Application of Xinjiang pomegranate peel ellagic acid in caries prevention

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of Xinjiang pomegranate rind ellagic acid in caries prevention, in-vivo and in-vitro experiments prove that the ellagic acid can effectively inhibit floating growth and biofilm formation of streptococcus mutans, streptococcus sobrinus and streptococcus sanguis, MICs of the ellagic acid are 4 mmol / L, 2 mmol / L and 1 mmol / L respectively, and MBICs of the ellagic acid are 8 mmol / L, 4 mmol / L and 1 mmol / L respectively; meanwhile, the ellagic acid can obviously inhibit the acid production capacity of the cariogenic bacteria, the generation of water-insoluble exopolysaccharides and the adhesion to hydroxyapatite; in a rat caries model, by locally applying 1-8 mmol / L ellagic acid, the number of streptococcus in the oral cavity can be reduced in a dose-dependent manner, the caries damage degree of smooth surfaces of molar teeth and fossa and fissure can be relieved, the effect of the ellagic acid is equivalent to that of positive control drugs chlorhexidine and sodium fluoride under the high dose (8 mmol / L), and the ellagic acid shows good biological safety. Therefore, ellagic acid can be used for preparing safe and effective anti-caries drugs or oral care products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the use of tannic acid from Xinjiang pomegranate peel in caries prevention. BACKGROUND

[0002] Caries is a common oral disease mediated by bacterial biofilm, and the main pathogenic bacteria include Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguinis. These cariogenic bacteria adhere to the tooth surface, metabolize sucrose to produce acid and synthesize water-insoluble extracellular polysaccharide, and build a dense biofilm structure, leading to continuous demineralization of hard dental tissues and formation of carious lesions.

[0003] At present, the chemical prevention of caries mainly relies on fluoride and chlorhexidine. Fluoride mainly enhances the acid resistance of enamel by promoting remineralization, but has limited effect on inhibiting the formation of cariogenic bacterial biofilm; although chlorhexidine can effectively inhibit bacteria in a broad spectrum, long-term use may cause tooth staining, abnormal taste and other adverse reactions, and its application compliance is restricted. Therefore, the development of new caries-preventing agents with high safety and both antibacterial and anti-biofilm properties has become a research hotspot.

[0004] Natural products are attracting attention due to their wide sources and small side effects. Pomegranate peel is a commonly used medicinal material in Uighur medicine, and its key active ingredient, tannic acid, has been reported to have antibacterial and anti-inflammatory activities. Although some studies have shown that tannic acid derived from other plants can inhibit the growth of Streptococcus mutans, whether tannic acid derived from Xinjiang pomegranate peel has broad-spectrum anti-caries effect, its inhibitory effect on multiple cariogenic bacteria (such as Streptococcus sobrinus and Streptococcus sanguinis), systematic inhibition of key virulence phenotypes (acid production, sugar production, adhesion) of biofilm, and actual anti-caries effect and safety in animals have not been systematically studied and reported. The prior art cannot reveal the overall potential and application value of tannic acid from Xinjiang pomegranate peel in the prevention and treatment of caries.

[0005] Therefore, the present application provides the use of tannic acid from Xinjiang pomegranate peel in caries prevention to solve the above problems. SUMMARY

[0006] The present application aims to solve the technical problems raised in the background art, and provides the use of tannic acid from Xinjiang pomegranate peel in caries prevention, which can effectively inhibit multiple cariogenic bacteria and systematically intervene in the formation of biofilm, acid production and adhesion, and has good safety.

[0007] The above-mentioned object of the present application is achieved in the following manner: The present application provides a use of ellagic acid in the preparation of a medicament or oral care product for preventing and / or treating dental caries.

[0008] Further, the use is for inhibiting planktonic growth and / or biofilm formation of cariogenic bacteria; the cariogenic bacteria are at least one of Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguis.

[0009] Further, the 50% minimum inhibitory concentration of the ellagic acid against planktonic state of Streptococcus mutans is 4 mmol / L, the 50% minimum inhibitory concentration of the ellagic acid against planktonic state of Streptococcus sobrinus is 2 mmol / L, and the 50% minimum inhibitory concentration of the ellagic acid against planktonic state of Streptococcus sanguis is 1 mmol / L.

[0010] Further, the use is for inhibiting acid production and / or water-insoluble exopolysaccharide production of cariogenic bacteria.

[0011] Further, the use is for inhibiting adhesion of cariogenic bacteria to hydroxyapatite surface.

[0012] Further, the use is for reducing the number of Streptococcus in the oral cavity of a mammal and / or reducing the degree of dental caries.

[0013] Further, the effective concentration of the medicament is 2 mmol / L to 8 mmol / L.

[0014] Further, the medicament is administered by oral coating.

[0015] The present application also provides a use of ellagic acid in the preparation of a product for inhibiting biofilm formation of cariogenic bacteria, the 50% minimum inhibitory concentration of the ellagic acid against biofilm formation of Streptococcus mutans is 8 mmol / L, the 50% minimum inhibitory concentration of the ellagic acid against biofilm formation of Streptococcus sobrinus is 4 mmol / L, and the 50% minimum inhibitory concentration of the ellagic acid against biofilm formation of Streptococcus sanguis is 1 mmol / L.

[0016] The present application solves the technical problems and has the following advantages: The traditional development idea of caries-preventing agents often focuses on strong bactericidal effect (such as chlorhexidine) or enhanced resistance of the tooth (such as fluoride); the present application breaks out of this traditional framework, moves the intervention point forward and expands it, and establishes a new strategy for preventing caries by simultaneously inhibiting multiple key virulence phenotypes such as bacterial adhesion, biofilm structure construction, and acid-producing metabolism. In addition, the oral cavity is a complex microecosystem, and studying the effect on Streptococcus mutans alone is not enough to evaluate its real potential for preventing caries. In the present application, three key cariogenic bacteria are systematically selected, and the large difference in sensitivity (MIC 50The experimental design complexity brought by the concentration of 1 to 4 mmol / L) accurately draws the bacteriostatic spectrum of ellagic acid, proves the broad spectrum, which needs a lot of comparison and screening work. Moreover, the scheme of the application is proved to be good in the in-vitro experiment, but may fail in the animal body due to factors such as saliva flushing, food residues and complex bacterial competition, a rat mixed bacterial infection model simulating the occurrence of human dental caries is successfully established, and in this complex environment, it is proved that the locally coated ellagic acid can effectively colonize pathogenic bacteria and significantly reduce caries damage; therefore, the scheme of the application no longer relies on the single bactericidal of the "antibiotic type", but through the multi-path intervention of "disrupting the enemy's offensive", effectively controls the occurrence and development of dental caries, and provides a new technical idea and an evidenced solution for the prevention and treatment of dental caries.

[0017] Compared with the prior art, the application has the following beneficial effects: 1. The scheme of the application first systematically proves that the ellagic acid (EA) of Xinjiang pomegranate peel is not through a single bacteriostatic pathway, but through the whole chain of "adhesion- colonization- pathogenesis" of synergistic inhibition of cariogenic bacteria to play the role of preventing dental caries, specifically, it has the following functions: (1) Inhibiting initial adhesion: effectively interfering with the adhesion of bacteria to the dental surface biomaterial (hydroxyapatite), reducing the "seeds" of plaque formation from the source.

[0018] (2) Disrupting the structure of biofilm: not only can inhibit the formation of biofilm (MBIC 50 1-8 mmol / L), but also can destroy the dense three-dimensional structure of the formed biofilm, significantly increase the proportion of dead bacteria, and fundamentally weaken the function of biofilm as a "pathogenic microenvironment".

[0019] (3) Cutting off energy supply and attack means: dose-dependent inhibition of the synthesis of water-insoluble extracellular polysaccharide (IEPS), destroying the "adhesive" and "granary" of the biofilm; at the same time, strongly inhibiting the acid-producing metabolism of bacteria, reducing the power of "attack weapons".

[0020] This "four-in-one" comprehensive action mechanism breaks through the limitation of single target of traditional caries prevention agents, and provides a more advanced and efficient ecological caries prevention new path.

[0021] 2. In the scheme of the application, it is clear that EA has strong inhibitory activity on three kinds of core cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguis), and the inhibitory effect shows a clear gradient (MIC 50 1-4 mmol / L), which shows that EA has broad-spectrum antibacterial potential and can cope with the complex cariogenic flora in the oral cavity; at the same time, the difference in efficiency of different bacteria provides a scientific basis for future precise dose adjustment according to the specific patient flora composition, realizes the leap from "extensive" to "precision".

[0022] 3. The present invention successfully bridges the huge gap between in vitro experiments and in vivo effectiveness. In a rat caries model, topical application of 4-8 mmol / L EA reduced the level of Streptococcus saliva in a manner comparable to the gold standard positive control (0.12% chlorhexidine, 1000 mg / L sodium fluoride). Furthermore, it showed superior potential to sodium fluoride in preventing pit and fissure caries. This in vivo experimental data convincingly demonstrates the outstanding effectiveness of EA in the complex environment of the real oral cavity, providing the most crucial support for its practical application.

[0023] 4. The present invention, through systematic safety evaluation, confirms that EA is non-hemolytic at effective concentrations, non-toxic to major organs and oral mucosa, and does not affect the normal growth and development of animals. This is in stark contrast to the side effects of chemical caries prevention agents such as chlorhexidine, such as staining and taste interference. Therefore, the present invention not only provides an effective means of caries prevention, but also meets consumers' core demands for "natural" and "safe" daily chemical products, and solves the long-standing contradiction in the prior art of "difficulty in balancing effectiveness and safety".

[0024] 5. This invention combines the core active ingredient (EA) of Xinjiang pomegranate peel, a traditional Uyghur medicinal material, with the prevalent dental caries problem in modern society. Through modern science and technology, its specific uses and mechanisms of action have been clarified. This greatly enhances the scientific connotation and economic value of Xinjiang's distinctive medicinal material, and provides a solid and irrefutable chain of theoretical and experimental evidence for developing it into oral care products (toothpaste, mouthwash, varnish, etc.) with clear efficacy claims (such as "anti-plaque" and "anti-caries"). It has huge market potential. Attached Figure Description

[0025] Figure 1 This invention relates to the effects of EA on the growth and acid production of three major cariogenic planktonic bacteria (A~C: under the action of EA). S. mutans , S. sobrinus , S. sanguinis 24-hour growth curve ( A 595 nm); D~F: Under the action of EA S. mutans , S. sobrinus , S. sanguinis (24-hour acid production curve); Figure 2 This invention relates to the effects of EA on the biofilm activity and structural morphology of three major cariogenic bacteria in this embodiment (A: crystal violet staining; B: CLSM microscopy image of live dead bacteria (40×); C: SEM microscopy image of biofilm structure (2000×, 5000×)). Figure 3is the influence of EA on the virulence factors of cariogenic bacteria biofilm in the embodiment of the present application (A: CLSM under the fluorescence staining image of biofilm and extracellular polysaccharide (40x); B: CLSM under the image of bacterial adhesion to saliva-coated HAP (100x)); Figure 4 is the in vivo caries prevention effectiveness of local application of EA in the embodiment of the present application (A: smooth surface caries Keyes score; B: pit and fissure caries Keyes score; C, E: stereomicroscope of the smooth surface and sagittal section of the dentition, different colored arrows point to different degrees of caries damage; D, F: the left graph is the enamel area identification graph (gray scale display) in the original Micro-CT image; the middle graph is the three-dimensional reconstruction graph of enamel (pseudo-color highlights structure); the right graph is the three-dimensional reconstruction graph of enamel separated alone; in the graph, the EA high, EA medium and EA low groups correspond to the experimental intervention concentrations of 8 mmol / L, 4 mmol / L and 2 mmol / L, respectively, and different superscript letters indicate significant differences between groups, P<0.05); Figure 5 is the in vivo biological safety evaluation of local application of EA in the embodiment of the present application (A: HE staining of oral mucosa and major organs (200x); B: hemolysis experiment results; C: body weight change of rats; D: alkaline phosphatase (ALP); E: aspartate aminotransferase (AST); F: alanine aminotransferase (ALT); G: creatinine (CREA); H: urea nitrogen (BUN). D-H: the red dotted line shows the normal reference range of serum biochemical indicators. In the graph, the EA high, EA medium and EA low groups correspond to the experimental intervention concentrations of 8 mmol / L, 4 mmol / L and 2 mmol / L, respectively, and different superscript letters indicate significant differences between groups, P<0.05). DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present application clearer, the following will combine the drawings of the present application with the embodiments to further describe the present application in detail. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. Figures 1-5

[0027] The implementation of the present application will be described in detail in combination with specific embodiments.

[0028] The present application provides the application of Punica granatum peel-derived ellagic acid in caries prevention, which can effectively inhibit multiple cariogenic bacteria, and systematically intervene in biofilm formation, acid production and adhesion, etc. key caries links, while having good safety. In addition, Punica granatum peel extract ellagic acid has shown significant caries prevention efficiency in in vitro and in vivo experiments.

[0029] The following is the specific implementation of the present application scheme: Materials and methods: ​

[0030] The experiment implementation time of the present application is from February 2025 to August 2025. All animal experiment procedures of the embodiments of the present application are approved by the Experimental Animal Research Ethics Committee of Xinjiang Medical University (Approval No.: IACUC-JT-20240415-16).

[0031] 1. Experimental materials Instruments: TS-2102C constant temperature shaker (Shanghai Tianrui Technology Co., Ltd., Shanghai, China); Multiskan GO full-wavelength enzyme marker (Thermo Fisher, USA); M320 F12 stereomicroscope (Leica, Germany); TCS-SP8 confocal laser scanning microscope (CLSM; Leica, Germany); scanning electron microscope (SEM; Thermo Fisher, USA); automatic tissue embedding machine (Leica, Germany); pathological microscope (Carl Zeiss, Germany).

[0032] 2. Reagents: brain heart infusion medium (BHI; BD biosciences, USA); Mitis Salivarius Agar (MAS; Beijing Luqiao Technology Co., Ltd., Beijing, China); microaerophilic gas production bag (Mitsubishi Gas Chemical Co., Ltd., Japan); LIVE / DEAD™ BacLight™ bacterial viability kit (SYTO®9 / PI) (Thermo Fisher, USA); a-glucan staining agent: Alexa Fluor™ 647-ConA fluorescent probe (Thermo Fisher, USA); murexide (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China); crystal violet (Beijing Solay Technology Co., Ltd., Beijing, China); chlorhexidine (0.12%, Shenzhen Nanyue Pharmaceutical Co., Ltd., Shenzhen, China); hydroxyapatite (HAP; Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China); ellagic acid (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China).

[0033] 3. Experimental animals: 48 specific pathogen free (SPF) Sprague-Dawley (SD) rats [all healthy males, body weight (40±5) g] were purchased from the Experimental Animal Center of Xinjiang Medical University and were raised in the SPF sterile environment of the Experimental Animal Center of Xinjiang Medical University.

[0034] 4. Preparation of the medicine solution Accurately weigh ellagic acid standard and dissolve it in dimethyl sulfoxide (DMSO) to prepare a stock solution (64 mmol / L). When using it, use BHI liquid medium. For biofilm experiments, use BHI liquid medium containing 1% sucrose (BHI-S) to serially dilute the solution to obtain eight drug-containing mediums with a gradient concentration of 32, 16, 8, 4, 2, 1, 0.5, and 0.25 mmol / L (final DMSO concentration ≤1%). Prepare the solution fresh each time.

[0035] 5. Strains culture The experimental strain was S. mutans UA159 (ATCC 700610) S. sobrinus (ATCC 6715) and S. sanguinis (ATCC 10556) (Guangdong Provincial Microbial Culture Collection Center). Frozen strains were revived on BHI plates, and single colonies were inoculated with BHI solution and cultured microaerophilically at 37 ℃, 5% O2, 10% CO2, and 85% N2 for 24 h (to reach the logarithmic growth phase). The bacterial suspension concentration was adjusted to OD using BHI solution. 630 =0.2 (approximately 1×10) 7 (CFU / mL) for later use.

[0036] 6. Planktonic bacteria experiment (1) Minimum inhibitory and bactericidal concentrations The 2-fold dilution method was used, mixing 0.25-16 mmol / L EA solution / BHI containing 1% DMSO with bacterial culture at a ratio of 10:1 (V / V), and incubating at 37 ℃ with microaerophilic shaking for 24 h. The culture was then analyzed using a microplate reader. A 595 nm, calculate the antibacterial rate. Antibacterial rate (%) = [1-( A 595 nm experimental group - A 595 (drug solution control group) / ( A 595 nm negative control group- A 595 [nm blank control group] × 100%. The lowest drug concentration at which the inhibition rate reaches 50% is the 50% minimum inhibitory concentration (MIC). 50 Take the MIC. 5020 μl of bacterial solution from wells of concentration 1 and above is evenly spread onto BHI agar plates. After incubation, the lowest drug concentration corresponding to a colony count ≤ 3 is taken as the minimum bactericidal concentration (MCB).

[0037] (2) Acid production capacity The method was the same as in 1.4.1, using BHI-S medium and expanding the concentration to 32 mmol / L. The pH of the culture medium was measured using a precision pH meter at 0, 1, 2, 6, 8, 12, 16, 18, 20, and 24 h, and the pH-time curve was recorded and the 24-h pH decrease (ΔpH) was calculated.

[0038] (3) Extracellular polysaccharide production The sulfuric acid-anthrone method was used: MIC, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC EA were co-cultured with bacterial culture for 24 h at 4℃ and 12000 rpm (R=10 cm) for 30 min. The supernatant was discarded, and the precipitate was repeatedly washed with 0.4 mol / L NaOH. The supernatants were combined. 200 μl of the supernatant was reacted with 600 μl of anthrone reagent at 95℃ for 10 min, and the concentration was determined. A 625 nm, the content of water-insoluble extracellular polysaccharides (IEPS) was calculated using the dextran standard curve.

[0039] 7. Biomembrane Experiment (1) Biofilm inhibition and scavenging concentrations The crystal violet staining method was used to determine the minimum biofilm inhibition concentration (MBIC): EA (0.25-16 mmol / L) was co-cultured with the bacterial culture for 24 h; the minimum biofilm reduction concentration (MBRC) was determined: the culture was first incubated for 24 h to form a mature biofilm, then replaced with fresh medium containing EA (0.5-32 mmol / L) and cultured for another 24 h. Both methods involved methanol fixation, crystal violet staining, and ethanol dissolution before measurement. A 595 nm. According to the formula: inhibition rate = [1-( A 595 nm experimental group - A 595 (drug solution control group) / ( A 595 nm negative control group- A595 MBIC = 100 x (ODnm of test sample - ODnm of blank control) / ODnm of blank control 50 and MBRC 50 .

[0040] (2) Live-dead staining and CLSM observation The BHI-S was diluted to the concentration of EA solution of MBIC, 1 / 2 MBIC and 1 / 4 MBIC as experimental groups, and the BHI-S containing 1% DMSO as a control group. In the confocal special culture dish, three kinds of bacterial suspensions and EA solution (v:v=1:10) were added to form 24h biofilm, and after gentle washing with sterile phosphate buffered saline (PBS), 500μl of SYTO®9 / PI (1:1) mixed staining solution was added, and the staining was carried out at 37°C for 15min in the dark. SYTO®9 can penetrate the intact cell membrane to dye live and dead bacteria as green, while PI can only enter the damaged dead bacteria and dye them as red through competitive binding, so as to distinguish live and dead bacteria. Under CLSM, 488nm / 535nm excitation wavelength was used, 40x objective was used for observation, 3 fields of view were randomly collected for each sample, and the three-dimensional structure of biofilm and the proportion of live and dead bacteria were analyzed.

[0041] (3) Sample preparation for scanning electron microscope The sterile glass was placed in a 12-well plate, and the biofilm was cultured under the above conditions, and then washed with PBS, fixed with 2.5% glutaraldehyde at 4°C overnight, dehydrated with ethanol gradient, critical point drying, ion sputtering gold plating, and observed under SEM at 2000 to 5000 times and collected images.

[0042] 8. Detection of biofilm virulence factors (1) Biofilm acid production The 24h biofilm was cultured in a 12-well plate, and then replaced with BHI-S containing EA (2MBIC, MBIC, 1 / 2MBIC, 1 / 4MBIC) and continued to culture for 24h, and the supernatant was collected, centrifuged at 8000 rpm (R=10cm) at 4°C for 30min, and the pH value of the supernatant was determined and ΔpH was calculated.

[0043] (2) Fluorescent staining of exopolysaccharide Alexa Fluor™ 647 was added to the bacterial suspension (1:1000), and the biofilm was formed by co-culturing with EA (MBIC, 1 / 2 MBIC, 1 / 4 MBIC, 1 / 8 MBIC) for 24h, and then washed with PBS and SYTO®9 restained for 15min. CLSM 650nm / 488nm dual-channel was used to collect EPS and bacterial distribution images.

[0044] (3) Bacterial adhesion experiment A hydroxyapatite-artificial saliva-coated model was established. SYTO®9-labeled bacteria were co-incubated with EA (MIC, 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, 1 / 16 MIC, 1 / 32 MIC) for 2 h. After washing, HAP was resuspended, and the adhesion rate was quantified using a fluorescence microplate reader (excitation 495 nm / emission 525 nm). The adhesion morphology was observed using CLSM.

[0045] 9. In vivo experiments (1) Animal grouping and modeling Forty-eight 17-day-old SPF-grade male SD rats were randomly divided into eight groups (n=6) using a random number table: a caries model group (intervention with distilled water); a solvent control group (negative control, EA solution solvent: intervention with 1% DMSO BHI); a chlorhexidine group (positive control, intervention with 0.12% chlorhexidine); a sodium fluoride group (positive control, intervention with 1000 mg / L sodium fluoride); three experimental groups (intervention with EA solution): high (8 mmol / L), medium (4 mmol / L), and low (2 mmol / L) dose groups of EA; a blank control group was fed a routine diet (without special intervention), while the other groups were fed a caries-inducing diet.

[0046] From 18-20 days of age, administer antibiotic solution containing 0.2 g / L potassium penicillin and 1.5 g / L streptomycin sulfate. From 21 days of age (except for the blank control group), switch to cariogenic feed No. 2000 and 5% sucrose solution, and inoculate the mouth twice daily for 3 consecutive days with the mixed bacterial solution. S. mutans : S. sobrinus : S. sanguinis = 1:1:1, 10 8 CFU / mL, 500 μl / animal).

[0047] (2) Intervention and detection From 27 to 47 days of age, oral medication was applied twice daily for ≥1 minute each time, followed by fasting and water restriction for 0.5 hours. Oral swabs were collected at 24, 30, 36, 42, and 48 days of age, diluted, and spread on MSA medium to count the number of streptococci in the oral cavity of each group of rats. At 48 days of age, rats in each group were deeply anesthetized using the veterinary compound anesthetic Shutai 50 (active ingredients: 125 mg / mL each of telastatin hydrochloride and zolazepam hydrochloride, Virbac, France). Euthanasia was performed by blood collection from the abdominal aorta. The jawbone was harvested for Micro-CT scanning, and the remaining teeth were stained with 0.4% ammonium purpurate and treated according to the modified Keyes method.

[16] Double-blind scoring was performed for both pit and fissure caries and smooth surface caries.

[0048] 10. Safety Evaluation Whole blood from the abdominal aorta of rats in the blank control group was used for an in vitro hemolysis test. To assess liver and kidney function, serum was prepared from whole blood in each group, and alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and blood urea nitrogen (BUN) were measured. Heart, liver, spleen, lung, kidney, and oral mucosa were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and histopathological changes were observed.

[0049] 11. Statistical Analysis Statistical analysis was performed using SPSS 26.0 software. Normality of continuous data was verified by the Shapiro-Wilk test, and homogeneity of variance was verified by the Levene test. Data that conformed to a normal distribution and had homogeneity of variance were denoted as follows: This indicates that one-way ANOVA was used for comparisons among multiple groups, and the LSD test was used for further pairwise comparisons; continuous data that do not conform to normal distribution or have unequal variances are expressed as median (interquartile range) [M(IQR)], and the Kruskal-Wallis H test was used for comparisons between groups. P A value <0.05 (two-tailed test) indicates that the difference is statistically significant.

[0050] The following are the results of the experiments in the above embodiments: 1. Effects of EA on the growth activity of airborne bacteria and caries-related virulence factors (1) EA inhibits the growth of three major cariogenic bacteria. EA showed effective inhibitory effects against all three oral streptococci (Table 3). S. sanguinis Highest antibacterial activity (MIC) 50 =1.0 mmol / L), S. sobrinus Second (MIC) 50 =2.0 mmol / L), for S. mutans Its activity is relatively low (MIC) 50 =4.0 mmol / L). S. mutans and S. sobrinus All MBCs are MIC 50 Twice that of EA. As EA concentration increased, the growth curves of each strain showed a clear concentration dependence, with high concentrations of EA (≥MIC) showing the highest growth rate. 50 This not only significantly delays the time it takes for bacteria to enter the logarithmic growth phase, but also greatly reduces the final culture size. A Values ​​(Table 1). At sub-inhibitory concentrations (e.g., 1 / 2 MIC)50 )The bacterial growth was still inhibited to some extent, indicating that EA still had potential antibacterial effect at lower concentrations.

[0051] (2) Effect of EA on acid production of 3 major cariogenic bacteria in planktonic state The acid production ability of bacteria is the core of its cariogenic virulence. After intervention by high concentration EA (≥ MIC 50 ), the speed and amplitude of pH decline were significantly slowed down (D-F), and the final pH value and ΔpH of the culture medium (Table 1) were significantly higher than those of the control group (G). Figure 1 <0.001). This indicates that EA can weaken the acid production ability of bacteria by inhibiting their growth and metabolic activity, thereby reducing the risk of hard tissue demineralization. P <0.001). This indicates that EA can weaken the acid production ability of bacteria by inhibiting their growth and metabolic activity, thereby reducing the risk of hard tissue demineralization.

[0052] Table 1 Final acid production (ΔpH) of 3 cariogenic bacteria after 24 h of EA treatment at different concentrations

[0053] Note: a indicates a significant difference compared with the negative control group P <0.05.

[0054] (3) Effect of EA on IEPS production of 3 major cariogenic bacteria in planktonic state EA inhibited the formation of IEPS of 3 major cariogenic bacteria in planktonic state, and significantly reduced the yield of IEPS synthesized by the three bacteria in a dose-dependent manner (Table 2). Even at sub-inhibitory concentration (1 / 2 MIC 50 ), the synthesis of IEPS was already significantly disturbed.

[0055] Table 2 Yield of water-insoluble exopolysaccharide (IEPS) of 3 cariogenic bacteria after 24 h of EA treatment at different concentrations

[0056] Note: Different superscript letters indicate significant differences between groups, P <0.05.

[0057] 2, Effect of EA on biofilm growth activity and caries-related factors (1) Effect of EA on biofilm formation and removal activity of 3 major cariogenic bacteria In terms of antibiofilm, the concentration of EA that inhibited biofilm formation was similar to its corresponding MBC value (Table 1, Figure 2 A, except S. sanguinis -MBIC 50 = 1 mmol / L). For the mature biofilm that had already formed, the removal ability of EA was significantly reduced, and the MBRC 50All ≥ 32 mmol / L. It suggested that EA could effectively inhibit planktonic growth and prevent biofilm formation, but it was more challenging to remove established biofilms.

[0058] Table 33 MIC of three major cariogenic bacteria 50 , MBC, MBIC 50 and MBRC 50 (mmol / L)

[0059] (2) Effect of EA on live and dead bacteria in biofilm of three major cariogenic bacteria Biofilm live and dead bacteria staining Control group biofilm was dense Figure 2 B), green fluorescence (SYTO®9+, live bacteria) dominated, suggesting that biofilm was vigorous. After EA intervention, green fluorescence was replaced by red fluorescence (PI+, dead bacteria), and red fluorescence signal was significantly enhanced. With increasing concentration, the proportion of red fluorescence increased, and the green fluorescence area gradually decreased and became sparse. It showed that EA not only inhibited the growth of biofilm, but also effectively penetrated the biofilm matrix. Under the treatment of high concentration EA (MBIC), the overall structure of biofilm lost integrity and became a loose aggregate dominated by dead bacteria.

[0060] (3) Effect of EA on the morphological structure of biofilm of three major cariogenic bacteria Control group biofilm showed a typical three-dimensional architecture Figure 2 C), bacteria were wrapped and tightly adhered by a large amount of extracellular matrix secreted by themselves. After EA intervention, the structure of biofilm was destroyed: with increasing EA concentration (1 / 2 MBIC 50 ), the extracellular matrix of biofilm decreased, the dense network structure disintegrated, and the internal bacteria lost the chain structure, and the remaining bacterial cell morphology shrunk and lysed. In the high concentration EA treatment group (MBIC 50 ), only scattered bacteria and extracellular matrix fragments were left. Morphologically, it was proved that EA had the effect of inhibiting biofilm formation and degrading matrix.

[0061] (4) Effect of EA on the acid-producing activity of biofilm of three major cariogenic bacteria Biofilm acid-producing activity determination (Table 4) reflected the key indicator of EA on the cariogenic activity of biofilm of three cariogenic bacteria. With increasing EA concentration (from 1 / 8 MBIC to 1 / 2 MBIC), the pH reduction (ΔpH) of the culture solution of each group of biofilm decreased significantly P <0.01). At 1 / 2 MBIC concentration, the acid-producing ability of biofilm was strongly inhibited, which was consistent with the trend of EA inhibiting planktonic acid production.

[0062] Table 4 Effect of EA on the acid-producing ability of biofilm of three cariogenic bacteria

[0063] Note: a Indicates a comparison with the negative control group P <0.05.

[0064] (5) Effects of EA on extracellular polysaccharide formation in three major cariogenic bacteria In the control group, the green fluorescence (SYTO®9+, labeled biomembrane) signal was dense ( Figure 3 A), coexisting tightly with a continuous network structure formed by red fluorescence (Alexa Fluor™ 647+, labeled α-glucan), exhibits a complete and dense structure. After EA intervention, S. mutans and S. sanguinis The intensity of the red EPS signal decreased sharply with increasing drug concentration, and the red signal almost disappeared completely in the high-concentration EA group. The green bacterial signal also became scattered and sparse. Combined with the experimental results in 2(2), it shows that EA can not only inhibit the survival and aggregation of bacteria, but also strongly interfere with the synthesis of extracellular polysaccharides or promote their degradation.

[0065] (6) Effect of EA on the ability of three major cariogenic bacteria to adhere to saliva and coat HAP CLSM microscopy showed bacterial adhesion to saliva-coated HAP control group ( Figure 3 B), it can be seen that the green fluorescently labeled bacteria adhere tightly to the surface of the hydroxyapatite group (dental enamel biomimetic model). After EA intervention, the fluorescence intensity per unit area of ​​green fluorescence on the HAP surface was significantly reduced (Table 5). The results further illustrate that EA can effectively interfere with the initial adhesion of bacteria to the tooth surface and has the potential to achieve effective intervention in the first step of plaque formation.

[0066] Table 5. Adhesion rate of hydroxyapatite (%) of three cariogenic bacteria under different concentrations of EA.

[0067] 3. Efficacy of EA in preventing tooth decay (1) EA significantly reduced the level of Streptococcus salivarius in rats. In in vivo experiments, streptococcal bacterial counts showed that EA significantly reduced streptococcal levels in the oral cavity of rats (Table 6). Compared with the caries model group and the solvent control group, the number of streptococci in the saliva of rats in all EA dose groups (especially the medium and high doses) was significantly reduced. P <0.05), with efficacy comparable to the chlorhexidine group. This indicates that EA can effectively inhibit the proliferation of cariogenic streptococci in the complex oral environment, reducing the microbial burden of caries.

[0068] Table 6. Changes in streptococcal counts in saliva of rats in different treatment groups over time (Log) 10 CFU / mL

[0069] Note: EA high, EA medium, EA low group in the table corresponds to the experimental intervention concentration 8 mmol / L, 4 mmol / L, 2 mmol / L, different superscript letters represent significant difference between groups, P <0.05).

[0070] (2) Keyes score under stereomicroscope and Micro-CT detection Keyes caries score Figure 4 B, C), EA medium (4 mmol / L), high (8 mmol / L) dose group of smooth surface caries score was significantly lower than the model group (P<0.05), the effect is close to the sodium fluoride control group. P <0.05), EA high dose group (8 mmol / L) showed the strongest protection against pit and fissure caries, the score was significantly lower than the sodium fluoride group (P<0.05), indicating that EA may have special potential in preventing pit and fissure caries. P <0.05), the stereomicroscope image provides intuitive morphological evidence for Keyes score (D, F). Micro-CT image and three-dimensional reconstruction (E, G) are used to observe the mineral density and structural integrity inside the tooth, and three-dimensional reconstruction more intuitively reflects the intact morphology of EA group teeth. Figure 4 Figure 4 4. Biocompatibility of EA in vivo The results of hemolysis experiment (B) showed that different concentrations of EA solution had no hemolytic effect. The health status of rats in each group was good, and the vitality was normal, and there was no death individual. During the treatment period, the body weight of rats in each group increased uniformly, and the results were shown in

[0071] C. HE staining proved the safety of EA to oral mucosa and main organs, and the cell morphology and structure were normal, and there was no obvious pathological change, and the results were shown in A. There was no significant difference in liver function index (D-F) and liver function index (G-H) between each treatment group and caries model group (P>0.05). The above results all showed that EA was a relatively safe method for preventing and treating the progression of dental caries. Figure 5 Figure 5 Figure 5 Figure 5 Figure 5 P >0.05). The above results all showed that EA was a relatively safe method for preventing and treating the progression of dental caries.

[0072] ​​​​​With the current state of the art, caries prevention is a key strategy for controlling dental caries. Natural drugs have become an ideal strategy for caries prevention due to their multi-target effects, such as antibacterial, anti-demineralization and promotion of remineralization. At present, a variety of natural extracts have been widely used in oral care products, such as toothpaste containing gall extract, tea polyphenol toothpaste, sipa gingival fluid and xylitol chewing gum, etc., which shows good transformation prospects. At the mechanism level, different natural ingredients show diverse intervention pathways. Effective components of Magnolia officinalis can intervene in the caries process through multiple targets: inhibit bacterial growth and acid production, reduce glucosyltransferase (GTF) activity to destroy biofilm, thereby achieving the effect of preventing caries. Tannins in Chinese sumac can inhibit the growth of various cariogenic bacteria by binding to the cell surface lipoteichoic acid, and the hydroxyl groups in tannins can promote early enamel caries remineralization. The tannin components in purple sage can inhibit the activity of GTF and lactate dehydrogenase (LDH) to block the acid production of cariogenic bacteria; at the same time, they can bind to proteins and calcium ions in demineralized enamel to form stable acid-resistant chelates, slow down the demineralization process and enhance the remineralization ability. Existing studies have found that the main component EA in Myrtus communis extract can prevent caries by inhibiting the activity of GTF, slowing down the pH drop caused by the glycolysis of Streptococcus mutans and reducing the production of lactic acid (and this process does not depend on the reduction of LDH activity). Pomegranate peel in Xinjiang is also rich in tannins and has strong antibacterial activity; through systematic in vitro and in vivo experiments, it is confirmed that the key active ingredient EA can affect the biological behavior of cariogenic bacteria in multiple ways, and it shows significant caries prevention activity in rat models. This component has antibacterial, biofilm destruction and virulence expression inhibition effects, which can effectively prevent caries, and the biological safety is good throughout the experiment.

[0073] Studies have confirmed that EA has a significant inhibitory effect on the growth and adhesion of periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum planktonic. The antibacterial biofilm efficacy of EA against Escherichia coli is mainly due to its ability to disrupt the tryptophan repressor protein binding protein A-driven bacterial redox homeostasis, and the inhibition of EA on the adhesion of E. coli is specific and not indirect. EA prevents the early colonization of Streptococcus agalactiae biofilm by inhibiting early adhesion and extracellular matrix accumulation, and its essence may lie in the process of biofilm adhesion and construction, rather than just resisting planktonic bacteria. EA shows significant antibacterial activity against Pseudomonas aeruginosa, and can inhibit biofilm formation, reduce bacterial adhesion and reduce EPS production, which may be closely related to its inhibition of polyphosphokinase 1 and synergistic effect on other key virulence factors. Activity screening and component analysis show that the GTF potent inhibitor in EA is mainly EA. Some scholars have found that EA from red fruit can inhibit the activity of GTF by non-competitive inhibition to inhibit S. mutansBiofilm formation. In the study of caries biofilm, the destruction of its structural integrity has become an important direction of intervention. Unlike the strategy of simply reducing the number of flora, directly disintegrating the three-dimensional structure of biofilm can fundamentally weaken its function as a pathogenic entity. The embodiments of the present application prove that EA has good bacteriostatic effect, and at the same time significantly inhibits IEPS synthesis and bacterial adhesion. Combined with the results of the experiments of the embodiments of the present application, it can be preliminarily inferred that EA may block IEPS generation by inhibiting GTF activity, effectively interfere with the adhesion and colonization process of bacteria, and then inhibit biofilm formation.

[0074] In the in vivo disease model of the above-mentioned embodiments of the present application, EA significantly inhibits the colonization and adhesion of streptococci in the oral cavity on the basis of maintaining good biological safety, effectively slows down the occurrence and development of caries lesions. It shows that EA has potential application value in the prevention and early intervention of dental caries. However, the complexity of the oral microenvironment requires more in-depth systematic study of the long-term clinical anticaries effect of EA. Compared with the traditional single inoculation of mutans streptococci dental caries animal model, the mixed infection model containing S. mutans 、 S. sanguinis and S. sobrinus used in the present application better simulates the initial colonization and development process of oral flora. The successful application of the multi-species model shows that the mechanism of EA in preventing dental caries does not depend on a single target of a specific strain, but plays a role by affecting the collective behavior of cariogenic flora and its microenvironment.

[0075] In summary, the above-mentioned embodiments of the present application system reveal that Xinjiang pomegranate peel ellagic acid (EA) can significantly prevent dental caries and has good biological safety in in vivo and in vitro models by interfering with the adhesion, biofilm formation and metabolic toxicity of cariogenic bacteria. These findings not only provide a new idea for the research of natural product in preventing dental caries, but also provide a theoretical basis for the development of eco-friendly anti-caries products.

[0076] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of ellagic acid in the preparation of a medicament or oral care product for preventing and / or treating dental caries.

2. Use according to claim 1, characterized in that, The use is to inhibit planktonic growth and / or biofilm formation of at least one of Streptococcus mutans, Streptococcus sobrinus and Streptococcus sanguis.

3. Use according to claim 2, characterized in that, The 50% minimum inhibitory concentration of the ellagic acid against Streptococcus mutans in planktonic state is 4 mmol / L, against Streptococcus sobrinus in planktonic state is 2 mmol / L, and against Streptococcus sanguis in planktonic state is 1 mmol / L.

4. Use according to claim 1, characterized in that, The use is to inhibit acid production and / or water-insoluble exopolysaccharide production of the cariogenic bacteria.

5. Use according to claim 1, characterized in that, The use is to inhibit adhesion of the cariogenic bacteria to hydroxyapatite surface.

6. Use according to claim 1, characterized in that, The use is to reduce the number of Streptococcus in the oral cavity of a mammal and / or to reduce the extent of dental caries lesions.

7. Use according to claim 6, characterized in that, The effective concentration of the medicament is 2 mmol / L to 8 mmol / L.

8. Use according to claim 7, characterized in that, The medicament is administered by oral coating.

9. Use of ellagic acid for the preparation of a product for inhibiting the formation of biofilm of cariogenic bacteria, characterized in that, The 50% minimum inhibitory concentration of the ellagic acid against biofilm formation of Streptococcus mutans is 8 mmol / L, against biofilm formation of Streptococcus sobrinus is 4 mmol / L, and against biofilm formation of Streptococcus sanguis is 1 mmol / L.

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