Application of secoiridoid compound in resisting oral pathogenic bacteria and harm thereof

Abstract

The invention discloses an application of a secoiridoid compound in resisting oral pathogenic bacteria and harm thereof, and belongs to the field of medicinal chemistry. The secoiridoid compounds are gentiopicroside and swertiamarin, and the secoiridoid compounds can be used for preparing medicines. The common oral pathogenic bacteria are staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, nocardia stellate and hemolytic streptococcus. The secoiridoid compounds gentiopicroside and swertiamarin show excellent inhibitory activity on oral cavity harm caused by detected oral pathogenic bacteria and porphyromonas gingivalis, can be used as bacteriostatic active ingredients to be applied to prevention and treatment of oral cavity microbial infection, and have the potential of being further researched and developed into novel oral cavity bacteriostatic agents.

Description

Technical Field

[0001] The invention discloses application of secoiridoid compounds in resisting oral pathogenic bacteria and their harm, and belongs to the field of medicinal chemistry. Background Art

[0002] The oral cavity is the second most complex microbial area after the intestines, with the temperature, humidity and nutritional conditions required for the growth of microorganisms. Under normal circumstances, these microorganisms can coexist with the body, but when the oral microecology is unbalanced, it will cause oral diseases such as caries, periodontal disease, oral mucosal disease, etc., which will bring great inconvenience to people's lives. Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Nocardia asteroides, hemolytic Streptococcus, Porphyromonas gingivalis, etc. are infectious bacteria in oral treatment and oral hazards. These microorganisms enter the body through the lungs, mouth, etc., causing respiratory diseases and oral diseases. Oral diseases can also cause systemic diseases such as diabetes, cardiovascular diseases, Alzheimer's disease, etc., which seriously affect human physical and mental health. Oral health issues are receiving more and more attention from people, and the manpower, time and financial resources consumed are increasing year by year, causing great trouble to people.

[0003] Most oral disease treatment drugs are antibiotics, and there are many types of oral microorganisms. While treating diseases, antibiotics can also kill normal cells, causing new problems. Long-term use of antibiotics can also lead to symptoms such as drug resistance of microorganisms in the body, reduced immunity, and imbalance of homeostasis. According to traditional Chinese medicine theory, oral diseases are closely related to the gastrointestinal tract, liver, gallbladder, kidney and other internal organs. When dampness and heat accumulate in the spleen, stomach, liver and gallbladder, they often disturb along the meridians, causing red, swollen and painful gums and pus. Blood heat can cause bleeding gums, bitter mouth and bad breath. Chinese herbal medicines with the effects of clearing heat, detoxifying, and aromatic dehumidification are reservoirs of bioactive compounds. Their extracts are rich in resources and are safe and friendly to humans and the environment. Therefore, using new strategies to find and develop new antibiotics from plants is one of the effective ways to solve the pathogens of oral diseases.

[0004] Secoiridoids are a class of compounds among cyclopentane monoterpene derivatives. They are formed by the ring cleavage of cycloiridoid compounds at C7 and C8. Their parent nucleus has a hemiacetal structure and is often combined with sugar to form glycosides. Gentiopicroside and scutopicroside are non-toxic scutoiridoid compounds. Gentiopicroside and scutopicroside are abundant in their plants and have simple extraction methods. Bioinformatics analysis has found that they are a natural drug resource with broad application and development prospects. Gentiopicroside can reduce the levels of 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA), increase the total antioxidant level (TAS), and synergistically fight tumors through signal pathways such as nerve growth factor NGF and pAkt / pS6. Scutopicroside has the effects of promoting the repair of damaged liver cells and improving liver circulatory disorders. Both have the effects of lowering blood sugar, anti-inflammatory, detoxification, and organ protection. Therefore, gentiopicroside and scutellariae marinus have potential application value in combating oral pathogens and can be used to develop new antibacterial agents for preventing and treating oral microbial infections. Summary of the invention

[0006] The purpose of the present invention is to provide a new use of a secoiridoid compound for oral pathogens and the harm caused by them.

[0007] To achieve the above object, the present invention provides the following technical methods:

[0008] The invention provides application of secoiridoid compounds as antibacterial active ingredients in preventing and treating oral pathogenic bacteria and in treating oral hazards.

[0009] Furthermore, the secoiridoid compounds are gentiopicroside and scutellariamarin.

[0010] Furthermore, the oral pathogens to be prevented and treated are Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Nocardia australis, and hemolytic Streptococcus.

[0011] Furthermore, the oral harm is oral harm caused by Porphyromonas gingivalis.

[0012] Beneficial effects:

[0013] Gentianopicrin and scutellariae glycoside have excellent antibacterial activity against oral pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Nocardia stellaria, and hemolytic Streptococcus, which is manifested in the antibacterial effect by affecting the permeability of cell membranes.

[0014] Gentianopicrin and gentiopicroside reduce the oral damage caused by Porphyromonas gingivalis by affecting the production of volatile sulfur-containing compounds.

[0015] Gentianopicrin and scutellariae marinus compounds are natural products that are not easy to develop resistance and have the potential to be further developed into new oral antibacterial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Effects of gentiopicroside on bacterial growth

[0017] Figure 2 Effects of Swertiamarin on Bacterial Growth DETAILED DESCRIPTION

[0018] In order to better understand the present invention, the above content of the present invention is further described in detail by the following specific examples. However, this should not be construed as limiting the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified. The specific experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0019] Gentianopicrin and scutellariamarin were purchased commercially, with CAS numbers of 20831-76-9 and 17388-39-5, respectively, and HPLC ≥ 98%.

[0020] Example 1 Determination of the activity of gentiopicroside and scutellaria baicalensis against oral pathogens

[0021] Drug preparation: prepare gentiopicroside and scutellaria baicalensis glycoside into a mother liquor of a certain concentration, filter and sterilize it for later use.

[0022] Inoculation and culture: The common oral pathogens used in the experiment were laboratory-preserved strains. Each strain was cultured at 37°C for one week, and activated Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Nocardia stellate, and hemolytic Streptococcus were selected and sterilized with high-pressure single colonies in fresh liquid culture medium at 37°C, 200r / min. OD was detected by microplate reader 610nm When the bacterial concentration is 1×10 6 CFU / mL, oleuropein and scutellaria baicalensis mother solutions were added respectively to make the concentrations of gentiopicroside and scutellaria baicalensis 640 μg / mL, and the culture was shaken at 37°C. Samples were taken and counted at 0h, 8h, 16h, 24h, 32h, and 48h to obtain a time-microorganism quantity curve.

[0023] Depend on Figure 1 , Figure 2It can be seen that with the extension of the action time, the bacterial growth of high-concentration gentiopicroside showed a downward trend, and the tested bacteria showed good inhibitory activity; with the extension of the action time, the bacterial growth of high-concentration scutellaria baicalensis glycoside showed a downward trend, and the tested bacteria showed good inhibitory activity. Take the high-concentration compound 1:1 dilution to prepare a solution, put the filter paper into the sterile tweezers to soak, stay for about 10s to filter out the excess liquid, press to make the filter paper with the drug fit with the bacterial culture medium, culture in the incubator for 48h, measure the size of the inhibition zone, calculate the inhibition rate (%), repeat 3 times for each concentration, and the inhibition rate of the compound is shown in Table 1.

[0024] Table 1 Inhibition rate of different compounds

[0025]

[0026] As shown in Table 1, when the concentration of gentiopicroside and scutellaria baicalensis was 320 μg / mL, the antibacterial effect on some test bacteria was excellent, and the inhibition rate of the tested bacteria was 51% to 84%. Within the above concentration range, the concentration was further reduced to continue testing to find the minimum inhibitory concentration, including 320, 160, 80, 40, 20, 10, and 0 μg / mL.

[0027] First, add 100uL of liquid culture medium to each well of the 96-well plate, add 100uL of gentiopicroside or scutellaria ... 6 CFU / mL. Place the 96-well plate in a 37°C incubator and culture for more than 18 hours, and observe the results. Use "-" to indicate negative, the well is clear, and use "+" to indicate positive, the well is turbid. Use "++" to indicate positive, the well is extremely turbid. The positive control is ciprofloxacin (5μg / mL), the control group is 100μL bacterial solution plus 100uL culture solution, and 200μL culture solution is the blank control. Set up 3 parallel experiments for each concentration, and the measurement results are shown in Table 2.

[0028] Table 2 Antibacterial effects of compounds at different concentrations

[0029]

[0030] It can be seen from Table 2 that for Staphylococcus aureus, the MIC of gentiopicroside is ≤40μg / mL, and the MIC of scutellaria serrata is ≤80μg / mL; for Pseudomonas aeruginosa, the MIC of gentiopicroside is ≤80μg / mL, and the MIC of scutellaria serrata is ≤80μg / mL; for Escherichia coli, the MIC of gentiopicroside is ≤40μg / mL, and the MIC of scutellaria serrata is ≤80μg / mL; for Nocardia asteroides, the MIC of gentiopicroside is ≤160μg / mL, and the MIC of scutellaria serrata is ≤160μg / mL; for hemolytic Streptococcus, the MIC of gentiopicroside is ≤80μg / mL, and the MIC of scutellaria serrata is ≤80μg / mL.

[0031] Example 2 Effects of gentiopicroside and scutellariae marin on cell membrane permeability of oral pathogenic bacteria

[0032] The bacterial cell membrane is an elastic semipermeable membrane composed of a phospholipid bilayer and inlaid proteins, which performs many important metabolic functions. When the cell membrane permeability increases, the macromolecules in the cell will flow into the culture medium. By adding different reagents during the bacterial culture process and measuring the content of macromolecules in the culture medium, we can determine the effect of the additives on the cell membrane permeability of each strain.

[0033] The exogenous protein-free culture medium is prepared, specifically, the exogenous protein-free culture medium includes NaHCO3, KNO3, aminoacid, Vitamin, NaCl and trace elements including ZnCl250μM / L and chelated iron ammonium ferric citrate 200mg / L.

[0034] The revived strains were selected and cultured in liquid culture medium for 15 h. The concentration of the culture medium was adjusted to 2.0 × 10 5 CFU / mL, placed in a 37℃ constant temperature culture for 24h, centrifuged at 150r / min, discarded the supernatant, and then added exogenous protein-free culture medium. At the same time, gentiopicroside and scutellaria baicalensis glycoside were added to the culture medium at a final concentration of 320μg / mL. Blank culture medium and drug-containing culture medium were used as controls and placed in a 37℃ constant temperature incubator for culture. The supernatant was taken at different time points, and the protein content of the supernatant was measured by BCA method. Three parallel wells were made for each group.

[0035] The results showed that when the concentrations of gentiopicroside and scutellaria baicalensis were both 320 μg / mL, the effects of gentiopicroside and scutellaria baicalensis on the tested pathogens were as follows: as time went on, the protein content in the supernatant increased. This indicated that the additives increased the permeability of bacterial cell membranes, causing ions and macromolecular substances to leak out, seriously affecting bacterial growth and reproduction, and achieving an antibacterial effect.

[0036] Example 3 Effects of gentiopicroside and scutellariae marin on the content of volatile sulfur compounds caused by Porphyromonas gingivalis

[0037] Bad breath is caused by the production of high concentrations of foul-smelling compounds when pathogenic microorganisms such as Porphyromonas gingivalis absorb and decompose nutrients in the human body. These gases are discharged from the body through the mouth and nose, seriously affecting physical and mental health. When cultured in vitro, Porphyromonas gingivalis can decompose sulfur-containing organic matter such as cystine, cysteine, and methionine to produce hydrogen sulfide. When hydrogen sulfide meets lead acetate test paper, it forms a black lead sulfide precipitate.

[0038] Medium configuration: Component I: 0.5% hemin solution filtered through a 0.22 μm filter, stored at 4°C away from light for later use; Component II: 1.8 g agar powder, 3.6 g BHI powder, 0.5 g yeast powder, 0.04 g / L cysteine ​​hydrochloride, fixed to 100 mL, sterilized by high pressure, stored in a 4°C refrigerator for later use. On a sterile operating table, component I: component II: sheep blood were mixed at a ratio of 1:100:10 to prepare BHI medium. Gentiopicroside and scutellaria baicalensis mother liquor were added to the BHI medium to make drug-carrying detection medium with concentrations of 640, 320, 160, 80, 40, 20, and 0 μg / mL, respectively.

[0039] Bacterial activation: Porphyromonas gingivalis was cultured in an anaerobic chamber at 37 degrees for 5-7 days.

[0040] Inoculation culture: Take 1 ml of Porphyromonas gingivalis solution (the concentration of the solution is 1×10 6 CFU / mL) into a sterile culture dish, then pour in the drug-containing test culture medium, mix quickly, and after the culture medium solidifies, culture it in a 37°C anaerobic box to set up a control group.

[0041] Lead sulfide detection: Paste lead acetate test paper in the culture dish and compare it with the standard colorimetric plate. The darker the black, the more lead sulfide is produced.

[0042] The test results showed that there was no significant change in the range of 0 to μg / mL. In the range of 160 to 640 μg / mL, as the drug concentration increased, the black precipitate decreased significantly. High concentrations of gentiopicroside and scutellariae marinus may affect bacterial growth by inhibiting the adhesion and colonization of pathogens, destroying the permeability barrier of bacterial cell membranes, and interfering with sugar metabolism, thereby reducing the production of its metabolite hydrogen sulfide and reducing the oral harm caused by it.

[0043] In summary, the secoiridoid ether terpenoid compounds gentiopicroside and scutellariamarin described in the present invention have good antibacterial activity against the tested oral pathogens, can reduce the harm caused by Porphyromonas gingivalis, and have further research and development value in preventing and treating oral microbial infections and oral protection.

Claims

1. Application of secoiridoid compounds as antibacterial active ingredients in combating oral pathogens and their hazards.

2. The use according to claim 1, characterized in that The secoiridoid compounds are gentiopicroside and scutellariamarin.

3. The use according to claim 2, characterized in that The oral pathogenic bacteria are Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Nocardia asteroides, and hemolytic Streptococcus.

4. The use according to claim 3, characterized in that The effective inhibition rates of the gentiopicroside and swertiamarin are 51-84%.

5. The use according to claim 3, characterized in that The effective use range of the gentiopicroside and scutellariamarin is 40 to 640 μg / mL.

6. The use according to claim 2, characterized in that The harm described is oral harm caused by Porphyromonas gingivalis.

7. The use according to claim 6, characterized in that The effective use range of the gentiopicroside and scutellariamarin is 160-640 μg / mL.

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