Natural product inhibitor aiming at enterococcus faecalis biological membrane as well as preparation method and application of natural product inhibitor

Abstract

The invention provides a natural product inhibitor aiming at an enterococcus faecalis biological membrane as well as a preparation method and application thereof, and the natural product inhibitor aiming at the enterococcus faecalis biological membrane is prepared from natural products in homology of medicine and food. The natural product inhibitor can inhibit the formation of an enterococcus faecalis biofilm and has a destructive effect on the formed mature biofilm, the adhesion of enterococcus faecalis is reduced, and the drug sensitivity of antibiotics can be increased by combining the natural product inhibitor with the antibiotics.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and relates to a natural product inhibitor against Enterococcus faecalis biofilm, a preparation method thereof, and an application thereof. Background Art

[0002] Root canal treatment is the most basic and optimal treatment method for diseases such as pulpitis and periapical periodontitis, aiming to eliminate these pathogens and eliminate pulp and periapical inflammation. In the steps of root canal preparation and disinfection, mechanical preparation and chemical disinfection are used to achieve maximum bacterial reduction. However, due to the complexity of the root canal system and the limitations of mechanical instruments, the success rate of treatment is between 70% and 95%. One of the main reasons for treatment failure is persistent root canal infection. Enterococcus faecalis is a Gram-positive facultative anaerobic bacterium associated with persistent root canal infection. Currently, clinical drugs used to eradicate bacterial infections in the root canal system include calcium hydroxide (CH), antibiotics (TAP: triple antibiotic paste), and chlorhexidine (CHX). Because this bacterium can survive continuously in a harsh environment and develop resistance to antibiotics and CHX in Enterococcus faecalis. One of the important reasons for the development of antibiotic resistance is the formation of biofilms. Summary of the Invention

[0003] Aiming at the problems in the prior art, the purpose of the present invention is to provide a natural product inhibitor against Enterococcus faecalis biofilm, a preparation method thereof, and an application thereof, which utilize natural products in medicine and food homology to destroy the biofilm of Enterococcus faecalis and are used in combination with antibiotics to reduce its drug resistance.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] The present invention provides a natural product inhibitor against Enterococcus faecalis biofilm, with pinosylvin as the main active ingredient.

[0006] The present invention also provides a preparation method of the above natural product inhibitor, including: mixing yam powder with an ethanol solution, placing it in a sealed container, soaking for several hours at normal temperature or under heating conditions, after the soaking is completed, filtering, evaporating and concentrating the obtained extract to remove most of the solvent to obtain a concentrated solution rich in pinosylvin, and finally extracting pinosylvin by column chromatography.

[0007] Preferably, the material-liquid ratio of the mixture of yam powder and the ethanol solution is 1:10 g / mL.

[0008] Preferably, soak for 12 hours at normal temperature or under heating conditions at 60°C.

[0009] Preferably, transfer the obtained extract to a rotary evaporator, and evaporate and concentrate it at 40-50°C under reduced pressure.

[0010] The present invention also provides the application of pinosylvin in inhibiting Enterococcus faecalis.

[0011] Preferably, the pinosylvin can inhibit the formation of Enterococcus faecalis biofilm and has a destructive effect on the formed mature biofilm.

[0012] Preferably, the pinosylvin can reduce the adhesiveness of Enterococcus faecalis.

[0013] Preferably, the combination of the pinosylvin and an antibiotic can increase the drug sensitivity of the antibiotic.

[0014] The beneficial effects of the present invention are as follows:

[0015] The present invention uses natural products in medicine and food homology to prepare a natural product inhibitor against Enterococcus faecalis biofilm. The natural product inhibitor can inhibit the formation of Enterococcus faecalis biofilm, has a destructive effect on the formed mature biofilm, reduces the adhesiveness of Enterococcus faecalis, and the combination of it and an antibiotic can also increase the drug sensitivity of the antibiotic. Description of the Drawings

[0016] Figure 1 . MIC of pinosylvin against Enterococcus faecalis.

[0017] Figure 2 . Effect of pinosylvin on the growth of Enterococcus faecalis.

[0018] Figure 3 . Effect of pinosylvin on the formation of Enterococcus faecalis biofilm.

[0019] Figure 4 . Effect of pinosylvin on the formed mature biofilm of Enterococcus faecalis.

[0020] Figure 5 . Schematic diagram of the operation of the checkerboard method for the synergy of pinosylvin and an antibiotic.

[0021] Figure 6 . Determination of the combined use of drugs and antibiotics by the checkerboard method. Detailed Description of the Invention

[0022] The present invention will be described in detail below in conjunction with the specific embodiments. 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.

[0023] Examples

[0024] I. Extraction of pinosylvin by solvent extraction method

[0025] 1. Material preparation: Select fresh or dried Chinese yams, crush them into powder to increase the contact area with the solvent.

[0026] 2. Soaking extraction: Mix yam powder with ethanol solution at a certain solid-liquid ratio (1:10 g / mL) and place it in a sealed container. Soak for 12 hours at room temperature or under water bath heating conditions (60 °C), and stir appropriately during this period to promote the dissolution of pinosylvin from yam powder into the solvent.

[0027] 3. Separation and concentration: After the soaking is completed, separate the extract from the residue by filtration. Transfer the obtained extract to a rotary evaporator and evaporate and concentrate it under reduced pressure at an appropriate temperature (such as 40 - 50 °C) to remove most of the solvent and obtain a concentrated solution rich in pinosylvin. Extract pinosylvin by column chromatography.

[0028] II. MIC of pinosylvin against Enterococcus faecalis

[0029] Add the seed solution of Enterococcus faecalis (E. faecalis) to the brain heart infusion (BHI) liquid medium at a ratio of 1% (v / v) (about 1 - 5×10 5 CFU / mL). Add pinosylvin to 1.6 mL of the bacterial solution and then dilute it using the two-fold dilution method to make the final concentrations 1600, 800, 400, 200, 100, 50 μg / mL. Transfer 200 μL of each mixed culture solution to a 96-well polystyrene microplate. After placing the 96-well plate in an incubator at 37 °C and statically culturing for 24 h, the concentration of the wells where the bacterial solution did not become turbid is the minimum inhibitory concentration (MIC). The results are as Figure 1 shown in Table 1. The MIC concentration of pinosylvin against Enterococcus faecalis is 100 μg / mL.

[0030] Table 1. MIC of pinosylvin against Enterococcus faecalis

[0031]

[0032] III. Effect of pinosylvin on the growth of Enterococcus faecalis

[0033] Add the seed solution of E. faecalis to the BHI liquid medium at a ratio of 1% (v / v). Then add pinosylvin to make the final concentrations 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC respectively, and then culture at 37 °C for 24 hours. DMSO is used as a negative control. Measure the OD value at 600 nm every hour with a microplate reader, and draw a growth curve with the measured data.

[0034] The results are as Figure 2 shown.

[0035] IV. Effect of pinosylvin on the biofilm formation of Enterococcus faecalis

[0036] Add the seed solution of E. faecalis to BHI liquid medium at a ratio of 1% (v / v). Then supplement with pinosylvin, and transfer 200 μL of the mixed culture solution to a 96-well polystyrene microplate. Add pinosylvin with final concentrations of 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC respectively, and use dimethyl sulfoxide (DMSO) as the negative control. The experiment was completed using the crystal violet staining method. After the 96-well plate was placed in an incubator at 37 °C and statically cultured for 24 h, gently remove the culture solution, and wash it 3 times with sterile phosphate buffer saline (PBS) to wash away the floating bacteria. Dry the moisture in the wells in an oven at 60 °C, fix the bottom biofilm with methanol for 15 min, remove the liquid and then dry it in an oven at 60 °C. Add 200 μL of 0.05% (w / v) crystal violet to each well and stain for 10 minutes. Gently remove the crystal violet solution, and rinse 3 times with PBS to remove the excess unattached crystal violet dye. After drying at 60 °C, add 200 μL of 95% ethanol to each well, and decolorize at 37 °C on a shaker at 180 rpm for 15 min. Take 150 μL of the decolorized solution and measure the OD value at 570 nm using a microplate reader.

[0037] The results are as Figure 3 shown.

[0038] V. Effect of pinosylvin on the mature biofilm formed by Enterococcus faecalis

[0039] Add the seed solution of E. faecalis to BHI liquid medium at a ratio of 1% (v / v). Take 200 μL of the mixture and add it to a 96-well plate, and place it in a constant temperature incubator at 37 °C for static culture for 24 h. Pour out the excess medium and wash it three times with sterile PBS. Dry the moisture in the wells in an oven at 60 °C, add the drug mixtures of 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC, and statically culture at 37 °C for 24 h. After the culture is completed, use the crystal violet staining method to determine the effect of pinosylvin on the mature biofilm formed by E. faecalis.

[0040] The results are as Figure 4 shown.

[0041] VI. Determination of the combination of drugs and antibiotics by checkerboard method

[0042] Pick a single colony of Enterococcus faecalis and inoculate it into 5 mL of BHI medium for activation for 17 h as the seed solution. Adjust the OD620 value of the seed solution to 0.5, and inoculate it into BHI medium at an inoculation amount of 0.1%, and mix evenly. Use the checkerboard method to determine the combined effect of pinosylvin and antibiotics.

[0043] Using the initial concentrations of pinosylvin (100 μg / mL) and the antibiotic gentamicin (250 μg / mL) measured during single drug use before as the initial concentrations when used in combination. Add drugs in different combinations to the BHI medium containing the bacterial solution, and perform gradient dilution using the two-fold dilution method. Take 200 μL of the mixed solution and add it to a 96-well plate, and culture at 37 °C and 150 rpm for 17 h, and measure OD620 with an enzyme-linked immunosorbent assay (ELISA) reader. The specific combination method of the combined drugs in the 96-well plate is shown in Figure 5 . The experiment was repeated three times. The calculation standard for the fractional inhibitory concentration (FIC) is: FIC = MIC of pinosylvin or the combined use of antibiotics / MIC of pinosylvin or antibiotics when used alone. The fractional inhibitory concentration index (FICI) = FIC of pinosylvin + FIC of antibiotics. FICI ≤ 0.5 indicates a synergistic effect, 0.5 < FICI ≤ 4 indicates no interaction, and FIC > 4 indicates an antagonistic effect. The experimental results are as shown in Figure 6 , and the calculated FICI results are shown in Table 2.

[0044] Table 2. Combination of lucidine and gentamicin

[0045]

[0046] Obviously, the above embodiments of the present invention are merely examples for more clearly explaining the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A natural product inhibitor of Enterococcus faecalis biofilm, with styracoside as the main active ingredient.

2. The method for preparing the natural product inhibitor according to claim 1, comprising: The yam powder is mixed with ethanol solution, placed in a sealed container, and soaked for several hours at room temperature or under heating conditions. After the soaking, it is filtered, and the obtained extract is evaporated and concentrated to remove most of the solvent to obtain a concentrated solution rich in red pine alkaloids. Finally, red pine alkaloids are extracted by column chromatography.

3. The preparation method according to claim 2, characterized in that: The solid-liquid ratio of the yam powder and the ethanol solution is 1:10 g / mL.

4. The preparation method according to claim 2, characterized in that: Soak for 12 hours at room temperature or at 60°C.

5. The preparation method according to claim 2, characterized in that: The obtained extract was transferred to a rotary evaporator and concentrated by evaporation at 40-50°C under reduced pressure.

6. Application of pine cone extract in inhibiting Enterococcus faecalis.

7. The use according to claim 6, characterized in that: The pinospermum officinale can inhibit the formation of Enterococcus faecalis biofilm and has a destructive effect on the formed mature biofilm.

8. The use according to claim 6, characterized in that: The pinospermum officinale can reduce the adhesion of Enterococcus faecalis.

9. The use according to claim 6, characterized in that: The combination of pinospermum officinale and antibiotics can increase the drug sensitivity of antibiotics.

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