Small-Scale Determination of Total Phenols, Tannins, and Flavonoids from Foliar Tissue Using Colorimetric Assays

Abstract

The quantification of plant secondary metabolites at levels higher than the population (i.e. community and ecosystem) requires the implementation of efficient, low-cost, and small-scale assays. We propose a modified protocol based on the Folin-Ciocalteu colorimetric assay that integrates the simultaneous quantitative determination of total phenols and total tannins from 40 µL of methanolic extract, and a modified aluminum chloride assay to quantify total flavonoids from 20 µL of methanolic extract. These miniaturized microplate colorimetric assays were tested and adjusted to work with as little as 50 mg of foliar tissue and to encompass the variation of secondary metabolite concentrations present in trees of 77 species, 58 genera, and 27 families from conserved and secondary tropical dry forest. At present, these new protocols are of great relevance to study ecological processes in highly diverse and strongly threatened ecosystems.

The Assembly of Tropical Dry Forest Tree Communities in Anthropogenic Landscapes: The Role of Chemical Defenses

“…Specifically, for the total phenol quantification, we used a modified version of the Folin-Ciocalteu colorimetric assay developed for microcentrifuge tubes and plate readers [32] to quantify total phenols (TP) [33]. We added 40 µL of supernatant to 960 µL of 95% methanol in a microtube and vortexed for 10 s (Appendix A).…”

“…For the analysis of total tannins, we used a modified version of a previously reported method [34] to quantify tannins (T) [33]. The remaining 900 µL aliquot from the phenol analysis was transferred to a new microtube containing 20 mg of polyvinylpyrrolidone (PVP), vortexed for 10 s, and incubated for 30 min (Appendix A).…”

“…After 1 min, we added 200 µL of NaOH (1M) and the volume was brought to 1 mL with dd H 2 O. The mixture was vortexed for 10 s, and 300 µL were transferred to a microplate to measure its absorbance at 510 nm [33]. A standard curve was prepared using (+)-catechin in concentrations from 20 to 200 mg/L.…”