontitis.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit 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 the claims of the present invention.

Claims

1. A compound antibacterial gel containing nine-section tea, characterized in that, The gel comprises, by weight, the following components: 2-8 parts of 90% ethanol eluent from *Tea japonica*, 0.5-2 parts of eugenol, 0.3-1 parts of gallic acid, 1-2 parts of carbomer 940, triethanolamine as a pH adjuster to adjust the pH of the gel to 6.5-7.0, 15-25 parts of glycerol, and purified water to make up to 100 parts; wherein, the 90% ethanol eluent from *Tea japonica* is obtained by extracting *Tea japonica* medicinal material with 90% ethanol, extracting the resulting extract with dichloromethane, and taking the dichloromethane layer concentrate and eluting it with 90% ethanol by silica gel column chromatography.

2. The nine-section tea compound antibacterial gel according to claim 1, characterized in that, The preparation of the 90% ethanol eluent of *Tea japonica* includes the following steps: pulverize the dried *Tea japonica* medicinal material, reflux extract with 8-12 times the amount of 90% ethanol 2-3 times, 1-2 hours each time, combine the extracts and concentrate under reduced pressure to obtain an extract; disperse the extract in water and extract sequentially with petroleum ether and dichloromethane, collect the dichloromethane extract; subject the dichloromethane extract to silica gel column chromatography, elute sequentially with a gradient of petroleum ether-ethyl acetate, then elute with 90% ethanol, collect the 90% ethanol eluent fraction, concentrate and dry under reduced pressure to obtain the final product.

3. The nine-section tea compound antibacterial gel according to claim 1, characterized in that, The amount of eugenol used is 0.8 to 1.5 parts.

4. The nine-section tea compound antibacterial gel according to claim 1, characterized in that, The amount of gallic acid used is 0.5 to 0.8 parts.

5. The nine-section tea compound antibacterial gel according to claim 1, characterized in that, The amount of the 90% ethanol eluent from the nine-section tea is 4 to 6 parts.

6. The preparation method of the nine-section tea compound antibacterial gel according to any one of claims 1 to 5, characterized in that, The process includes the following steps: (1) Gel matrix preparation: Carbomer 940 is dispersed in purified water and allowed to swell at room temperature for 8-16 hours to obtain a uniform and transparent gel matrix; (2) Active component solution preparation: 90% ethanol eluent of *Tea japonica*, eugenol and gallic acid are dissolved in glycerol and stirred until completely dissolved to obtain an active component solution; (3) Mixing and preparation: Under continuous stirring, the active component solution is slowly added to the gel matrix and stirred evenly. Triethanolamine is added dropwise to adjust the pH to 6.5-7.0, and stirring is continued until the gel system is uniform and transparent; (4) Degassing and filling: The gel is degassed under vacuum for 10-20 minutes and then filled.

7. The preparation method according to claim 6, characterized in that, In step (1), the ratio of carbomer 940 to purified water is 1:40 to 1:60, and the swelling temperature is 20 to 30°C.

8. The preparation method according to claim 6, characterized in that, In step (3), the stirring speed is 200~500 r / min and the stirring time is 20~40 min.

9. The preparation method according to claim 6, characterized in that, In step (4), after degassing, the process also includes pasteurization at 60°C for 30 minutes.

10. The use of the nine-section tea compound antibacterial gel according to any one of claims 1 to 5 in the preparation of periodontal pocket topical drug delivery formulations.