Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

The presented protocol is used for correctly screening and analyzing bioactive candidates in natural products to control oral biofilms. It can also be adapted for applications in other biofilm research fields. This most starch screening and analysis method make it possible to remove bioactive components simultaneously.

Dental caries is a biofilm diet derived, highly prevalent, chronic disease. This protocol can help to identify natural products to control biofilms and consequently, dental caries. Begin by preparing the extraction solvent with a hydroalcoholic mixture.

You sample concentrations between 50 and 100 milligrams per milliliter of the extraction solvent and perform 15-minute ultrasound assisted extractions in microtubes. Repeat the extraction three times. After each extraction step, centrifuge the sample to decap the solid residue and remove the supernatant.

Combine the supernatants from each extraction step and filter them. Save the aliquots for chemical analysis and bioassays. For fractionation, dilute the crude extract sample to obtain a 100 milligram per milliliter solution, using the initial extraction solvent.

Then, transfer one milliliter of this sample to a preconditioned solid-phase extraction cartridge with one gram of Absorbent, and a capacity of six milliliters. Perform fractionation, using around three dead volumes of each extraction LUN, and collect one fraction by LUN composition. Save aliquots for chemical analysis and bioassays.

Remove the solvent under vacuum, nitrogen flow, or lyophilization, and register the weight and yield. Reconstitute the dry matter with the best possible solvents. Calculate the solvent concentration for the stock solution, using the formula in the text manuscript.

Reactivate the microbial strain of S.mutans UA 159 on blood agar, and culture it in liquid culture medium. Perform a one to 20 dilution of the initial culture using the same culture medium, and incubate it until it reaches the mid-log growth phase. Prepare the inoculum with a defined population in TYEG for anti-microbial assays and TYE with 1%sucrose for biofilm assays.

For anti-microbial activity, define the concentration of the sample for analysis, and add it to a 96-well plate. Include a set of controls in each plate, as described in the text manuscript. Using TYEG, adjust the volume to 100 microliters, inoculate 100 microliters of microbial culture, and incubate the plate for 24 hours at 37 degrees Celsius in 5%carbon dioxide.

Analyze the bacterial growth according to turbidity by visual inspection of the wells. Inoculate an aliquot of the desired dilution in specific agar plates in duplicates, and incubate the plates. After the incubation, perform colony counts.

Weigh hydroxyapatite beads in microtubes and sterilize them. Then wash the beads, using adsorption buffer Ab.Add 500 microliters of saliva into the microtubes to form a salivary film, and incubate at 37 degrees Celsius with 24 rotations per minute for 40 minutes. Remove the saliva supernatant and wash the beads three times with Ab to prepare them for downstream assays.

Add 500 microliters of the sample or the control into each microtube containing beads with salivary pellicle, and incubate them at 24 rotations per minute and 37 degrees Celsius for 30 minutes. Wash the beads three times with Ab.Add 500 microliters of microbial culture to each microtube, and incubate the tubes in similar conditions for one hour. Then wash the unbound cells three times with Ab buffer.

Resuspend each sample with one milliliter of Ab buffer, and centicade at seven Watts for 30 seconds. Serially dilute aliquots of each suspension, tenfold, and plate them on specific agar plates. Incubate the plates for 48 hours at 37 degrees Celsius in 5%carbon dioxide, and count the colonies.

For data analysis, input the raw data for the bioassays into a spreadsheet, and calculate the log of microbial growth inhibition by each treatment, and the log percentage of microbial growth inhibition, compared with the control. Correct the optical density of the readings, and calculate the percentage of biomass inhibition. The chemical profile of biological screening for the quantity of Clerodane-type diterpenes and glycosylated flavonoids in three varieties of C.sylvestris extracts, namely sylvestris, intermediate, and lingua, are shown here.

After analysis of the raw data, four extracts showed a favorable response. The chromatographic data of these four extracts show the simultaneous presence of Clerodane-type diterpenes and glycosylated flavonoids. In addition, they include the same biome and variety.

Calculated percent CFU of treated planktonic cells, percent biomass of the treated biofilms, and percent CFU of the treated biofilm are shown here. No crude extract significantly affected the removal of S.mutans cells adhered to the salivary pellicle. The adhesion of S.mutans cells to the glucan matrix was weakened by three of the crude extracts.

Since each plant species requires optimized and specific chemical analysis methods, the best solvent fraction and analytical method must be selected from previous reports or defined by experimental design. It is critical to prepare formulations with the most active crude extract and fractions and test them on complex models to prevent the development of biofilms and cavities.