We express our gratitude to N&#250;cleo de Bioensaios, Bioss&#237;ntese e Ecofisiologia de Produtos Naturais (NuBBE) of the Chemistry Institute of UNESP, Araraquara/SP for providing the laboratories for preparing plant material. We also thank the Applied Microbiology Laboratory of the Department of Dental Materials and Prosthodontics, UNESP, Araraquara/SP. This research was supported by a research grant from the S&#227;o Paulo Research Foundation (FAPESP #2013/07600&#8211;3 to AJC) and scholarships plus overhead funds (FAPESP #2017/07408&#8211;6 and FAPESP #2019/23175-7 to SMR; #2011/21440&#8211;3 and #2012/21921&#8211;4 to PCPB). The National Council for Scientific and Technological Development in association with FAPESP provided additional support (INCT CNPq #465637/2014&#8211;0 and FAPESP #2014/50926&#8211;0 to AJC).

1. Collection of Plant Material
<ol>
	<li>Plant material
	<ol>
		<li>Record the access to the plant material on electronic platforms that regulate access to genetic heritage in the country where the collection will take place. For example, in Brazil, register with the National System for the Management of Genetic Heritage and Associated Traditional Knowledge &#8211; SisGen (website https://sisgen.gov.br/paginas/login.aspx).</li>
		<li>Collect samples of the plant material of interest (e.g., leaves, stems, roots, flower...

No conflicts of interest declared.

The main challenges related to the work with natural crude extracts comprise their complex composition and the inadequacies of classic bio-guided isolation studies. Although this process is slow, it is effective and has led to major findings in NP research. To rationalize, prioritization-driven studies are needed to rationalize. Thus, the use of modern chemical profiling approaches for the analysis of CE and dereplication before isolation are important to characterize the studied material and especially useful to avoid r...

The earliest models of medicine used in societies were based on natural products (NPs). Since then, humans have been searching for new chemicals in nature that can be transformed into drugs1. This search caused a continuous improvement of technologies and methods for ethnobotanical screening1,2,3. NPs offer a rich source of structurally diverse substances, with a wide range of biological activities useful for developing alternative or adjuvant therapies. However, the inherent complex plant matrix makes the isolation and identification of the active compounds a challenging and time-consuming task4.
NPs-based drugs or formulations can be used to prevent and/or treat several conditions affecting oral, including dental caries4. Dental caries, one of the most prevalent chronic diseases globally, derives from the interaction of sugar-rich diet and microbial biofilms (dental plaque) formed on the tooth surface that leads to demineralization caused by organic acids derived from microbial metabolism, and if not treated, leads to teeth loss5,6. Although other microorganisms may be associated7, Streptococcus mutans is a critical cariogenic bacterium because it is acidogenic, aciduric, and an extracellular matrix builder. This species encodes multiple exoenzymes (e.g., glycosyltransferases or Gtfs) that use sucrose as a substrate8 to build the extracellular matrix rich in exopolysaccharides, which are a virulence determinant9. Also, the fungus Candida albicans can drive up the production of that extracellular matrix7. Albeit fluoride, administered in various modalities, remains the basis for preventing dental caries10, new approaches are needed as adjuvants to increase its effectiveness. In addition, the available anti-plaque modalities are based on the use of broad-spectrum microbicidal agents (e.g., chlorhexidine)11. As an alternative, NPs are potential therapies for controlling biofilms and preventing dental caries12,13.
The further advance in the discovery of new bioactive compounds from plants includes necessary steps or approaches such as: (i) the use of reliable and reproducible protocols for sampling, considering that plants often show intraspecific variability; (ii) the preparation of comprehensive extracts and their respective fractions in small scale; (iii) the characterization and/or dereplication of their chemical profiles thought the acquisition of multidimensional data such as GC-MS, LC-DAD-MS, or NMR, for example; (iv) the use of viable and high-yield models to assess bioactivity; (v) the selection of potential new hits based on multivariate data analysis or other statistical tools; (vi) to perform the isolation and purification of the targeted compounds or promising candidates; and (vii) the validation of the corresponded biological activities using the isolated compounds2,14.
Dereplication is the process of rapidly identifying known compounds in crude extract and allows differentiating novel compounds from those that have already been studied. Besides, this process prevents isolation when bioactivity has already been described for certain compounds, and it is particularly helpful to detect &#8220;frequent hitters&#8221;. It has been used in different untargeted workflows ranging from major compound identification or the acceleration of activity-guided fractionation up to the chemical profiling of collections of extracts. It can be fully integrated with metabolomic studies for the untargeted chemical profiling of CE or the targeted identification of metabolites. All of this ultimately leads to prioritizing extracts before the isolation procedures1,15,16,17.
Therefore, in the present manuscript, we describe a systematic approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions. It includes four multidisciplinary steps: (1) collection of plant material; (2) preparation of crude extracts (CE) and fractions (CEF), followed by their chemical profile analysis; (3) bioassays; and (4) biological and chemical data analyses (Figure 1). Thus, we present the protocol developed to analyze of the antimicrobial and antibiofilm activities of Casearia sylvestris extracts and fractions against Streptococcus mutans and Candida albicans13, as well as the procedures for the phytochemical characterization and data analysis. For simplicity, the focus here is to demonstrate the approach for screening natural compounds using the bacterium.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61773/61773fig01v2.jpg" /> 
Figure 1: Flow-chart of the Systematic Approach to identify active molecules from plants extracts and fractions. <a href="https://www.jove.com/files/ftp_upload/61773/61773fig01v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>96-well microplates&#160;</td>
			<td>Kasvi</td>
			<td>Flat bottom</td>
			<td></td>
		</tr>
		<tr>
			<td>Activated carbon</td>
			<td>LABSYNTH</td>
			<td>Clean up and/or fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical mill</td>
			<td>Ika LabortechniK</td>
			<td>Model A11 Basic</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood agar plates</td>
			<td>Laborclin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic column C18</td>
			<td>Phenomenex Kinetex</td>
			<td>150 &#215; 2.1 mm, 2.6 &#181;m, 100&#194;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>Vehicle solution</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate reader</td>
			<td>Biochrom Ez</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J. T. Baker</td>
			<td>For extraction and fractionation steps, and mobile phase composition</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>Vehicle solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>J. T. Baker</td>
			<td>Fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Software</td>
			<td>La Jolla</td>
			<td>GraphPad Prism7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>J. T. Baker</td>
			<td>Fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>J. T. Baker</td>
			<td>For extraction step</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer (a freeze dryer)</td>
			<td>Savant</td>
			<td>Modulyo</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Millipore</td>
			<td>LAC</td>
			<td>0.22 &#181;m x 13 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Quimis</td>
			<td>Model G816&#8201;M20</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyamide solid phase extraction cartridge</td>
			<td>Macherey-Nagel</td>
			<td>Clean up and/or fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Merck</td>
			<td>40&#8211;63&#8201;&#956;m, 60&#8201;&#194;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Synth</td>
			<td>0,89% in water</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid phase extraction cartridges (SPE)</td>
			<td>Macherey-Nagel</td>
			<td>Clean up and/or fractionation step</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UHPLC-DAD</td>
			<td>Dionex</td>
			<td>Ultimate 3000 RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>UNIQUE</td>
			<td>Model USC 2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

systematic approach to identify novel antimicrobial and antibiofilm molecules from plants' extracts and fractions to prevent dental caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

We provide an example of using a systematic approach to screen the biological activity of plant extracts and fractions to identify potentially active molecules for possible new anti-caries therapies: antimicrobial and antibiofilm activities of Casearia sylvestris extracts from distinct Brazilian biomes against Streptococcus mutans and Candida albicans13.
Background 
Complex interactions between specific oral microorganism...

Watch this Scientific Journal Video about Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries at JoVE.com

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants&#039; Extracts and Fractions to Prevent Dental Caries

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

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

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

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.

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6.0K visualizações.  São Paulo State University (UNESP). O protocolo apresentado é utilizado para triagem e análise correta de candidatos bioativos em produtos naturais para controle de biofilmes orais. Também pode ser adaptado para aplicações em outros campos de pesquisa de biofilm. Este método de triagem e análise mais amido permite remover componentes bioativos simultaneamente.A cárie dentária é uma dieta biofilm derivada, altamente prevalente, doença crônica. Este protocolo pode ajudar a identificar produtos naturais para con...