Streptococcus mutans inhibitor based on natural compound and preparation method and application thereof
By extracting and purifying dihydrobamate from vine tea, an efficient and safe Streptococcus mutans inhibitor was prepared, which solved the problems of limited inhibition effect, insufficient safety and difficulty in long-term use in the prior art, and achieved significant inhibition of the formation of oral pathogenic bacteria biofilm and improving oral health.
Patent Information
- Application Number
- CN202510184060.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2025-05-30
AI Technical Summary
The existing oral capsule inhibitors have limited inhibitory effects, insufficient safety and difficulty in long-term use during use.
Using dihydrobamate as the main active ingredient, a highly efficient and safe Streptococcus mutans inhibitor was prepared by extracting and purifying the compound from vine tea. This inhibitor is prepared by ultrasonic assisted extraction and reduced pressure concentration technology, and has the ability to significantly inhibit the formation of oral pathogenic bacteria biofilms.
Dihydrobayllin can significantly inhibit the formation and metabolic activity of Streptococcus mutans biofilm, destroy the microstructure of the biofilm, inhibit the acid production ability and the production of extracellular polysaccharides, reduce the aggregation rate of bacteria and virulence gene expression, thereby providing long-term and efficient oral care effects.
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Figure CN120053423A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and relates to a Streptococcus mutans inhibitor based on natural compounds, its preparation method and application. Background Art
[0002] There are usually various bacteria in the oral cavity, which play an important role in maintaining oral health and balance. The normal flora in the oral cavity includes streptococci, lactobacilli, etc., which help to maintain the oral acid-base balance and inhibit the growth of pathogenic microorganisms. When oral hygiene is poor, such as the accumulation of dental plaque, it may lead to the overgrowth of pathogenic bacteria such as Streptococcus mutans and Actinomyces, and these bacteria can cause dental caries and periodontal diseases.
[0003] Streptococcus mutans is a Gram-positive coccus and is one of the streptococci with the largest proportion in the natural oral flora and is one of the main components of dental plaque. At present, the treatment of Streptococcus mutans infection mainly relies on antibiotics. However, the presence of the bacterial biofilm makes it difficult for antibiotics to penetrate, thus greatly reducing the effect of traditional antibiotic therapy. In addition, biofilm formation also promotes the spread of drug-resistant genes in the bacterial population, further exacerbating the problem of drug resistance. Summary of the Invention
[0004] Aiming at the problems of limited inhibitory effect, insufficient safety and difficulty in long-term use existing in the use of existing oral biofilm inhibitors, the purpose of the present invention is to provide a Streptococcus mutans inhibitor based on natural compounds, its preparation method and application. By using natural compounds as oral biofilm inhibitors, an efficient and safe solution is provided, which can significantly inhibit the formation of bacterial biofilms in the oral cavity, improve oral health, and is suitable for long-term use, overcoming the deficiencies of the prior art.
[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:
[0006] The present invention provides a Streptococcus mutans inhibitor based on natural compounds, with dihydromyricetin as the main active ingredient.
[0007] Preferably, the purity of dihydromyricetin is not less than 95%.
[0008] The present invention also provides a preparation method of the above Streptococcus mutans inhibitor, including: mixing the crushed Ampelopsis grossedentata with ethanol, performing ultrasonic-assisted extraction, filtering the extract to remove impurities, and then performing reduced pressure concentration to obtain a crude extract of dihydromyricetin.
[0009] Preferably, the crushed Ampelopsis grossedentata is mixed with ethanol at a solid-liquid ratio of 1:20 (g / mL); the ultrasonic frequency for ultrasonic-assisted extraction is 20 kHz, the extraction temperature is controlled at 30°C, and the extraction time is 2 - 3 hours.
[0010] The present invention also provides the use of dihydromyricetin in the preparation of oral care products, and the oral care products include oral biofilm inhibitors.
[0011] Preferably, the dihydromyricetin can significantly inhibit the formation and metabolic activity of Streptococcus mutans biofilm and disrupt the microstructure of the biofilm.
[0012] Preferably, the dihydromyricetin can inhibit the acid production ability of Streptococcus mutans.
[0013] Preferably, the dihydromyricetin can inhibit the production amount of extracellular polysaccharide (EPS) in Streptococcus mutans biofilm.
[0014] Preferably, the dihydromyricetin can inhibit the aggregation rate of Streptococcus mutans.
[0015] Preferably, the dihydromyricetin can inhibit the expression of virulence genes gtfB, gtfC, and luxS of Streptococcus mutans.
[0016] The beneficial effects of the present invention are as follows:
[0017] The present invention uses dihydromyricetin extracted from Ampelopsis grossedentata as the main active ingredient to prepare a product with significant activity in inhibiting the formation of oral pathogenic bacteria biofilm. This product has the following excellent properties:
[0018] Powerful biofilm inhibition: Dihydromyricetin can effectively inhibit the formation of biofilms of various oral pathogenic bacteria, reduce the accumulation of dental plaque, and thus prevent the occurrence of oral diseases such as dental caries and periodontal diseases.
[0019] Natural and safe: As a natural extract, dihydromyricetin is safe and harmless to the human body and is suitable for long-term oral care use.
[0020] Good stability: High-purity dihydromyricetin shows good stability in oral care products and is not easily inactivated by external factors. Description of the Drawings
[0021] Figure 1 : LC-MS analysis results of Ampelopsis grossedentata extract.
[0022] Figure 2:Inhibitory effect of dihydromyricetin on the biofilm formation of S. mutans at sub-minimum inhibitory concentrations (sub-MICs). (a) Determination of S. mutans biofilm biomass at 24 h (crystal violet staining method); (b) Viable cell count of S. mutans biofilm at 24 h (CFU counting method, data logarithmically transformed); (c) Evaluation of the metabolic activity of S. mutans biofilm formed at 24 h (MTT method); (d) Observation of the microstructure of S. mutans biofilm at 24 h by scanning electron microscopy (SEM); (e) Observation of biofilm images after treatment with different concentrations of dihydromyricetin by confocal laser scanning microscopy (CLSM) (scale bar = 20 μm).
[0023] Figure 3 :Effect of different concentrations of dihydromyricetin on acid production by S. mutans. The abscissa in the figure represents the concentration of dihydromyricetin, with three treatment groups (125 μg / mL, 62.5 μg / mL, and 31.25 μg / mL) and one control group (DMSO) set; the ordinate represents the pH value.
[0024] Figure 4 :Effect of different concentrations of dihydromyricetin on the production of exopolysaccharides (EPS) in S. mutans biofilms. The abscissa in the figure represents the concentration of dihydromyricetin, with three treatment groups (125 μg / mL, 62.5 μg / mL, and 31.25 μg / mL) and one control group (DMSO) set; the ordinate represents the absorbance, which is used to quantify the effect on the production of exopolysaccharides by Streptococcus mutans.
[0025] Figure 5 :Effect of different concentrations of dihydromyricetin on the aggregation of S. mutans. The abscissa in the figure represents the concentration of dihydromyricetin, with three treatment groups (125 μg / mL, 62.5 μg / mL, and 31.25 μg / mL) and one control group (DMSO) set; the ordinate represents the aggregation rate of S. mutans, which is used to quantify the strength of its aggregation.
[0026] Figure 6 :Effect of dihydromyricetin (125 μg / mL) treatment on the expression of virulence genes. Using S. mutans 16S rRNA as an internal reference. Detailed implementation manners
[0027] The present invention will be described in detail below in conjunction with the specific implementation manners. The following specific examples are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.
[0028] Examples
[0029] Raw material preparation: Select high-quality Ampelopsis grossedentata as raw material to ensure no impurities and mildew.
[0030] Crushing treatment: Crush the Ampelopsis grossedentata raw material to 80 mesh to improve the extraction efficiency.
[0031] Selection of extraction solvent: Ethanol is selected as the extraction solvent because it has good solubility for dihydromyricetin.
[0032] Extraction operation: The crushed Ampelopsis grossedentata is mixed with ethanol at a solid-liquid ratio of 1:20 (g / mL), and ultrasonic-assisted extraction is carried out. Ultrasonic treatment helps to break the cell wall and accelerate the release of active ingredients. The ultrasonic frequency of ultrasonic-assisted extraction is 20 kHz, the extraction temperature is controlled at 30 °C, and the extraction time is 2 - 3 hours.
[0033] Filtration and concentration: After the extract is filtered to remove impurities, vacuum concentration is carried out to obtain the crude extract of dihydromyricetin.
[0034] Component percentage or weight range: In the present invention, the purity requirement of dihydromyricetin is not less than 95% to ensure its biological activity and medicinal effect. In the final product, the content of dihydromyricetin can be adjusted according to specific application requirements, but generally it is recommended to keep it within the effective concentration range to exert the best inhibitory effect on the biofilm activity.
[0035] Analyze the chemical components of the Ampelopsis grossedentata extract by liquid chromatography-mass spectrometry (LC-MS), such as Figure 1 shown, and the results show that dihydromyricetin is a compound in the Ampelopsis grossedentata extract.
[0036] Based on the dihydromyricetin prepared in this example, the following tests its effect on S. mutans strains through different experiments. Among them, the S. mutans strains are from the State Key Laboratory of Oral Diseases, Sichuan University, and the purity of dihydromyricetin is 97%, which is dissolved in DMSO.
[0037] Test Example 1
[0038] The purpose of this test is to study the effects of different concentrations of dihydromyricetin on the biofilm inhibition, metabolic activity and colony formation ability of S. mutans. The specific operation is as follows:
[0039] Prepare S. mutans bacterial solution: Anaerobically culture the S. mutans strains in BHI medium until the OD600 is 0.5.
[0040] Configure dihydromyricetin solution: Use DMSO to configure a dihydromyricetin solution with a concentration of 100 mg / mL and dilute it to the required concentrations (such as 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL).
[0041] Inoculation and culture: Add 200 μL of brain heart infusion (BHIS) medium containing 1% sucrose into each well of a 96-well plate, inoculate the S. mutans bacterial solution, and add dihydromyricetin solutions at different concentrations, then culture anaerobically for 24 hours.
[0042] Biofilm staining and quantification: Remove the medium, wash 3 times with PBS, add crystal violet staining solution and stain for 10 minutes, then wash with PBS to remove the excess staining solution, add 95% ethanol for decolorization, and finally measure the OD590 value.
[0043] MTT assay for metabolic activity: After the culture is completed, add MTT solution to each well, continue to culture for 3 hours, then add DMSO to dissolve MTT, and measure the OD590 value.
[0044] CFU counting: Dilute the cultured bacterial solution and spread it on a BHI agar plate, and count the number of colonies after anaerobic culture for 48 hours.
[0045] Figure 2 (a) shows that with the increase in the concentration of dihydromyricetin, the biomass of the biofilm decreases significantly, indicating that dihydromyricetin has a concentration-dependent inhibitory effect on the formation of S. mutans biofilm. Figure 2 (b) shows that the number of viable bacteria in the dihydromyricetin treatment group is significantly lower than that in the control group, further confirming its inhibitory effect on S. mutans biofilm. Figure 2 (c) shows that with the increase in the concentration of dihydromyricetin, the metabolic activity of the biofilm gradually decreases, indicating that dihydromyricetin not only inhibits the formation of the biofilm but also affects its metabolic function. Figure 2 (d) shows that the biofilm structure in the control group is dense and complete, while the biofilm structure in the dihydromyricetin treatment group becomes loose and irregular, and the bacteria are sparsely distributed, further confirming the destructive effect of dihydromyricetin on the biofilm structure. Consistent with the SEM results, Figure 2 (e)'s CLSM images also show that the biofilm thickness in the dihydromyricetin treatment group decreases, the bacterial density decreases and the distribution is uneven.
[0046] The results show that dihydromyricetin can significantly inhibit the formation and metabolic activity of S. mutans biofilm and destroy its microstructure at sub-minimum inhibitory concentration, providing strong support for the application of dihydromyricetin in preventing dental caries.
[0047] Experimental example 2
[0048] The purpose of this experiment is to study the effects of different concentrations of dihydromyricetin on the acid-producing ability of S. mutans. The specific operation is as follows:
[0049] The S. mutans strain was cultured in environments with different concentrations of dihydromyricetin (including the DMSO control group, and treatment groups of 31.25 μg / mL, 62.5 μg / mL, and 125 μg / mL), and the changes in pH value within 120 minutes were monitored.
[0050] As Figure 3 shown, in the control group, S. mutans rapidly produced acid, resulting in a significant decrease in pH value. In the dihydromyricetin treatment groups, especially the high-concentration treatment group (such as 125 μg / mL), the rate of pH value decrease was significantly slowed down, indicating that dihydromyricetin effectively inhibited the acid-producing ability of S. mutans, and this inhibitory effect showed concentration dependence. This finding is of great significance for understanding the mechanism of dihydromyricetin in preventing dental caries, because acid production by S. mutans is one of the key factors leading to tooth demineralization and dental caries formation.
[0051] Experimental Example 3
[0052] The purpose of this experiment was to study the effects of different concentrations of dihydromyricetin on the production of exopolysaccharides (EPS) in the S. mutans biofilm. EPS is an important component of the S. mutans biofilm and plays a key role in the stability and pathogenicity of the biofilm. The specific operation was as follows:
[0053] The S. mutans strain was cultured in environments with different concentrations of dihydromyricetin (including the DMSO control group, and treatment groups of 31.25 μg / mL, 62.5 μg / mL, and 125 μg / mL) to form a biofilm. Subsequently, the anthrone-sulfuric acid method was used to determine the amount of EPS produced in each group of biofilms.
[0054] The results are as Figure 4 shown. It can be clearly seen that as the concentration of dihydromyricetin increased, the amount of EPS produced gradually decreased. Especially at a concentration of 125 μg / mL, the amount of EPS produced decreased significantly, and this inhibitory effect was concentration-dependent. This finding is of great significance for understanding the mechanism of action of dihydromyricetin in disrupting the structure of the S. mutans biofilm and reducing its pathogenicity. By inhibiting the production of EPS, dihydromyricetin may help reduce the attachment of S. mutans to the tooth surface and the formation of biofilms, thereby preventing the occurrence of dental caries.
[0055] Experimental Example 4
[0056] The purpose of this experiment was to study the effects of different concentrations of dihydromyricetin on the aggregation of S. mutans. The results are as Figure 5 shown. The specific operation was as follows:
[0057] The S. mutans strain was cultured in an environment with different concentrations of dihydromyricetin (including DMSO control group, 31.25 μg / mL, 62.5 μg / mL, and 125 μg / mL treatment groups) to form biofilms. Subsequently, the aggregation rate of S. mutans was measured using the aggregation method.
[0058] Control group (DMSO): S. mutans showed a relatively high aggregation rate under DMSO treatment, reflecting its natural aggregation ability.
[0059] 125 μg / mL treatment group: When the concentration of dihydromyricetin reached 125 μg / mL, the aggregation rate of S. mutans decreased significantly, indicating that dihydromyricetin at this concentration had a strong inhibitory effect on the aggregation of S. mutans.
[0060] 62.5 μg / mL and 31.25 μg / mL treatment groups: As the concentration of dihydromyricetin decreased, the aggregation rate of S. mutans rebounded to some extent but was still lower than that of the control group, indicating that dihydromyricetin at these two concentrations still had a certain inhibitory effect on the aggregation of S. mutans, but the effect was weaker than that at the 125 μg / mL concentration.
[0061] As can be seen from the figure, as the concentration of dihydromyricetin increased, the aggregation rate of S. mutans gradually decreased, showing an obvious concentration-dependent effect. This further confirmed the inhibitory effect of dihydromyricetin on the aggregation of S. mutans. This finding provided strong support for the potential application of dihydromyricetin as an anti-biofilm agent in the prevention and treatment of dental caries.
[0062] Test Example 5
[0063] The purpose of this experiment was to study the effect of dihydromyricetin (125 μg / mL) on the expression of virulence genes of S. mutans. The specific operations were as follows:
[0064] Inoculation and culture: Add BHIS medium containing 125 μg / mL dihydromyricetin to the culture flask, inoculate the S. mutans bacterial solution, and culture anaerobically for 24 hours.
[0065] RNA extraction and reverse transcription: Collect the bacterial cells, extract total RNA, and reverse transcribe it into cDNA. The RNA extraction kit was from Shanghai Promega Bioproducts Co., Ltd., and the reverse transcription kit was the PrimeScript FAST RT reagent Kit with gDNA Eraser kit from TaKaRa, Japan. The synthesis method of cDNA was as follows:
[0066] 1. Genomic DNA removal reaction
[0067] <Each reaction>
[0068] Reagent Dosage 8×gDNA Eraser Premix 2 μl <![CDATA[RNA sample *1 > <![CDATA[RNase Free H 2 O]]> Total 16 μl
[0069] 42°C for 2 min (or room temperature for 5 min *2 )
[0070] *1: In a 20-μl reverse transcription reaction system, up to 1 μg of total RNA can be used for the TB Green qPCR method, and up to 2 μg of total RNA can be used for the probe qPCR analysis method. The reverse transcription reaction can also be scaled up proportionally according to requirements.
[0071] *2: When reacting at room temperature, it can be extended to 30 minutes.
[0072] 2. Reverse transcription reaction
[0073] Add 4 μl of 5×RT Premix to the reaction solution 1 on ice each time, gently mix well, and immediately perform the reverse transcription reaction.
[0074] <One reaction>
[0075] Reagent Dosage Reaction solution of Step 1 16 μl 5×RT Premix 4 μl Total 20 μl
[0076] 37°C for 10 min
[0077] 85°C for 5 sec
[0078] 4°C *3
[0079] *3: When the synthesized cDNA needs to be stored for a long time, store it at -20°C or lower temperature.
[0080] RT-qPCR detection of gene expression: Design primers targeting the gtfB, gtfC, and luxS genes, perform RT-qPCR reactions, and measure the expression levels of each gene. The primers were synthesized by Hainan Qingke Biotechnology Co., Ltd., the MIX was from TaKaRa, Japan, and the qRT-PCR adopted the standard two-step PCR amplification procedure:
[0081] Hold: Pre-denaturation
[0082] Cycle: 1
[0083] 95°C for 30 sec
[0084] 2Step PCR reaction
[0085] Cycles: 40
[0086] 95°C for 5 sec
[0087] 60°C for 10 sec.
[0088] The results are as Figure 6 shown. The expression levels of gtfB, gtfC, and luxS genes of S. mutans in the dihydromyricetin treatment group were all significantly down-regulated, indicating that dihydromyricetin can inhibit the expression of virulence genes of S. mutans, thereby weakening its pathogenicity.
[0089] Obviously, the above-mentioned embodiments of the present invention are merely examples for more clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or variations can be made. It is impossible to enumerate all the implementation methods here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.
Claims
1. Streptococcus mutans inhibitor based on natural compounds, with dihydromyricetin as the main active ingredient.
2. The mutans streptococcus inhibitor according to claim 1, characterized in that The purity of the dihydromyricetin is not less than 95%.
3. The method for preparing the mutans streptococcus inhibitor according to claim 1 or 2, comprising: The crushed rattan tea is mixed with ethanol and subjected to ultrasonic-assisted extraction. The extract is filtered to remove impurities and then concentrated under reduced pressure to obtain a crude extract of dihydromyricetin.
4. The preparation method according to claim 3, characterized in that: The crushed rattan tea is mixed with ethanol at a solid-liquid ratio of 1:20 g / mL; the ultrasonic frequency of ultrasonic-assisted extraction is 20 kHz, the extraction temperature is controlled at 30° C., and the extraction time is 2-3 hours.
5. Use of dihydromyricetin in the preparation of an oral care product, wherein the oral care product comprises an oral film inhibitor.
6. The use according to claim 5, characterized in that: The dihydromyricetin can significantly inhibit the formation and metabolic activity of the biofilm of Streptococcus mutans and destroy the microstructure of the biofilm.
7. The use according to claim 5, characterized in that: The dihydromyricetin can inhibit the acid production ability of Streptococcus mutans.
8. The use according to claim 5, characterized in that: The dihydromyricetin can inhibit the production of extracellular polysaccharides in the biofilm of Streptococcus mutans.
9. The use according to claim 5, characterized in that: The dihydromyricetin can inhibit the aggregation rate of Streptococcus mutans.
10. The use according to claim 5, characterized in that: The dihydromyricetin can inhibit the expression of virulence genes gtfB, gtfC and luxS of Streptococcus mutans.