Application of Xinjiang medicine mulberry extract in preparation of medicine for treating or preventing periapical periodontitis

Abstract

The invention relates to the technical field of medicines, and particularly discloses application of a Xinjiang medicine mulberry extract in preparation of a medicine for treating or preventing periapical periodontitis, which is technically characterized in that the Xinjiang medicine mulberry extract is found and proved to have a remarkable inhibition effect on periapical periodontitis key pathogenic bacterium enterococcus faecalis for the first time, the minimum inhibitory concentration (MIC) is 0.604 mg / mL, and the Xinjiang medicine mulberry extract has a remarkable inhibition effect on the periapical periodontitis key pathogenic bacterium enterococcus faecalis. The minimum bactericidal concentration (MBC) is 1.875 mg / mL, and a biological membrane can be effectively inhibited and removed; in-vivo experiments of a rat periapical periodontitis model prove that the extract can remarkably reduce the number of bacteria in root canals and relieve periapical bone destruction, the curative effect is equivalent to that of calcium hydroxide, and the extract is non-toxic to main organs and high in biocompatibility. The invention provides a new candidate and a clear technical scheme for developing an efficient and low-toxicity natural plant source root canal disinfectant.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of Xinjiang mulberry extract in the preparation of drugs for inhibiting Enterococcus faecalis and for treating or preventing periapical periodontitis. Background Technology

[0002] Periapical diseases of the dental pulp are common oral diseases, usually resulting from mixed infections of various microorganisms, primarily anaerobic bacteria. Currently, root canal treatment is the main method for controlling infection and inflammation. However, due to the complexity of the root canal system and the limitations of existing disinfectants, such as phenolic compounds, sodium hypochlorite, and calcium hydroxide, which have problems like cytotoxicity and drug resistance, recurrent infections and treatment failures are common. Therefore, developing novel, highly effective, and low-toxicity root canal disinfectants is a research hotspot in the field of endodontics.

[0003] Enterococcus faecalis ( Enterococcus faecalis It is a dominant pathogen in persistent periapical periodontitis and root canal treatment failure. It has a strong ability to form biofilms and is resistant to environmental conditions. It is prone to developing resistance to common root canal disinfectants such as calcium hydroxide, and may even form a more robust biofilm in a highly alkaline environment, which is a challenge in clinical treatment.

[0004] Mulberry (Morus alba) from Xinjiang is a medicinal and edible plant in traditional Uyghur medicine. Its active ingredients include flavonoids, phenolic acids, and alkaloids, exhibiting anti-inflammatory and antioxidant pharmacological activities. Current research mainly focuses on the inhibitory effects of mulberry or leaf extracts on caries-related bacteria (such as Streptococcus mutans) or their anti-inflammatory effects. However, whether mulberry extract from Xinjiang has an inhibitory effect on key pathogens of periapical diseases (Enterococcus faecalis) is currently unclear, and there is a lack of relevant animal studies to support its in vivo efficacy and safety.

[0005] Therefore, this application proposes the use of Xinjiang mulberry extract in the preparation of drugs for the treatment or prevention of periapical periodontitis, aiming to provide a novel natural root canal disinfectant that is effective against Enterococcus faecalis and has good biocompatibility, in order to solve the above-mentioned problems. Summary of the Invention

[0006] The purpose of this invention is to solve the technical problems mentioned in the background section and to provide the application of Xinjiang mulberry extract in the preparation of drugs for treating or preventing periapical periodontitis. The above-mentioned objective of the present invention is achieved as follows: One aspect of the present invention is the application of Xinjiang mulberry extract in the preparation of a drug for inhibiting Enterococcus faecalis.

[0007] Furthermore, the preparation method of the Xinjiang mulberry extract is as follows: take mulberry leaf raw material, use ethanol as solvent for heating and reflux extraction, and after concentration and drying, obtain mulberry leaf extract powder.

[0008] Further, the ethanol is 95% ethanol, the material-liquid ratio is 1:3, the heating reflux extraction temperature is 70 DEG C, and the extraction time is 3 hours.

[0009] Further, the medicine is used for treating or preventing periapical periodontitis caused by Enterococcus faecalis.

[0010] Further, the medicine is a root canal disinfectant.

[0011] Further, the minimum bacteriostatic concentration of the Xinjiang mulberry extract on Enterococcus faecalis is 0.604 mg / mL, and the minimum bactericidal concentration is 1.875 mg / mL.

[0012] Further, the minimum concentration of the Xinjiang mulberry extract required for inhibiting 50% Enterococcus faecalis biofilm formation is 1.875 mg / mL, and the minimum concentration required for removing 50% formed Enterococcus faecalis biofilm is 3.75 mg / mL.

[0013] The scheme of the present application also provides a pharmaceutical composition for treating or preventing periapical periodontitis, comprising an effective amount of Xinjiang mulberry extract and pharmaceutically acceptable adjuvants.

[0014] Further, the dosage form of the pharmaceutical composition is a root canal disinfectant paste, an irrigation fluid, a nanoemulsion or a hydrogel.

[0015] Further, the Xinjiang mulberry extract is prepared by a method comprising the following steps: taking mulberry leaf raw materials, using 95% ethanol as a solvent, performing 70 DEG C heating reflux extraction for 3 hours at a material-liquid ratio of 1:3, and obtaining mulberry leaf extract powder after concentrating and drying the extract.

[0016] The difficulty and significance of the present application in solving the technical problems are as follows: At present, the mainstream root canal disinfectant (such as calcium hydroxide) in the prior art has a fatal paradox of "inducing drug resistance" and "promoting biofilm formation". For the prior art, it is not only necessary to solve "effectiveness", but also to overcome the side effect of "the more you use, the more difficult it is to treat". This is a typical clinical treatment bottleneck problem. Since the target pathogenic bacteria Enterococcus faecalis is a recognized "biofilm master" and "persistent colonizer in root canal", it is more difficult to develop a drug that not only can kill planktonic bacteria, but also can penetrate and disintegrate the mature biofilm structure of the bacteria than to inhibit ordinary bacteria. The present application first determines the quantitative efficacy of Xinjiang medicine mulberry extract against Enterococcus faecalis and biofilm and in-vivo safety through systematic in-vivo and in-vitro experiments, fills the research gap in this specific field, and provides a complete and rigorous research paradigm (from extraction, in-vitro efficacy evaluation to animal model verification) for screening and verifying natural drugs against specific oral pathogenic bacteria from the treasure trove of traditional medicine. The present application not only means the birth of a potential new drug, but also verifies a feasible path for combining traditional national medical wisdom and modern precision medical needs to develop characteristic natural drugs, which has a breakthrough, demonstration and broad transformation prospect.

[0017] Compared with the prior art, the present application has the following beneficial effects: 1. The scheme of the present application first discloses the inhibition effect of Xinjiang medicine mulberry extract on Enterococcus faecalis, and proves through rigorous in-vitro experiments that Xinjiang medicine mulberry extract has a clear inhibitory and killing effect on the key pathogenic bacteria Enterococcus faecalis of periapical periodontitis, and can effectively inhibit the formation of biofilm and remove the formed biofilm, which provides direct experimental basis for developing Xinjiang medicine mulberry extract into an anti-Enterococcus faecalis drug.

[0018] 2. In the scheme of the present application, the MIC, MBC, MBIC50 and MBEC50 and other key pharmacodynamic parameters of Xinjiang medicine mulberry extract against Enterococcus faecalis are determined for the first time. These specific values constitute a technical feature that is different from the existing vague description, and provide a scientific basis for drug dosage design.

[0019] 3. The scheme of the present application proves through in-vivo experiments on a rat periapical periodontitis model that Xinjiang medicine mulberry extract can significantly reduce the number of Enterococcus faecalis in the root canal and alleviate periapical bone destruction in vivo, and its curative effect is comparable to that of the commonly used drug calcium hydroxide. At the same time, no toxic reaction is found in the histological examination of the main organs, indicating that it has excellent biocompatibility and solves the problem of high toxicity of existing chemical disinfectants.

[0020] 4. The present application expands the application of Xinjiang medicine mulberry extract from caries prevention to the more difficult periapical periodontitis treatment field, especially for stubborn Enterococcus faecalis infection, and provides a natural drug candidate with high efficiency, low toxicity and good clinical application potential. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 This is an embodiment of the present invention. E. faecalis Crystal violet staining of biofilms (A: Different concentrations of drugs on Enterococcus faecalis) E. faecalis The role of biofilm formation; B: The effect of different drug concentrations on the removal of mature Enterococcus faecalis biofilm). Figure 2 The mulberry extract in the embodiments of the present invention has the effect on... E. faecalis Growth curve of the effect; Figure 3 This is an embodiment of the present invention. E. faecalis Laser confocal microscopy results of staining with live and dead bacteria; Figure 4 This invention relates to the animal model creation and analysis in the embodiments of the present invention (A:ad shows the Enterococcus faecalis after 24 hours of intervention). E. faecalis Typical fluorescence microscopic images (×100) of live bacteria, dead bacteria, and their combinations in biofilms; a: negative control group; b: low concentration of Morus nigra extract group: 1 / 4 MBEC 50 c: Medium concentration of mulberry extract group: 1 / 2 MBEC 50 ;d: High-concentration mulberry extract group: MBEC 50 B: 3D reconstructed images of each group; C: Analysis results of biofilms in each group using ImageJ software. Figure 5 HE staining of the major organs of rats in this embodiment of the invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0024] Example: The present invention provides the application of Xinjiang mulberry extract in the preparation of drugs for the treatment or prevention of periapical periodontitis, aiming to overcome the shortcomings of existing root canal disinfectants and provide a novel natural root canal disinfectant that is effective against Enterococcus faecalis and has good biocompatibility.

[0025] The specific experimental implementation of the present invention is as follows: 1. Materials and Methods 1.1 Preparation method of mulberry leaf extract 100g of mulberry leaf raw material was weighed and processed using the ethanol extraction method. 95% ethanol was added as solvent at a material-to-liquid ratio of 1:3, and the mixture was heated in a 70℃ water bath under reflux for 3 hours. After extraction, the extract was filtered, and the filtrate was collected. This extraction process was repeated three times, and the resulting filtrates were combined. The combined extract was concentrated using a rotary evaporator, and the 95% ethanol solvent was recovered, ultimately yielding a paste-like product. The paste was then freeze-dried to obtain a powdered mulberry leaf extract.

[0026] 1.2. Procedure for the resuscitation, culture, and identification of Enterococcus faecalis The Enterococcus faecalis used in this experiment ( E. faecalis, (ATCC29212) was purchased from Guangdong Microbial Culture Center. The cryopreservation tubes were rapidly thawed in a 37°C water bath. Using a sterile pick, the thawed culture solution was inoculated onto the surface of BHI solid medium. Microaerophilic conditions (5% O2, 85% N2, 10% CO2) were established, and colony growth characteristics were observed after 24 hours of aseptic incubation. Randomly selected single colonies were Gram-stained and examined microscopically (staining showed purple cells) to confirm a pure culture and absence of contamination. A single colony was picked from the culture dish and inoculated into a glass tube containing 15 ml of BHI liquid medium, and incubated overnight at 37°C with 5% CO2. The OD value (optical density) of the original bacterial suspension was measured using a 0.5 McFarland turbidimetric tube, and adjusted by serial dilution with appropriate amounts of BHI liquid medium. When the OD600 value reached 0.5 (approximately equivalent to 1.5 × 10⁻⁶), the culture was considered complete. 8 A concentration of CFU / mL indicates that the cell is in the logarithmic growth phase. E. faecalis The bacterial culture was diluted 10-fold with BHI medium to a concentration of 1×10⁻⁶. 7 CFU / mL concentration is available for reference.

[0027] 1.3 Determination of MIC and MBC of Xinjiang mulberry extract against Enterococcus faecalis standard strain Xinjiang mulberry extract solution was prepared by serial dilution in sterile BHI liquid medium using a two-fold dilution method. Calcium hydroxide solution was set up as a positive control group, physiological saline as a negative control group, and BHI medium without bacterial suspension as a blank control group. A concentration of 1×10⁻⁶ was used. 8 CFU / mL E. faecalis Standard strains were inoculated at a ratio of 1:100 into freshly prepared BHI liquid medium and shaken to achieve a final concentration of 1×10⁻⁶. 6After the CFU / mL level of each experimental group was used as a backup, the bacterial suspension was mixed with different concentrations of drug solution in a 96-well plate, and the mixed system was placed in a standard anaerobic environment (85% N2, 10% H2, 5% CO2) at 37°C for 24 hours. After that, the OD of each well was measured by an enzyme-labeled instrument. 600 When the drug concentration was the lowest and the OD value changed by ≤0.05, it was determined as the MIC value. From all groups with a concentration higher than the MIC, 20 μL was taken and uniformly coated on the surface of the BHI solid culture medium. The same conditions were maintained for continuous culture for 24 hours. After observation, the lowest drug concentration with ≤3-5 colonies was recorded, and this concentration was the MBC value.

[0028] 1.4, Determination of the effect of Xinjiang mulberry extract on E. faecalis standard strain MBIC 50 MBIC 50 is the minimum drug concentration that inhibits 50% of biofilm formation. E. faecalis The standard strain was resuscitated and cultured to the logarithmic growth phase, centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the resuspension was performed in BHI medium to adjust the concentration to OD 600 =0.5. The experimental groups included Xinjiang mulberry extract intervention group, calcium hydroxide as positive control group, and normal saline as negative control group. The bacterial suspension was mixed with the drug-containing medium in a test tube at a volume ratio of 1:3, and cultured under the conditions of 85% N2, 10% H2, 5% CO2, and 160 rpm for 24 hours. The supernatant was gently aspirated, the plate was washed with sterile PBS, and methanol was fixed for 15 min. After washing the plate, the biofilm was stained with 0.1% crystal violet for 5 min, and then the plate was dried in a dark and ventilated place. 200 μL of 95% ethanol was added to each well, and the OD=600 nm absorbance was measured after incubation at room temperature for 30 min.

[0029] 1.5, Determination of the effect of Xinjiang mulberry extract on E. faecalis standard strain MBEC 50 MBEC 50 is the minimum drug concentration that clears 50% of the formed biofilm. E. faecalis The standard strain was resuscitated and cultured to the logarithmic growth phase, centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the resuspension was performed in BHI medium to adjust the concentration to OD 600 ​​=0.5, Experimental groups: Xinjiang mulberry extract intervention group, calcium hydroxide as positive control group, and physiological saline as negative control group. 200 μL of bacterial suspension was sequentially added to the wells of different groups and incubated for 24 h. The supernatant was aspirated, and the plates were carefully washed with sterile PBS. 200 μL of the respective drug solution was added to the wells of each group, and incubation continued for 24 h. The plates were then carefully washed with sterile PBS. After fixation with methanol for 15 min, the plates were washed, and then each well was stained with 0.1% crystal violet for 5 min, followed by washing. The plates were then dried in a dark and ventilated place. 200 μL of 95% ethanol was added to each well, and the plates were incubated at room temperature with shaking for 30 min. The absorbance at 600 nm was measured to determine the immediate biomass of the biofilm.

[0030] 1.6. Extracts from Xinjiang mulberry trees on... E. faecalis Influence of growth curve The extract of Xinjiang mulberry was dissolved in BHI liquid medium using a two-fold dilution method. Calcium hydroxide was used as the positive control, physiological saline as the negative control, and BHI medium without bacterial suspension as the blank control. A concentration of 1×10⁻⁶ was used. 8 CFU / mL E. faecalis The standard strain was inoculated into freshly prepared BHI liquid medium at a ratio of 1:100, and thoroughly shaken to achieve a final bacterial concentration of 1×10⁻⁶. 6 CFU / mL; the bacterial suspension and drug solution were inoculated into 96-well plates and cultured under standard anaerobic conditions (85% N2, 10% H2, 5% CO2) at 37°C in a constant temperature shaker at 160 rpm for 24 h. During the 24 h of culture, the absorbance at 600 nm was measured using a microplate reader at time points of 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, and 24 h.

[0031] 1.7. Extracts from Xinjiang mulberry trees on... E. faecalis The standard strain has formed a biofilm scavenging effect. Place sterile cell smears into 6-well plates, inject 2 mL of the mixed bacterial suspension, and incubate for 24 h. Experimental groups: MBEC obtained from previous experiments. 50 For reference, participants were divided into a low-concentration intervention group (1 / 4 MBEC) of Xinjiang mulberry extract. 50 ), Xinjiang mulberry extract medium concentration intervention group (1 / 2 MBEC) 50 ), Xinjiang mulberry extract high-concentration intervention group (MBEC) 50) and negative control group (0.9% sodium chloride solution). After the biofilm was formed, 2 mL of the corresponding drug was added to each plate, and incubation was continued for 24 h. The plates were carefully washed twice with preheated sterile PBS. Fluorescent staining solution was prepared: 3 pL SYTO9 and 3 pL PI staining solution were added to 3 mL sterile PBS and mixed well in the dark. 500 pL of the prepared staining solution was added to each well of the slide, and incubation was continued for 15 min at 37°C in the dark. The wells were carefully rinsed with preheated sterile PBS, and the slide was removed using sterile instruments, placed on a glass slide, and observed under an inverted fluorescence microscope.

[0032] 1.8, Establishment of a rat experimental periapical periodontitis model After approval by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University [Approval No: IACUC-JT-202401119-31], 24 6-7-week-old male SD rats were selected and divided into ① experimental group: Xinjiang mulberry extract (MBEC 50 ), n=6; ② positive control group: calcium hydroxide paste; n=6 ③ model group: no intervention after modeling; n=6 ④ blank control group: no modeling and other interventions; n=6. The rats were fasted for 12 hours before modeling, and the sterile operating environment was maintained. A mixture of 10% Lumingning and Shu Tai (1:1) was injected into the hind leg muscles of the anesthetized rats (10 mg / kg). The rats were fixed in a supine position, the upper molar region was exposed, and a portable electric dental drill was used to open the pulp of the right upper first molar of the rat. A 6# stainless steel K file was used to explore and clear the root canal, remove the crown and pulp, and prepare it to 10#. After disinfection with 5.25% NaClO and 17% ethylenediaminetetraacetic acid (EDTA), the root canal was dried with a sterile paper tip, and 10 pL of bacterial solution was introduced into the root canal with the help of a 6# stainless steel K file. The opening was tightly sealed with glass ionomer, and the rat's saliva and excess bacterial solution were promptly removed during the operation. The rats were deprived of water for 2 h and food for 24 h after the pulp was opened. After the animals recovered without complications, they were continued to be housed for 4 weeks. After the success of the periapical periodontitis model was confirmed by Micro-CT, the drug was sealed in the root canal for 2 weeks. After the intervention, Micro-CT was used to observe the efficacy again. E. faecalis

[0033] 1.9, Evaluation of antibacterial activity in root canal The bacterial samples in the root canal of each group of rats before and after sealing for 2 weeks were taken for colony counting. The collected specimens were ultrasonically suspended in 500 pL of sterile PBS. The bacterial suspension was diluted according to a gradient of 1:10. 50 pL of the diluted solution was evenly spread on the surface of BHI solid culture medium using a bacterial spreader. Three concentration gradients were selected for each sample, and three plates were prepared for each concentration gradient. After incubation at 37°C under anaerobic conditions for 48 h, colony counting was performed by two people using double recording. The final results were averaged for comparison between groups.

[0034] ​1.10. Histological analysis After the drug intervention period in each group was completed, the rats were euthanized after general anesthesia. The maxilla was dissected and separated under sterile conditions, and excess soft tissues such as the gingiva were removed. Three-dimensional scanning of the maxilla was performed using high-resolution Micro-CT, and the analysis area was selected as the root apex of the first molar of the rat maxilla. At the same time, organs such as heart, liver, kidney, spleen, and lungs of each group of rats were collected for subsequent HE staining experiments.

[0035] 2. Statistical Analysis Methods Experimental data are expressed as mean ± standard deviation ( The t-test was used for comparisons between groups, and P < 0.05 indicates that the difference is statistically significant.

[0036] 3. Results 3.1. The effects of Xinjiang mulberry extract on... E. faecalis MIC, MBC, MBIC 50 and MBEC 50 Measurement Xinjiang mulberry extract for E. faecalis The MIC value reached 0.604 mg / mL, and the MBC value was 1.875 mg / mL. This extract can inhibit [the growth of certain substances] within a certain range. Figure 1 The formation of biofilms also has a certain effect on the removal of existing biofilms, such as... E. faecalis As shown, for E. faecalis MBIC of Xinjiang mulberry extract 50 It is 1.875 mg / m², and its MBEC 50 The concentration was 3.75 mg / mL.

[0037] 3.2. The effects of Xinjiang mulberry extract on... Figure 2 Influence of growth curve like E. faecalis As shown, E. faecalis The control group entered the logarithmic phase at 4-8 hours and the plateau phase at 8 hours. The absorbance was inhibited to varying degrees at drug concentrations of 1 / 4 MIC and 1 / 2 MIC, and the inhibitory effect was significant at concentrations of MIC and 2 MIC.

[0038] 3.3. Staining of live and dead bacteria to determine the effect of Xinjiang mulberry extract on... Figure 3 The influence of biomembrane structure like Figure 4As shown, the green fluorescence corresponding to SYTO9 reflects the distribution of live bacteria within the biofilm, the red fluorescence corresponding to PI indicates the distribution of dead bacteria, and Merge represents all bacteria in the biofilm. After 24 hours of drug treatment, the biofilm in the negative control group was dense and concentrated, with a larger area occupied by live bacteria. The proportion of dead bacteria was higher in the mulberry extract treatment group, and the overall structure of the biofilm was more loose. As the drug concentration gradually increased, the proportion of live bacteria in the drug-treated group continuously decreased. These results indicate that mulberry extract can effectively inhibit the proliferation of Enterococcus faecalis biofilm and destroy its original structure.

[0039] 3.4 Animal modeling and model validation like Figure 4 As shown in AC, a rat periapical periodontitis model was successfully established, and Micro-CT reconstruction was performed on the periapical region of rats in each group. E. faecalis The bone volume fraction (BV / TV) and trabecular bone separation (Tb.Sp) in the periapical region were measured to analyze the effect of the drug on alveolar bone resorption in periapical periodontitis. Compared with the model group, the BV / TV values ​​of the Xinjiang mulberry extract group and the calcium hydroxide group were lower than those of the model group (P<0.05), while the Tb.Sp values ​​of the two groups were significantly higher than those of the model group (P<0.05).

[0040] Table 1. Quantitative CFU count of bacteria in the root canal ( (CFU / ml)

[0041] * indicates that P < 0.05 compared to the model group.

[0042] 3.5 Comparison of bacterial colonies within root canals Two weeks after establishing the rat periapical periodontitis model and two weeks after medication treatment, bacteria were collected from the root canals for culture. The relevant results are shown in Table 1. Before medication, there was no significant difference in the CFU quantitative detection values ​​of bacteria in the root canals among the groups. After two weeks of medication intervention, the detection results of the mulberry extract group, calcium hydroxide group and model group showed significant differences (P < 0.05), while there was no significant difference in the detection data between the mulberry extract group and calcium hydroxide group (P > 0.05).

[0043] 3.6. Organ HE staining HE-stained sections of the heart, liver, spleen, lungs, and kidneys of rats in all groups showed uniformity, and the histological structures of each organ were consistent. This result fully confirms that the extract of Xinjiang mulberry has good biocompatibility in animals.

[0044] The significant advancement of this invention lies in: Calcium hydroxide is the most commonly used root canal disinfectant in clinical practice, but its strong alkaline environment not only makes it difficult to completely eradicate Enterococcus faecalis, but also may induce drug resistance and promote the formation of a more robust biofilm. Therefore, it is crucial to develop new disinfectants that can effectively combat such stubborn bacteria. The mulberry extract provided in the present solution exhibits comparable antibacterial and tissue repair efficacy in vitro and in vivo as calcium hydroxide, and no toxicity reactions have been found, highlighting its potential as a natural alternative. The loose structure of the biofilm observed in the staining of live and dead bacteria may be related to the expression of biofilm-related virulence genes inhibited by the mulberry extract, which has been reported in studies on mulberry ketone G against Streptococcus mutans biofilm. The results of the rat periapical periodontitis animal experiment further extend the bacterial experiment. Micro-CT analysis shows that the area of periapical lesions in the rat of the mulberry group is significantly reduced compared to the model group, and its effect on periapical tissue repair is comparable to that of calcium hydroxide. This repair effect may be attributed to its antibacterial effect, but it may also be related to the regulation of the periapical immune microenvironment and the influence on bone metabolism balance, especially E. faecalis Such bacteria with virulence factors, strong biofilm formation ability, and the ability to colonize in root canals and cooperate with other bacteria to cause immune escape and destruction of the apical region. Current research has found that, E. faecalis , Porphyromonas gingivalis and other pathogens can regulate the immune microenvironment and programmed death of cells in the apical region through NF-ĸB and Wnt / β-catenin pathways, thereby disrupting bone homeostasis. The mulberry extract may inhibit ​ , and other pathogen-associated molecular patterns triggered excessive inflammatory response, down-regulate osteoclast activity, and may promote osteogenic differentiation through related signaling pathways, thereby facilitating the restoration of bone homeostasis. This may be the key to its superiority over single-antibacterial disinfectants, and it is worth further studying. The HE staining sections of the main organs of the experimental group rats in the above examples of the present invention did not show any abnormal pathological changes, indicating that the mulberry extract has good biological safety in the repair of periapical tissue in a complex in vivo environment, providing a safety basis for its clinical translation, and it is expected to be developed into an ideal root canal disinfectant with antibacterial and anti-inflammatory and biocompatibility.

[0045] In summary, the above examples of the present invention demonstrate that the Xinjiang mulberry extract provided in the present invention has significant antibacterial, bactericidal, and anti-biofilm activity in vitro against Enterococcus faecalis; in a rat periapical periodontitis model, it exhibits comparable in vivo antibacterial and lesion repair-promoting efficacy to the commonly used clinical drug calcium hydroxide, and has excellent biological safety. Therefore, Xinjiang mulberry extract can be used to prepare a drug for treating or preventing periapical periodontitis caused by Enterococcus faecalis, especially as a root canal disinfectant, and has good development and application prospects. The drug can be formulated into a paste, an irrigation solution, a nanoemulsion, or a hydrogel, etc.

[0046] The above merely describes preferred embodiments of the present application, and is not used to limit the present application, any modification, equivalent replacement and improvement within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of Xinjiang Morus alba L. extract in the preparation of a medicament for inhibiting Enterococcus faecalis.

2. Use according to claim 1, characterized in that, The Xinjiang Morus alba L. extract is prepared by heating reflux extraction of Morus alba L. raw material with ethanol as solvent, and the extract is concentrated and dried to obtain Morus alba L. extract powder.

3. Use according to claim 2, characterized in that, The ethanol is 95% ethanol, the solid-liquid ratio is 1:3, the heating reflux extraction temperature is 70°C, and the extraction time is 3 hours.

4. Use according to any one of claims 1 to 3, characterized in that, The medicament is used for treating or preventing periapical periodontitis caused by Enterococcus faecalis.

5. Use according to claim 4, characterized in that, The medicament is a root canal disinfectant.

6. Use according to claim 5, characterized in that, The minimum inhibitory concentration of the Xinjiang Morus alba L. extract on Enterococcus faecalis is 0.604 mg / mL, and the minimum bactericidal concentration is 1.875 mg / mL.

7. Use according to claim 5, characterized in that, The minimum concentration of the Xinjiang Morus alba L. extract required to inhibit 50% Enterococcus faecalis biofilm formation is 1.875 mg / mL, and the minimum concentration required to remove 50% of the formed Enterococcus faecalis biofilm is 3.75 mg / mL.

8. A pharmaceutical composition for treating or preventing apical periodontitis, characterized by, The medicament composition comprises an effective amount of Xinjiang Morus alba L. extract and pharmaceutically acceptable excipients.

9. The pharmaceutical composition of claim 8, wherein, The dosage form of the medicament composition is a root canal disinfectant paste, an irrigation solution, a nanoemulsion or a hydrogel.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that, The Xinjiang Morus alba L. extract is prepared by a method comprising the following steps: taking Morus alba L. raw material, using 95% ethanol as solvent, heating reflux extraction at 70°C for 3 hours at a solid-liquid ratio of 1:3, and then concentrating and drying the extract to obtain Morus alba L. extract powder.

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