Composition capable of inhibiting oral pathogenic bacteria and application thereof

Abstract

The invention discloses a composition capable of inhibiting oral pathogens and application thereof, and the composition capable of inhibiting oral pathogens is characterized in that the composition is composed of isopsoralen chalcone and psoralen flavanone methyl ether in a mass ratio of (1-10): 1; experiments prove that the composition disclosed by the invention can generate a relatively strong inhibition effect on four oral pathogens, namely porphyromonas gingivalis, actinobacillus actinomycetes, prevotella intermedia and fusobacterium nucleatum subsp.polymorpha. Compared with the two raw materials, the two raw materials in the combined group play a synergistic role, and a better antibacterial effect is played under the condition of a lower dosage.

Description

Technical Field

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

[0002] Oral bacteria contain more than 700 species of microorganisms, which is the second largest microbial group after the intestine. These microorganisms live in teeth, tongue, gingival sulcus or saliva, and are closely related to oral health. The special and unique microenvironment of the oral cavity makes oral bacteria have unique abundance composition and characteristics. Screening antibacterial products suitable for oral characteristics is of great value in maintaining oral health and preventing and treating oral diseases. [1-5] Microorganisms not only have significant species specificity, but also have significant differences in drug sensitivity and resistance between strains due to different living environments. Except for broad-spectrum antibiotics, it is impossible to determine their activity against specific bacterial species by simple analogy.

[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 occurrence and development of oral diseases. Their abnormal proliferation can lead to oral diseases such as bad breath, 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, relieving asthma, warming the spleen and stopping diarrhea. It 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 chalcone and psoralea corylifolia methyl ether are flavonoid components in psoralea corylifolia and are known compounds. Isopsoralea chalcone has inhibitory effects on gram-positive staphylococci such as Staphylococcus aureus, Staphylococcus epidermidis, and Methicillin-resistant Staphylococcus aureus.

[12] Psoralea corylifolia methyl ether showed weak antibacterial activity against Gram-positive Staphylococcus and fungi, and no inhibitory activity against Gram-negative bacteria was reported.

[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 components and pharmacological effects of Psoralea corylifolia. Chinese Journal of Experimental Traditional Chinese Medicine Formulae. 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):4387-92.11.Alam,F et al.Psoralea corylifoliaL:Ethnobotanical,biological,and chemical aspects:A review.Phytotherapyresearch:PTR.2018,32(4):597-615.

[0017] 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.

[0018] 13. Wang, Summary of the invention

[0019] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a composition capable of inhibiting oral pathogens.

[0020] The second object of the present invention is to provide the use of the above composition in preparing a product for inhibiting oral pathogens.

[0021] The technical solution concept of the present invention is as follows:

[0022] A composition capable of inhibiting oral pathogens, the composition consists of isopsoralea chalcone and psoralea corylifolia methyl ether in a mass ratio of 1 to 10:1.

[0023] The mass ratio of isopsoralea chalcone to psoralea corylifolia methyl ether is preferably 1:1.

[0024] The application of the composition in preparing products for inhibiting oral pathogens.

[0025] Oral pathogens are Porphyromonas Gingivalis, Aggregatibacter Actinomycetemcomitans, Prevotella Intermedia or Fusobacterium Nucleatum Subsp.Polymorphum.

[0026] Experiments have shown that the composition of the present invention can have a strong inhibitory effect on four oral pathogens, namely Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Fusobacterium nucleatum polymorpha. Compared with the two raw materials, the two raw materials in the combination group play a synergistic role and exert a better antibacterial effect at a lower dosage. DETAILED DESCRIPTION

[0027] The following described embodiments are some embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments 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 embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0028] The CAS number of the isopsoralea corylifolia chalcone in the present invention is: 20784-50-3;

[0029] The CAS number of psoralea corylifolia is: 19879-30-2.

[0030] Isopsoralea chalcone and psoralea corylifolia methyl ether can also be prepared by extraction, see the literature (J Nat Prod. 1996, 59 (7): 671-2).

[0031] Porphyromonas Gingivalis (ATCC BAA-308) was purchased from Beijing Biobw Biotechnology Co., Ltd. on September 4, 2023, website: https: / / www.biobw.org / China-strain / bio-53046.html.

[0032] 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.

[0033] 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.

[0034] Fusobacterium Nucleatum Subsp. Polymorphum (ATCC10953) was purchased from Beijing Biobw Biotechnology Co., Ltd. on September 4, 2023, website: https: / / www.biobw.org / China-strain / bio-53041.html.

[0035] Example 1

[0036] A composition capable of inhibiting oral pathogens comprises isopsoralea chalcone and psoralea corylifolia methyl ether in a mass ratio of 1:1.

[0037] Example 2

[0038] A composition capable of inhibiting oral pathogens comprises isopsoralea chalcone and psoralea corylifolia methyl ether in a mass ratio of 10:1.

[0039] Example 3 (control)

[0040] A composition capable of inhibiting oral pathogens comprises isopsoralea chalcone and psoralea corylifolia methyl ether in a mass ratio of 1:10.

[0041] Example 4

[0042] A composition capable of inhibiting oral pathogens comprises isopsoralea chalcone and psoralea corylifolia methyl ether in a mass ratio of 5:1.

[0043] Example 5

[0044] Evaluation of the anti-oral pathogenic effect of isopsoralea chalcone, psoralea corylifolia methyl ether and the combination of Examples 1-3

[0045] Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Fusobacterium nucleatum polymorpha were cultured in Gifu Anaerobic Medium (GAM, Qingdao Hibo Biotechnology) at 37°C under the conditions of 80% N2-10% CO2-10% H2. 600 nm ) was 0.45-0.5, the bacterial solution was diluted 70 times with GifuAnaerobic Medium and then added to a 96-well plate at a volume of 100 μl / well.

[0046] The experimental group was composed of different concentrations of isopsoralea chalcone, psoralea corylifolia methyl ether and the composition prepared in Examples 1-3. The drugs in the experimental group were prepared into a mother solution with a concentration of 10 mg / ml using dimethyl sulfoxide (DMSO).

[0047] The positive control group was chlorhexidine, whose minimum inhibitory concentration (MIC) in the experimental test was 1 μg / mL;

[0048] The negative control group consisted of wells containing only bacterial solution, which grew well throughout the evaluation process;

[0049] The positive control group and the negative control group met the experimental expectations, indicating that the method operation is feasible and credible;

[0050] The mother liquor of the experimental group drug was diluted to the experimental concentration with GAM medium, including 1, 2, 4, 8, 16, 32, and 64 micrograms per milliliter.

[0051] There were 3 duplicate wells in each group, and anaerobic culture was carried out for 36 - 60 hours (the growth rates of each bacterium are different, and the culture times are different. All experiments were uniformly cultured until the OD of the negative control group 600 nm was 0.3 - 0.4). The 96 - well plate was taken out, and the OD 600 nm reading value was measured with an enzyme - linked immunosorbent assay (ELISA) reader, and the inhibition rate was calculated {Inhibition rate = [1 - (experimental group OD600nm - GAM OD600nm ) / (negative control group OD600nm - GAM OD600nm )]*100%}.

[0052] Determination of the minimum inhibitory concentration (MIC): The equal - dilution method was used. The concentrations of isobavachalcone, bavachinin, and the compositions of Examples 1 - 3 were set to 1, 2, 4, 8, 16, 32, and 64 micrograms per milliliter respectively. The lowest concentration with an inhibition rate greater than 90% was taken as the MIC.

[0053] FIC index = MIC (isobavachalcone concentration in the composition) / MIC (isobavachalcone)+MIC (bavachinin concentration 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.

[0054] As shown in Table 1, the MICs of isobavachalcone against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum polymorphum were 8, 8, 8, and 32 micrograms per milliliter respectively. The MICs of bavachinin against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum polymorphum were 8, 8, 8, and 64 micrograms per milliliter 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) had the best effect and showed a synergistic antibacterial effect against the four pathogenic bacteria (Table 2), which has important application value in the development of products for inhibiting oral pathogenic bacteria.

[0055] Table 1 The effects of isobavachalcone, bavachinin, and the compositions on inhibiting oral pathogenic bacteria

[0056]

[0057] Table 2 The combined effect of isopsoralea chalcone and psoralea corylifolia methyl ether composition in inhibiting oral pathogens

[0058]

[0059] 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 for oral problems such as caries, periodontitis, halitosis, etc. caused by abnormal proliferation of oral pathogens, so as to restore oral health of patients.

Claims

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

1.

2. The composition according to claim 1, characterized in that The mass ratio of the isopsoralea chalcone to the 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.

4. The use according to claim 3, characterized in that The oral pathogens are Porphyromonas Gingivalis, Aggregatibacter Actinomycetemcomitans, Prevotella Intermedia or Fusobacterium Nucleatum Subsp. Polymorphum.

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