Composition capable of inhibiting oral pathogens and application thereof

Abstract

The present invention discloses a composition capable of inhibiting oral pathogens and its use. The composition is characterized in that it comprises isopsoralea chalcone and psoralea corylifolia methyl ether in a mass ratio of 1 to 10:1. Experiments have shown that the composition has a strong inhibitory effect on four strains of oral pathogens: Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum polymorpha. Compared with two raw materials, the two raw materials in the combination of the present invention work synergistically, achieving a superior antibacterial effect at a lower dosage.

Description

Technical Field

[0001] The invention belongs to the field of daily chemicals or oral application products with disinfectant marks, and particularly relates to a composition capable of inhibiting oral pathogens and an application thereof. Background Art

[0002] Oral bacteria contain approximately 700 species of microorganisms, making them the second largest microbial community after the intestines. These microorganisms live in areas such as teeth, tongue, gingival sulcus, or saliva, and are closely related to oral health. The oral cavity's special and unique microenvironment gives oral bacteria a unique abundance composition and characteristics. Screening antibacterial products that are suitable for oral characteristics is of great value in maintaining oral health and preventing and treating oral diseases. [1-5] Microorganisms not only exhibit significant species specificity, but also exhibit significant differences in drug sensitivity and resistance between strains due to their diverse habitats. With the exception of broad-spectrum antibiotics, it is impossible to determine their activity against specific bacterial species through simple analogies.

[0003] Four oral Gram-negative pathogens, Porphyromonas Gingivalis (ATCC BAA-308), Aggregatibacter Actinomycetemcomitans (ATCC 43717), Prevotella Intermedia (ATCC 49046), and Fusobacterium Nucleatum Subsp. Polymorphum (ATCC 10953), play an important role in the development and progression of oral diseases. Their abnormal proliferation can lead to oral diseases such as bad breath, dental caries, or periodontitis. [2,5,6] .

[0004] Psoralea corylifolia is a commonly used Chinese medicine in clinical practice. It is warm in nature, pungent and bitter in taste, and enters the kidney and spleen meridians. It has the effects of warming the kidney and supporting yang, promoting qi and relieving asthma, warming the spleen and stopping diarrhea, and can be used externally to eliminate wind and remove spots. Current research shows that the main chemical components of Psoralea corylifolia are flavonoids, coumarins, terpenoids, etc. [7-9] Among them, flavonoids are reported to inhibit Gram-positive bacteria [10,11] .

[0005] Isopsoralea corylifolia chalcone and psoralea corylifolia flavonoid methyl ether are flavonoid components of Psoralea corylifolia and are known compounds. Isopsoralea corylifolia chalcone has inhibitory effects on Gram-positive Staphylococcus aureus, Staphylococcus epidermidis, and Methicillin-resistant Staphylococcus aureus.

[12] Psoralea corylifolia methyl ether shows weak antibacterial activity against Gram-positive Staphylococci and fungi, and has no inhibitory activity against Gram-negative bacteria.

[13] .

[0006] References:

[0007] 1.Mombelli, A. Microbial colonization of the periodontal pocket and its significance for periodontal therapy. Periodontology 2000.2018,76(1):85-96.

[0008] 2.Haque, MM et al. Advances in novel therapeutic approaches for periodontal diseases. BMC oral health. 2022, 22(1):492.

[0009] 3.Balta,MG et al.Host Modulation and Treatment of PeriodontalDisease.Journal of dental research.2021,100(8):798-809.

[0010] 4.Cekici,A et al.Inflammatory and immune pathways in the pathogenesis of periodontal disease.Periodontology 2000.2014,64(1):57-80.

[0011] 5.Balta,MG et al.Host Modulation and Treatment of PeriodontalDisease.Journal of dental research.2021,100(8):798-809.

[0012] 6. Yousefi, L et al. Oral spirochetes: Pathogenic mechanisms inperiodontal disease. Microbial pathogenesis. 2020,144:104193.

[0013] 7. Lu Yaqi et al. Research progress on chemical composition and pharmacological effects of Psoralea corylifolia. Chinese Journal of Experimental Traditional Chinese Medicine. 2019, 25(3): 180-189.

[0014] 8.Lim,S et al.Estrogenic activities of Psoralea corylifolia L.seedextracts and main constituents.Phytomedicine.2011,18(5):425-30.

[0015] 9.

[0016] 10.Yin,S et al.Antibacterial prenylflavone derivatives from Psoraleacorylifolia,and their structure-activity relationship study.Bioorganic&medicinal chemistry.2004,12(16):4387-92.

[0017] 11.Alam, F et al. Psoralea corylifolia L: Ethnobotanical, biological, and chemical aspects: A review. Phytotherapy research: PTR. 2018, 32 (4): 597-615.

[0018] 12.Sun,L et al.Exploration of Antimicrobial Ingredients in Psoraleacorylifolia L.Seed and Related Mechanism against Methicillin-ResistantStaphylococcus aureus.Molecules(Basel,Switzerland).2022,27(20):6952.

[0019] 13. Wang, Summary of the Invention

[0020] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a composition capable of inhibiting oral pathogens.

[0021] The second object of the present invention is to provide the use of the above composition in preparing a product for inhibiting oral pathogens.

[0022] The technical solution concept of the present invention is as follows:

[0023] A composition capable of inhibiting oral pathogens comprises isopsoralea corylifolia chalcone and psoralea corylifolia methyl ether in a mass ratio of 1 to 10:1.

[0024] The mass ratio of isopsoralea corylifolia chalcone to psoralea corylifolia methyl ether is preferably 1:1.

[0025] Application of the above composition in preparing products for inhibiting oral pathogens.

[0026] Oral pathogens include Porphyromonas Gingivalis, Aggregatibacter Actinomycetemcomitans, Prevotella Intermedia, or Fusobacterium Nucleatum Subsp. Polymorphum.

[0027] Experiments have demonstrated that the composition of the present invention exhibits a strong inhibitory effect against four oral pathogens: Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum polymorpha. Compared to single ingredients, the combination exhibits a synergistic effect, achieving a superior antibacterial effect at a lower dosage. DETAILED DESCRIPTION

[0028] The following examples are some examples of the present invention, rather than all examples, and are only used to illustrate the present invention, but should not be considered as limiting the scope of the present invention. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0029] The CAS number of the isopsoralea corylifolia chalcone in the present invention is: 20784-50-3;

[0030] The CAS number of psoralea corylifolia methyl ether is: 19879-30-2.

[0031] Isopsoralea chalcone and psoralea corylifolia methyl ether can also be prepared by extraction, see the literature (J Nat Prod. 1996, 59 (7): 671-2).

[0032] Porphyromonas Gingivalis (ATCC BAA-308) was purchased from Beijing Biobw Biotechnology Co., Ltd. on September 4, 2023. The website is: https: / / www.biobw.org / China-strain / bio-53046.html.

[0033] Aggregatibacter Actinomycetemcomitans (ATCC 43717) was purchased from Beijing Biobw Biotechnology Co., Ltd. on September 4, 2023, website: https: / / www.biobw.org / China-strain / bio-53034.html.

[0034] Prevotella intermedia (ATCC 49046) was purchased from Beijing Biobw Biotechnology Co., Ltd. on September 4, 2023, website: https: / / www.biobw.org / ATCC / bio-79965.html.

[0035] Fusobacterium nucleatum subsp. Polymorphum (ATCC10953) was purchased from Beijing Biobowei Biotechnology Co., Ltd. on September 4, 2023, website: https: / / www.biobw.org / China-strain / bio-53041.html.

[0036] Example 1

[0037] A composition capable of inhibiting oral pathogens comprises isopsoralea corylifolia chalcone and psoralea corylifolia methyl ether in a mass ratio of 1:1.

[0038] Example 2

[0039] The invention discloses a composition capable of inhibiting oral pathogens. The composition comprises isopsoralea corylifolia chalcone and psoralea corylifolia dihydroflavonoid methyl ether in a mass ratio of 10:1.

[0040] Example 3 (control)

[0041] A composition capable of inhibiting oral pathogens comprises isopsoralea corylifolia chalcone and psoralea corylifolia flavonoid methyl ether in a mass ratio of 1:10.

[0042] Example 4

[0043] A composition capable of inhibiting oral pathogens comprises isopsoralea corylifolia chalcone and psoralea corylifolia methyl ether in a mass ratio of 5:1.

[0044] Example 5

[0045] Evaluation of the anti-oral pathogenic effects of isopsoralea corylifolia chalcone, psoralea corylifolia flavonoid methyl ether, and the combination of Examples 1-3

[0046] Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Fusobacterium nucleatum polymorpha were cultured in Gifu Anaerobic Medium (GAM, Qingdao Haibo Biological) at 37°C under the conditions of 80% N2-10% CO2-10% H2. 600nm ) was 0.45-0.5, the bacterial solution was diluted 70-fold with GifuAnaerobic Medium and then added to a 96-well plate at a volume of 100 μl / well.

[0047] The experimental groups were prepared with different concentrations of isopsoralea corylifolia chalcone, psoralea corylifolia methyl ether, and the compositions prepared in Examples 1-3. The experimental group drugs were prepared with dimethyl sulfoxide (DMSO) to a stock solution with a concentration of 10 mg / ml.

[0048] The positive control group was chlorhexidine, whose minimum inhibitory concentration (MIC) was 1 μg / mL;

[0049] The negative control group consisted of wells containing only bacterial solution, which grew well throughout the evaluation process;

[0050] The positive control group and the negative control group met the experimental expectations, indicating that the method operation is feasible and credible;

[0051] The mother liquor of the experimental group drug was diluted to the experimental concentration with GAM medium, including 1, 2, 4, 8, 16, 32, 64 micrograms / ml.

[0052] There are 3 duplicate wells in each group, and anaerobic culture is carried out for 36 - 60 hours (the growth rates of each bacterium are different, and the culture times are different. All experiments are uniformly cultured until the OD of the negative control group 600nm is 0.3 - 0.4), take out the 96-well plate, and use an enzyme-labeled instrument to detect the OD 600nm reading value, and calculate the inhibition rate {inhibition rate = [1 - (experimental group OD600nm - GAM OD600nm ) / (negative control group OD600nm - GAM OD600nm ) * 100%}.

[0053] Determination of the minimum inhibitory concentration (MIC): The equal dilution method was used, and the concentrations of isobavachalcone, bavachinin, and the compositions of Examples 1 - 3 were set to 1, 2, 4, 8, 16, 32, 64 micrograms / ml respectively. The lowest concentration with an inhibition rate greater than 90% was taken as the MIC.

[0054] FIC index = MIC(concentration of isobavachalcone in the composition) / MIC(isobavachalcone) + MIC(concentration of bavachinin in the composition) / MIC(bavachinin). When the FIC index < 0.5, the two drugs have a synergistic effect; when 0.5 < FIC index < 1.0, the two drugs have an additive effect; when 1.0 < FIC index < 2.0, the two drugs have no relevant effect; when the FIC index > 2.0, the two drugs have an antagonistic effect.

[0055] As shown in Table 1, the MICs of isobavachalcone against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum polymorphum are 8, 8, 8, 32 micrograms / ml respectively. The MICs of bavachinin against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum polymorphum are 8, 8, 8, 64 micrograms / ml respectively. The compositions of Examples 1 and 2 both showed lower MICs than the monomers. Among them, Example 1 (the mass ratio of isobavachalcone to bavachinin is 1:1) has the best effect and shows a synergistic antibacterial effect against the four pathogenic bacteria (Table 2), and has important application value in the development of products related to inhibiting oral pathogenic bacteria.

[0056] Table 1 Effects of isobavachalcone, bavachinin, and the composition on inhibiting oral pathogenic bacteria

[0057]

[0058] Table 2 The combined effect of isopsoralea corylifolia chalcone and psoralea corylifolia flavonoid methyl ether composition in inhibiting oral pathogens

[0059]

[0060] The composition of Example 1, Example 2 or Example 4 of the present invention is used as an active ingredient and is combined with acceptable excipients according to conventional techniques to prepare different dosage forms, such as tablets, powders or pastes, which are conveniently used to treat oral problems such as caries, periodontitis, and bad breath caused by abnormal proliferation of oral pathogens, thereby restoring oral health to patients.

Claims

1. A composition for inhibiting oral pathogens, characterized in that The composition consists of isopsoralea corylifolia chalcone and psoralea corylifolia dihydroflavonoid methyl ether in a mass ratio of 1 to 10:

1.

2. The composition according to claim 1, wherein The mass ratio of the isopsoralea corylifolia chalcone to psoralea corylifolia methyl ether is 1:

1.

3. Use of the composition of claim 1 or 2 in the preparation of a product for inhibiting oral pathogens; The oral pathogens are Porphyromonas Gingivalis, Aggregatibacter Actinomycetemcomitans, Prevotella Intermedia or Fusobacterium Nucleatum Subsp. Polymorphum.

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