The subject of this article brings together two researchers who have a common interest in specialized metabolites involved in plant chemical defenses. Dr. Thomas Michel is analytical chemist at the Institute of Chemistry of Nice (Université Côte d’Azur, Nice, France) where he develops analytical tools for chemical ecology and for discovery of bioactive compounds from plant biodiversity. He employs HPTLC for analytical goals and TLC for preparative separations. Dr. Anne- Violette Lavoir, ecologist at the Institute Sophia Agrobiotech (INRAe, Université Côte d’Azur, Antibes, France) works on the chemical interaction between plants and insects (community ecology, chemical ecology). In this work, the authors employed HPTLC for secondary metabolites screening of different plant extracts in an ecological context.

Ecosystems are complex systems, forming a vast network in which different actors – the living beings – interact with each other. The main trophic network is the “green world” [1,2], in which the chlorophyllous plants are the primary producers. They bring organic matter and energy into the network through the fixation of mineral matter by photosynthesis. In this green world, fresh plants are the basis as they are consumed by herbivores, which will in turn be consumed by predators. The “brown world” [1,2] is based on the litter, mostly formed from plant parts (e.g. leaves, bark, stems) that dry out and fall to the ground (inert organic matter). The litter is consumed by detritus feeders (or decomposers), which will then also be consumed by their predators. As litter are previously fresh leaves, the basis of the green and brown world is linked through the so-called plantlitter continuum.

A change in plant or litter chemistry would impact not only their consumers, but all the trophic levels above. For instance, plants could modify their chemical defenses such as secondary metabolites, impacting all their consumers. Because of the plant-litter continuum, any modification in the chemistry of the fresh plants will in turn modify the chemistry of the litter and consequentially the brown trophic network.

In this context, we studied the variation in content of secondary metabolites at five stages of the plant-litter continuum (young plants, mature plants, fresh litter, old litter and decomposer feces), from four plant species (Bromus erectus, Pilosella officinarum, Potentilla grandiflora and Sanguisorba minor). Each stage and plant species were sampled by our collaborators on grasslands of Les Causses (South of France) and each grassland was either fertilized or not. Our main objective was to compare the global chemistry of the four plant species throughout the plant-litter continuum and to see if the fertilization may modify such pattern.

The great diversity of phytochemicals and their small quantities in plant species, and the number of samples to investigate led us to set up a large-scale HPTLC analysis. HPTLC was chosen because it offers numerous advantages such as versatility, capacity to run several samples in parallel and low solvent consumption. Additionally, detection using various derivatization reactions can provide crucial information about the nature of chemical substances present in samples. Furthermore, sample preparation in HPTLC is simple while other chromatographic techniques (e.g. GC and HPLC) usually require a time-consuming and expensive sample preparation prior to the analyses. Therefore, HPTLC is suitable as a high-throughput fingerprinting method in an ecological context.

Quercetin, asiaticoside and betuline are dissolved in methanol at different concentration levels.

20mg of ground plant are extracted in an ultrasonic bath with 1.6 mL ethanol – water 7:3. Extracts are then filtered and stored at –20 °C before HPTLC analysis.

HPTLC plates silica gel 60 F₂₅₄ (Merck), 20 x10 cm

Automatic TLC Sampler (ATS 4), application as bands, 16 tracks, band length 8.0 mm, distance from left edge 15.0 mm, distance from lower edge 8.0 mm, application volume 2.0 μL for sample solutions and 2.0 μL for standard solutions

In the Automatic Developing Chamber (ADC 2) with ethyl acetate – formic acid – acetic acid – water 100:11:11:26 (polyphenols), chloroform – methanol – water 5:4:1 (glycosylated terpenoids), or hexane – ethyl acetate – water 4:1:0.5 (hydrophobic terpenoids) after chamber saturation (filter paper) and activation at 33% relative humidity for 10 min using a saturated solution of magnesium chloride, migration distance of 70 mm (from the lower edge), drying for 5 min

All plates are immersed into specific reagents for 2 s using the Chromatogram Immersion Device. For phenolic compounds, the plate is immersed into NP reagent (1.25 g diphenyl borinic acid ethylamino ester in 250 mL of ethyl acetate) and then into PEG reagent (10% polyethylene glycol 400 in dichloromethane). For terpenoids, the plate is immersed into anisaldehyde-sulfuric acid reagent (0.5 mL anisaldehyde, 10 mL glacial acetic acid, 85 mL methanol, 5 mL sulfuric acid) and heated on the TLC Plate Heater at 105 °C for 5 min.

TLC Visualizer under UV 254 nm, UV 366 nm, and white light

TLC Scanner 4 and winCATS, slit dimension 5.00 mm x 0.20 mm, scanning speed 50 mm/s, spectra recording from 190 to 450 nm, measurement after derivatization in fluorescence mode at 366/>400 nm with Hg lamp (polyphenols), in absorbance mode at 400 nm (polar terpenoids), and at 600 nm (apolar terpenoids) with tungsten lamp, evaluation via peak area and calibration curves of specific compounds (i.e. quercetin, asiaticoside, betuline)

Data are statistically analyzed by a Scheirer-Ray- Hare test using R software.