A natural product inhibitor against enterococcus faecalis biofilm and preparation method and application thereof

Abstract

The application provides a natural product inhibitor for Enterococcus faecalis biofilm and a preparation method and application thereof. The natural product inhibitor for Enterococcus faecalis biofilm is prepared by using natural products in medicinal food homology. The natural product inhibitor can inhibit the formation of Enterococcus faecalis biofilm, has a destructive effect on the mature biofilm which has been formed, reduces the adhesion of Enterococcus faecalis, and can increase the drug sensitivity of antibiotics when used in combination with the antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a natural product inhibitor targeting Enterococcus faecalis biofilm, its preparation method, and its application. Background Technology

[0002] Root canal treatment is the most basic and optimal treatment for diseases such as pulpitis and periapical periodontitis, aiming to eliminate these pathogens and relieve inflammation of the pulp and periapical region. During 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). This is because the bacteria can survive persistently in harsh environments and develop resistance to antibiotics and CHX in Enterococcus faecalis. One important reason for antibiotic resistance is the formation of a biofilm. Summary of the Invention

[0003] To address the problems in the prior art, the present invention aims to provide a natural product inhibitor against Enterococcus faecalis biofilm, its preparation method, and its application. This invention utilizes a natural product that is both food and medicine to disrupt the biofilm of Enterococcus faecalis and, when used in combination with antibiotics, reduces its drug resistance.

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

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

[0006] The present invention also provides a method for preparing the above-mentioned natural product inhibitor, comprising: mixing yam powder with ethanol solution, placing it in a sealed container, soaking it for several hours at room temperature or under heating conditions, filtering it after soaking, evaporating and concentrating the obtained extract to remove most of the solvent, obtaining a concentrated solution rich in pinocembrin, and finally extracting pinocembrin by column chromatography.

[0007] Preferably, the ratio of yam powder to ethanol solution is 1:10 g / mL.

[0008] Preferably, the soaking is carried out for 12 hours at room temperature or under heating conditions of 60°C.

[0009] Preferably, the obtained extract is transferred to a rotary evaporator and concentrated by evaporation at 40–50°C under reduced pressure.

[0010] This invention also provides the application of pinocembrin in inhibiting Enterococcus faecalis.

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

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

[0013] Preferably, the combination of the red pinocembrin with antibiotics can increase antibiotic sensitivity.

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

[0015] This invention utilizes natural products derived from food and medicine to prepare natural product inhibitors targeting Enterococcus faecalis biofilms. These natural product inhibitors can inhibit the formation of Enterococcus faecalis biofilms and disrupt existing mature biofilms, reducing the adhesion of Enterococcus faecalis. When used in combination with antibiotics, they can also increase antibiotic susceptibility. Attached Figure Description

[0016] Figure 1 MIC of pinococcus faecalis.

[0017] Figure 2 The effect of red pinocembrin on the growth of Enterococcus faecalis.

[0018] Figure 3 The effect of red pinocembrin on biofilm formation of Enterococcus faecalis.

[0019] Figure 4 The effect of red pinocembrin on the mature biofilm formed by Enterococcus faecalis.

[0020] Figure 5 Schematic diagram of the synergistic chessboard method of red pinocembrin and antibiotics.

[0021] Figure 6 The checkerboard method was used to determine the combined use of drugs and antibiotics. Detailed Implementation

[0022] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0023] Example

[0024] I. Extraction of Pinus julibrissin by Solvent Extraction

[0025] 1. Material preparation: Select fresh or dried yam and crush it into powder to increase the contact area with the solvent.

[0026] 2. Soaking and Extraction: Mix yam powder with ethanol solution at a certain material-to-liquid ratio (1:10 g / mL) and place in a sealed container. Soak for 12 hours at room temperature or under water bath heating conditions (60℃), stirring appropriately during the process to promote the dissolution of pinocembrin from the yam powder into the solvent.

[0027] 3. Separation and Concentration: After soaking, the extract and residue are separated by filtration. The obtained extract is transferred to a rotary evaporator and concentrated under reduced pressure at an appropriate temperature (e.g., 40–50°C) to remove most of the solvent, yielding a concentrated solution rich in pinocembrin. Pinocembrin is then extracted using column chromatography.

[0028] II. MIC of pinococcus faecalis

[0029] Add Enterococcus faecalis seed culture (approximately 1-5 × 10⁻⁶) to Brain Heart Infusion (BHI) liquid medium at a ratio of 1% (v / v). 5 CFU / mL). Add pinocembrin to 1.6 mL of bacterial culture, then dilute using a two-fold dilution method to achieve final concentrations of 1600, 800, 400, 200, 100, and 50 μg / mL. Transfer 200 μL of each mixed culture to a 96-well polystyrene microplate. After incubating the 96-well plate at 37°C for 24 hours, the concentration in the well where the bacterial culture did not become cloudy is the minimum inhibitory concentration (MIC). Results are as follows. Figure 1 As shown in Table 1, the MIC concentration of pinococcus faecalis against Enterococcus faecalis is 100 μg / mL.

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

[0031]

[0032] III. Effects of Pinus julibrissin on the growth of Enterococcus faecalis

[0033] Seed culture of *E. faecalis* was added to BHI liquid medium at a ratio of 1% (v / v). Then, pinocembrin was added to final concentrations of 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC, respectively, and the medium was incubated at 37°C for 24 hours. DMSO was used as a negative control. The OD value at 600 nm was measured every hour using a microplate reader, and growth curves were plotted using the measured data.

[0034] The results are as follows Figure 2 As shown.

[0035] IV. Effects of Pinus julibrissin on biofilm formation of Enterococcus faecalis

[0036] Seed culture of *E. faecalis* was added to BHI liquid medium at a ratio of 1% (v / v). Then, pinene was added, and 200 μL of the mixed culture medium was transferred to 96-well polystyrene microplates. The final concentrations of pinene added were 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC, with dimethyl sulfoxide (DMSO) as a negative control. The experiment was performed using crystal violet staining. After incubating the 96-well plates at 37°C for 24 h, the culture medium was gently removed, and the plates were washed three times with sterile phosphate-buffered saline (PBS) to remove airborne bacteria. The wells were dried in a 60°C oven, and the bottom biofilm was fixed with methanol for 15 min. After removing the liquid, the plates were dried in a 60°C oven. 200 μL of 0.05% (w / v) crystal violet was added to each well for staining for 10 min. The crystal violet solution was gently removed, and the plates were rinsed three times with PBS to remove excess unattached crystal violet dye. After drying at 60℃, add 200 μL of 95% ethanol to each well and decolorize in a shaker at 37℃ and 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 follows Figure 3 As shown.

[0038] V. Effects of pinocembrin on mature biofilms formed by Enterococcus faecalis

[0039] Seed culture of *E. faecalis* was added to BHI liquid medium at a ratio of 1% (v / v). 200 μL of the mixture was added to a 96-well plate and incubated statically at 37°C for 24 h. Excess medium was discarded, and the plate was washed three times with sterile PBS. The wells were dried in a 60°C oven, and mixtures of 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC solutions were added. The plates were then incubated statically at 37°C for 24 h. After incubation, the effect of pinocembrin on the mature biofilm formed by *E. faecalis* was determined using crystal violet staining.

[0040] The results are as follows Figure 4 As shown.

[0041] VI. Checkerboard method for determining the combined use of drugs and antibiotics

[0042] A single colony of *Enterococcus faecalis* was picked and inoculated into 5 mL of BHI medium for activation for 17 h to obtain a seed culture. The OD620 value of the seed culture was adjusted to 0.5, and then inoculated into BHI medium at a rate of 0.1%, and mixed thoroughly. The combined effect of pinocembrin and antibiotics was determined using the checkerboard method.

[0043] The initial concentrations of pinosylvin (100 μg / mL) and the antibiotic gentamicin (250 μg / mL) measured during single drug use before were used as the initial concentrations when used in combination. Different combinations of drugs were added to the BHI medium containing the bacterial solution, and gradient dilution was carried out 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-labeled instrument. The specific combination method of the combined drugs in the �6-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 antibiotic when used in combination / MIC of pinosylvin or antibiotic when used alone. The fractional inhibitory concentration index (FICI) = FIC of pinosylvin + FIC of antibiotic. 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 gracine and gentamicin

[0045]

[0046] Obviously, the above embodiments of the present invention are merely examples for more clearly explaining the present invention, rather than limitations on the implementation modes of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is impossible to list 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. Application of red pinocembrin combined with the antibiotic gentamicin in the preparation of drugs that inhibit Enterococcus faecalis.

Images