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

Summary

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.

Abstract

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 "adhesion strength" 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’ glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Introduction

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 screening^1,2,3. 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 com....

Protocol

1. Collection of Plant Material

1. Plant material
   1. 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 – SisGen (website https://sisgen.gov.br/paginas/login.aspx).
   2. Collect samples of the plant material of interest (e.g., leaves, stems, roots, flower.......

Discussion

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.......

Materials

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