Identification of natural bioactive compounds to promote the biodiversity of French Riviera
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1 Identification of natural bioactive compounds to promote the biodiversity of French Riviera Elise Sarrazin, PhD Laboratoire de Chimie des Molécules Bioactives et des Arômes (LCMBA) UMR CNRS/UNS 6001, Institut de Chimie de Nice
2 What is biodiversity? Neologism proposed at UN Earth Summit of Rio de Janeiro in Biodiversity (also called biological diversity) is the degree of variation of life forms within a given ecosystem, biome, or an entire planet
3 Biodiversity, a great chance of development for the région Provence-Alpes-Côte d Azur (PACA) Manche Océan Atlantique Mer Méditerranée - The Mediterranean Basin is one of the 34 biodiversity «hot spots». - The PACA region is situated at a phytogeographical corner between the Alps and the Mediterranean Sea. The PACA region contains 2/3 of the biodiversity of Metropolitan France. In particular, the Maritime Alps are the most important endemic center of the whole Alps.
4 Laboratoire des Molécules Bioactives et des Arômes Main research topics Analysis of natural extracts (historically great expertise in the field of Flavors and Fragrances) Discovery of new molecules from natural extracts (plants, flowers, fruits, roots, ) Focused properties: organoleptic (odor, flavor) bioactive (antioxidant, antimicrobial) Our work strategy is always divided into three steps 1. Extraction 2. Fractionation (purification) 3. Characterization (identification) Presentation of two current projects
5 1. Experimental methodology to isolate bioactive compounds from natural matrices Infusion Distillation
6 1. Experimental methodology to isolate bioactive compounds from natural matrices
7 1. Experimental methodology to isolate bioactive compounds from natural matrices Pressure Ultrafiltration Separation based on size Permeate Retentate Membrane Ion-exchange Separation based on electrical charge Sources:
8 1. Experimental methodology to isolate bioactive compounds from natural matrices Solid-Phase Extraction (SPE) Separation based on chemical affinity Sample Introduction Water rinsing Elution with solvant 1 Elution with solvant 2 F1 F2 F3 Source:
9 1. Experimental methodology to isolate bioactive compounds from natural matrices
10 1. Experimental methodology to isolate bioactive compounds from natural matrices NMR: Atom linking and environment Mass spectrometry (MS): Molecular mass of the compound and raw formula Intens. x
11 Laboratoire des Molécules Bioactives et des Arômes Presentation of 2 current projects aiming at promoting local biodiversity - Identification of new bioactive compounds dedicated to oral care. - Financial support from the Local Council of Maritimes Alpes - Development of original and natural preservatives for cosmetics and nutraceutics - Labelled by the PASS Cluster (APRF 2009) -Collaborations: Laboratory of Oral Microbiology (LOM) Solidages - Collaborations : Sofia Cosmétiques NATUREX
12 2. Solidago Project: Introduction Project launched in January 2010 (PhD thesis of Lise Laurençon) Development of a mouthwash to prevent senior and medicated patients from candidosis Objective: Inhibiting the pathogen form (hyphae) of Candida albicans while preserving its commensal form (yeast-like), as well as the other bacteria of the oral biofilm. Yeast Non-pathogen form Hyphae (filament) Pathogen form A local plant Solidago virgaurea (Asteraceae) was shown to present interesting results and was selected to be further investigated.
13 2. Solidago Project: Solidago virgaurea subspecies Solidago virgaurea virgaurea Height: up to 1m Ramified floral scape Europe (0 to 1500 m) Solidago virgaurea alpestris Height: up to 40cm No ramification Alpine areas (1600 to 2700 m) Various publications + Phytotherapy patents: Antiviral (H1N1), coagulative, fongicide, natural latex (cosmetics) No phytochemical study But plant available in the Maritim Alps
14 2. Solidago Project: Experimental Protocol Performed Fractionation step-by-step to isolate the active molecules Plant
15 2. Solidago Project: Experimental Protocol Performed Fractionation step-by-step to isolate the active molecules Characterization of the chemical composition of the fractions using HPLC (High Performance Liquid Chromatography) HPLC prep Pure active compounds Fractionation Active fraction Decoction Plant Raw aqueous extract Bioassays after 24h and 48h incubation: - Hemolysis in agarose gel - Counting of Candida colonies - Study of the percentage of Candida filament formed
16 2. Solidago Project: Experimental Protocol Performed Fractionation step-by-step to isolate the active molecules Characterization of the chemical composition of the fractions using HPLC (High Performance Liquid Chromatography) Fractionation HPLC prep Active fraction Pure active compounds Identification in progress Decoction Plant Raw aqueous extract Optimization of the extraction process
17 3. NATUBAVAL Project : Introduction Project launched in January 2011 (PhD Theses of Florence Merck and Audrey Kerdudo) Inventory of the medicinal and aromatic plants of the PACA region and screening for bioactive plants (preservative: antimicrobial, and/or antioxydant) Objective: identify original natural bioactive compounds in order to propose new preservatives for cosmetics and nutraceutics. Two activity tests: - chemical : focused on antioxydant activity - microbiological: antimicrobial activity
18 3. NATUBAVAL Project:Screening for antioxydant compounds Colorimetric test using DPPH (2,2-diphenyl-1-picrylhydrazyl) Free radical = anti-oxydant Experimental strategy: Fractions obtained from an active plant extract 1. Screening for anti-oxydant extracts using DPPH test 2. Fractionation guided by DPPH test 3. Isolation of the active compounds E
19 3. NATUBAVAL Project:Screening for antimicrobial compounds 4 microorganisms selected from European Pharmacopy Aspergillus niger (fungus) Candida albicans (yeast) Staphylococcus aureus (bacteria Gram +) Pseudomonas aeruginosa (bacteria Gram -) Dilution method: Homogenous dispersion of the plant extract in a microorganism-selected medium using 96-well plates. Turbidity taken as an indicator of bacterial density. Turbidity measured after 24h, 48h and 72h of incubation Picture frombiopreserv Grade of microbial inhibition related to the turbidity of the medium and measured by spectrophotometry Initial medium Microorganismselected medium + plant extract 72h incubation Final medium 2 possibilities Turbidity increased = no influence of the extract Turbidity unchanged or decreased = influence of the extract
20 3. NATUBAVAL Project: work in progress 200 plants selected 25 plants already studied 3 interesting plants Isolation and identification in progress
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22 Aknowledgements Université de Nice Sophia Antipolis CNRS Public Institutions Natubaval Project SOFIA Cosmétiques Naturex Acphytaroma Biopreserv Nixe Local Authorities Solidago Project Pole PASS Conseil Général des Alpes Maritimes Conseil Régional PACA Solidages Laboratoire de Microbiologie Orale Biophyto
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