Application of 23-acetyl alisol B in preparation of streptococcus mutans inhibitor
By using 23-Acetyl Alassium B to prepare Streptococcus mutans inhibitors, the problem of drug resistance proliferation of Streptococcus mutans was solved, effective inhibition of Streptococcus mutans and blocking of biological membranes was achieved, and new directions for anti-caries drug development were provided.
Patent Information
- Application Number
- CN202510933609.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-08
- Publication Date
- 2025-08-22
Smart Images
Figure CN120514713A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicine, and in particular relates to an application of 23-acetyl alismatol B in the preparation of a mutans streptococcus inhibitor. Background Art
[0002] Streptococcus mutans, a facultative anaerobic Gram-positive bacterium belonging to the Streptococcus family, is a common oral pathogen and a major cause of dental caries. It can cause a variety of oral diseases, including enamel demineralization, caries, and pulpitis. Early in life, the pathogen adheres to tooth enamel and secretes numerous glycosyltransferases. In the presence of sucrose, these enzymes break down sucrose to produce large amounts of glucans, which, primarily insoluble components, form a biofilm attached to the tooth surface. Subsequently, the pathogen continuously produces acid, forming an acidic biofilm microenvironment and ultimately initiating dental caries. gtfB and gtfC are core virulence genes for biofilm formation and cariogenicity, directly influencing adhesion and plaque stability. gtfD primarily provides metabolic support, aiding the pathogen's survival in the dynamic oral environment. The synergistic action of these three genes enables S. mutans to effectively utilize sucrose, establish a cariogenic biofilm, and resist host defense mechanisms.
[0003] Streptococcus mutans ATCC700610 is widely present in the human oral cavity, colonizing within dental plaque biofilms. It is the primary pathogenic microorganism that causes dental caries in humans and can also be transmitted through saliva. The bacterium is highly adaptable in the oral environment and is currently one of the most frequently detected cariogenic bacteria worldwide, possessing significant oral public health implications. In current clinical dental caries prevention and treatment, although topical antimicrobial agents such as chlorhexidine can temporarily suppress cariogenic bacteria, long-term use leads to the increasingly serious problem of selective proliferation of drug-resistant strains of Streptococcus mutans. Currently, the field of oral medicine is actively exploring alternative therapies such as microecological modulators and natural antimicrobial substances. This shift has led to a focus on the development of novel anti-caries agents targeting oral cariogenic bacteria, creating an urgent need to identify preventive and treatment options that effectively inhibit pathogenic bacteria while complying with antibiotic stewardship requirements. Summary of the Invention
[0004] The purpose of the present invention is to provide the use of 23-acetyl alismol B in the preparation of Streptococcus mutans inhibitors, aiming to solve the problems raised in the background technology.
[0005] In order to solve the above problems, the present invention is achieved by providing a use of 23-acetyl alismatol B in the preparation of a Streptococcus mutans inhibitor.
[0006] Preferably, the 23-acetyl alismatol B is used to inhibit the growth of Streptococcus mutans.
[0007] Preferably, the 23-acetyl alismatol B is used to inhibit the formation of Streptococcus mutans biofilm.
[0008] Preferably, the 23-acetyl alismatol B is used to inhibit the expression of gtfB, gtfC, and gtfD genes of Streptococcus mutans.
[0009] Preferably, the MIC value of the 23-acetyl alismol B against Streptococcus mutans is 4 μg / mL.
[0010] Preferably, the mutans Streptococcus is Streptococcus mutans ATCC700610.
[0011] Another object of the present invention is to provide a Streptococcus mutans inhibitor, comprising a pharmaceutically acceptable carrier and 23-acetyl alismatol B as an active ingredient.
[0012] Preferably, the concentration of 23-acetyl alismatol B is 4-16 μg / mL.
[0013] Another object of the present invention is to provide a use of 23-acetyl alismol B or the above-mentioned Streptococcus mutans inhibitor in the preparation of a drug for preventing and treating Streptococcus mutans infection.
[0014] The present invention provides the use of 23-acetyl alismol B in the preparation of a Streptococcus mutans inhibitor. 23-acetyl alismol B can inhibit the activity and growth of Streptococcus mutans, inhibit the gene transcription levels of gtfB, gtfC, and gtfD of Streptococcus mutans, and reduce the formation of Streptococcus mutans biofilm, thereby providing a new candidate compound and a corresponding theoretical basis for the prevention and treatment of Streptococcus mutans infection and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is the growth curve of Streptococcus mutans after treatment with 23-acetyl alismatol B; Figure 2 This is a biofilm image of Streptococcus mutans after treatment with 23-acetyl alismatol B; Figure 3 This is a graph showing gene expression levels in Streptococcus mutans after treatment with 23-acetyl alismol B; Figure 4 This is a bacterial live / dead staining image of Streptococcus mutans after treatment with 23-acetyl alismatol B; Figure 5 This is a diagram of the cytotoxicity assay of Streptococcus mutans after treatment with 23-acetyl alismatol B. DETAILED DESCRIPTION
[0016] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present 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 only used to explain the present invention and are not intended to limit the present invention.
[0017] 23-Acetyl alismatol B, molecular formula: C 32 H 50 O5, molecular weight: 514.74, as a representative active ingredient of the genus Alisma, it has multiple pharmacological activities, including anti-inflammatory, hepatoprotective, anti-fibrotic, antibacterial, and lipid metabolism regulation. The present invention provides a preliminary analysis of the inhibitory activity of 23-acetyl alismatol B against Streptococcus mutans ATCC700610, aiming to provide a theoretical basis for the application and development of this compound in the field of oral antibacterial. 23-acetyl alismatol B is derived from plants of the Alismataceae family, such as oriental Alisma and Alisma, which are commonly used in traditional medicine to treat edema, hyperlipidemia, and inflammatory-related diseases. Pharmacological studies have shown that 23-acetyl alismatol B can not only regulate lipid metabolism, but also has significant anti-inflammatory and antioxidant activities, and has an inhibitory effect on pathological processes such as liver fibrosis. In addition, 23-acetyl alismatol B exhibits inhibitory effects on a variety of pathogenic microorganisms, and its antibacterial mechanism may be related to its interference with microbial cell membrane stability or metabolic pathways. The present invention focuses on the inhibitory effect of 23-acetyl alismol B on Streptococcus mutans to assess its feasibility as a potential anti-caries agent. Streptococcus mutans is a major oral cariogenic bacterium, and its ability to form biofilms is a key factor in the development of dental caries. By analyzing the inhibitory effect of 23-acetyl alismol B on the growth and biofilm formation of Streptococcus mutans and the transcription levels of related genes, a scientific basis can be provided for the development of novel, naturally derived oral antimicrobial agents, and new research directions can be explored for the medicinal value of Alisma plants.
[0018] Specifically, in one embodiment of the present invention, a use of 23-acetyl alisol B in the preparation of a Streptococcus mutans inhibitor is provided. Specifically, 23-acetyl alisol B is used to inhibit the growth of Streptococcus mutans, inhibit the formation of Streptococcus mutans biofilms, and inhibit the expression of the gtfB, gtfC, and gtfD genes of Streptococcus mutans. The MIC value of 23-acetyl alisol B against Streptococcus mutans is 4 μg / mL; the Streptococcus mutans is Streptococcus mutans ATCC 700610, but is not limited thereto.
[0019] In another embodiment of the present invention, a Streptococcus mutans inhibitor is provided, comprising a pharmaceutically acceptable carrier and 23-acetyl alisol B as an active ingredient. The concentration of 23-acetyl alisol B is 4-16 μg / mL. The carrier may be, but is not limited to, physiological saline, buffer, liposomes, nanoparticles, or the like.
[0020] In another embodiment of the present invention, there is provided a use of 23-acetyl alissol B or the above-mentioned Streptococcus mutans inhibitor in the preparation of a drug for preventing and treating Streptococcus mutans infection.
[0021] In the examples of the present invention, MIC assays, growth curve assays, crystal violet staining assays, bacterial live / dead staining assays, qRT-PCR assays, and cytotoxicity assays verified that 23-acetyl alismatol B can inhibit the activity of Streptococcus mutans ATCC700610, inhibit the gene transcription levels of gtfB, gtfC, and gtfD of Streptococcus mutans ATCC700610, and reduce the formation of biofilms of Streptococcus mutans ATCC700610.
[0022] Specifically, in order to demonstrate the efficacy of 23-acetyl alismatol B, some examples are listed below to conduct verification experiments on the efficacy of 23-acetyl alismatol B.
[0023] Example 1: This example is a minimum inhibitory concentration (MIC) test, which is as follows: The MIC value of 23-acetylalismol B against Streptococcus mutans ATCC700610 was determined using the standard dilution method published by the American Association for Clinical and Laboratory Standards. Bacterial suspension was incubated overnight in BHI medium. 100 μL of the adjusted bacterial suspension was added to each well of a 96-well plate (inoculation size was 1 × 10 6 CFUs / mL), then add the same volume of diluted 23-acetylalismol B and mix by pipetting to achieve final concentrations of 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, 256 μg / mL, 512 μg / mL, 1024 μg / mL, 2048 μg / mL, and 4096 μg / mL. The plates were incubated anaerobically at 37°C in a 5% CO2 incubator. The bacterial growth on the 96-well plates was observed the next day. The concentration of compound that inhibited bacterial growth with the naked eye was determined as the MIC value of 23-acetylalismol B against S. mutans ATCC700610. The results show that the minimum MIC value of 23-acetylalismol B against S. mutans ATCC700610 was 4 μg / mL.
[0024] Example 2: This example is a growth curve test, which is as follows: Streptococcus mutans ATCC700610 was cultured overnight and inoculated with various concentrations of 23-acetyl alisinol B in fresh BHI cultures at 37°C for 24 hours. Groups were set up: ATCC700610 without 23-acetyl alisinol B, and ATCC700610 with 4 μg / mL, 16 μg / mL, and 64 μg / mL of 23-acetyl alisinol B. 0.2% chlorhexidine (CHX) was used as a positive control. During the incubation period, the OD values of the bacterial samples were measured every hour. 600nm The results are plotted with time as the horizontal axis. Figure 1 shown.
[0025] from Figure 1 It can be seen that the curves with the addition of 23-acetyl alismatol B are close to the CHX positive control curve, and are distant from the curve without the addition of 23-acetyl alismatol B, indicating that the addition of different concentrations of 23-acetyl alismatol B affects the growth of Streptococcus mutans ATCC700610.
[0026] Example 3: This example is a bacterial biofilm determination test, which is as follows: Dilute Streptococcus mutans ATCC700610 into a 96-well plate using BHIS medium to a bacterial concentration of 2 × 10 6 CFU / mL. Different concentrations of 23-acetyl alismatol B were added to each well, with 3 replicates per group, and the final drug solution concentration ratios were 0, 4, 16, and 64 µg / mL, respectively. After anaerobic incubation at 37°C for 12 h, the 96-well ELISA plate was removed, excess culture medium was removed, and the plate was washed three times with 200 µL PBS to remove loosely attached planktonic bacteria. The biofilm in the well was fixed with 4% (wt / vol) paraformaldehyde and kept in the dark for 15 min. 0.1% (wt / vol) crystal violet-ethanol solution was then added to each well for staining for 30 min. After removing the solution, the plate was washed three times with 200 µL PBS to remove the floating color. Anhydrous ethanol was added to dissolve the dye, and an equal volume of the solution in each well was transferred to a new 96-well plate, and the OD was measured using a spectrophotometer. 575nm The test results are as follows. Figure 2 shown.
[0027] from Figure 2 It can be seen that the purple color in the wells with the addition of 23-acetyl alismol B is lighter, indicating that the biofilm that can bind to crystal violet is reduced, indicating that 23-acetyl alismol B can inhibit the formation of biofilm of Streptococcus mutans ATCC700610 when treated with Streptococcus mutans ATCC700610 at concentrations of 4, 16, and 64 μg / mL.
[0028] Example 4: This example is a qRT-PCR test for detecting gene expression levels, as follows: Streptococcus mutans ATCC700610 was cultured using BHIS, and 23-acetyl alismatol B was added to adjust the concentration to 64 μg / mL, and incubated anaerobically at 37°C for 12 h. The bacteria were collected by centrifugation (4°C, 4000 rpm) and then treated with 100 μL of lysozyme (30 mg / mL) for 30 min. Total RNA was extracted and the purified RNA was dissolved in 80 μL of RNase-Free water. The bacterial RNA was then reverse transcribed to generate cDNA. qRT-PCR was performed using a 96-well plate. After obtaining the data, the relative gene expression was calculated using the 2-ΔΔCt method. The experimental results are shown in Figure 2. Figure 3 shown.
[0029] from Figure 3 It can be seen that after treating Streptococcus mutans ATCC700610 with 23-acetyl alismol B, the expression of gtfB, gtfC, and gtfD genes can be significantly inhibited.
[0030] Example 5: This example is a bacterial live / dead staining test, as follows: As described above, bacterial biofilms were cultured in 96-well plates and a gradient of 23-acetylalismol B (64 μg / mL, 16 μg / mL, 4 μg / mL, and 0) was set. After 12 h of growth, the bacterial solution was discarded, and the floating bacteria on the surface were rinsed with PBS and dried. The biofilms were observed using an inverted fluorescence microscope. The biofilms were stained using a bacterial viability detection kit, and the molecular probes SYTO9 and PI were used to label live and dead Streptococcus mutans, respectively. The experimental results are shown in Figure 2. Figure 4 shown.
[0031] Figure 4 Reflects the green (live bacteria) and red (dead bacteria) fluorescence intensity. Figure 4 As can be seen, in the negative control, the biofilm was evenly distributed, relatively dense, and completely covered. After treatment with 23-acetyl alismatol B, the biofilm was highly dispersed and significantly loosened. 23-acetyl alismatol B reduced the surface area covered by the biofilm, resulting in a significant reduction in biofilm biomass.
[0032] Example 6: This example is a cytotoxicity assay, specifically as follows: Human oral keratinocytes were used for cytotoxicity assays. Human oral keratinocytes were cultured in DMEM medium supplemented with streptomycin-penicillin to prevent contamination. The cells were incubated at 37°C and 5% CO2, with the medium replaced every 2-3 days. Different concentrations of 23-acetyl alismol B and 1×10 5 Cells were placed in the wells of a 96-well plate. Control culture medium was added accordingly, and the cell status was assessed using a cytotoxicity kit (CCK-8). After 24 hours, the 96-well plate was washed twice with sterile PBS. 100 μL of culture medium containing 10% CCK-8 was added to each well for 3 hours, and the cell proliferation rate was determined by measuring the optical density absorbance at a wavelength of 450 nm using a microplate reader. The experimental results are shown in Figure 2. Figure 5 shown from Figure 5 It can be seen that 23-acetyl alismatol B does not cause toxicity to human oral keratinocytes at an experimental concentration of 16 μg / mL.
[0033] In summary, the examples of the present invention demonstrate that 23-acetyl alismatol B can be used to inhibit Streptococcus mutans and does not produce toxicity to human oral keratinocytes within a certain concentration range; 23-acetyl alismatol B can inhibit the activity and growth of Streptococcus mutans, inhibit the gene transcription levels of gtfB, gtfC, and gtfD of Streptococcus mutans, and reduce the formation of Streptococcus mutans biofilm, providing a new candidate compound and a corresponding theoretical basis for the prevention and treatment of Streptococcus mutans infection and clinical application.
[0034] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.
Claims
1. Application of 23-acetyl alismatol B in the preparation of Streptococcus mutans inhibitors.
2. The use according to claim 1, characterized in that The 23-acetyl alismatol B is used for inhibiting the growth of Streptococcus mutans.
3. The use according to claim 1, characterized in that The 23-acetyl alismatol B is used for inhibiting the formation of mutans streptococcus biofilm.
4. The use according to claim 1, characterized in that The 23-acetyl alismatol B is used for inhibiting the expression of gtfB, gtfC and gtfD genes of Streptococcus mutans.
5. The use according to claim 1, characterized in that The MIC value of the 23-acetyl alismol B against Streptococcus mutans is 4 μg / mL.
6. The use according to any one of claims 1 to 5, characterized in that The mutans streptococcus is mutans streptococcus ATCC700610.
7. A mutans streptococcus inhibitor comprising a pharmaceutically acceptable carrier, characterized in that: Also included is 23-acetyl alismatol B as an active ingredient.
8. The mutans Streptococcus inhibitor according to claim 7, characterized in that The concentration of the 23-acetyl alismatol B is 4-16 μg / mL.
9. Use of 23-acetyl alismol B or the Streptococcus mutans inhibitor according to any one of claims 7 to 8 in the preparation of a medicament for preventing and treating Streptococcus mutans infection.