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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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 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.......

Representative Results

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.......

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.......

Acknowledgements

We express our gratitude to Núcleo de Bioensaios, Biossíntese 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 São Paulo Research Foundation (FAPESP #2013/07600–3 to AJC) and scholarships plus overhead funds (FAPESP #2017/07408–6 and FAPESP #2019/23175-7 to SMR; #2011/21440–3 and #2012/21921–4 to PCPB). The National Council for Scientific a....

Materials

NameCompanyCatalog NumberComments
96-well microplates KasviFlat bottom
Activated carbonLABSYNTHClean up and/or fractionation step
Analytical millIka LabortechniKModel A11 Basic
Blood agar platesLaborclin
Chromatographic column C18Phenomenex Kinetex150 × 2.1 mm, 2.6 µm, 100Â
Dimethyl sulfoxide Sigma-AldrichVehicle solution
ELISA plate readerBiochrom Ez
EthanolJ. T. BakerFor extraction and fractionation steps, and mobile phase composition
EthanolSigma-AldrichVehicle solution
Ethyl acetateJ. T. BakerFractionation step
GraphPad SoftwareLa JollaGraphPad Prism7
HexaneJ. T. BakerFractionation step
IncubatorThermo Scientific
IsopropanolJ. T. BakerFor extraction step
Lyophilizer (a freeze dryer)SavantModulyo
Nylon MilliporeLAC0.22 µm x 13 mm
Orbital shakerQuimisModel G816 M20
Polyamide solid phase extraction cartridgeMacherey-NagelClean up and/or fractionation step
Silica gelMerck40–63 μm, 60 Â
Sodium Chloride (NaCl)Synth0,89% in water
Solid phase extraction cartridges (SPE)Macherey-NagelClean up and/or fractionation step
TryptoneDifco
UHPLC-DADDionexUltimate 3000 RS
Ultrasonic bathUNIQUEModel USC 2800
Yeast extractDifco

References

  1. Newman, D. J., Cragg, G. M. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. Journal of Natural Products. 83 (3), 770-803 (2020).
  2. Wolfender, J. L., Litaudon, M., Touboul, D., Queiroz, E. F.

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AntimicrobialAntibiofilmNatural ProductsDental CariesBiofilmPlant ExtractsFractionationS MutansAnti microbial AssayBiofilm Assay

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