Advancing quantification methods for polyphenols in brown seaweeds-applying a selective qNMR method compared with the TPC assay.

INTRODUCTION: Brown seaweeds are a sustainable biomass with a potential for various industrial applications. Polyphenols are an important contributor to this potential. OBJECTIVE: The aim was total quantification of polyphenols in brown seaweeds from different tidal zones, using a selective 1 H quantitative NMR (qNMR) method, comparing the results with the colorimetric Folin-Ciocalteu total phenolic content (TPC) assay. METHOD: qNMR was performed with integration of selected peaks in the aromatic region (7-5.5 ppm). Deselection of non-polyphenolic 1 H signals was based on information from 2D (1 H-13 C, 1 H-15 N) NMR spectra. 13 C NMR phlorotannin characterisation facilitated the average number of protons expected to be found per aromatic ring used for the 1 H quantification. RESULTS: Selective qNMR and the TPC assay showed similar results for the three sublittoral growing species from the Laminariaceae; lower amounts for Laminaria hyperborea and Laminaria digitata (qNMR: 0.4%-0.6%; TPC: 0.6%-0.8%, phloroglucinol equivalents (PGE), dry weight (DW)) and higher amounts for Saccharina latissima (qNMR: 1.2%; TPC: 1.5%, PGE, DW). For the eulittoral Fucaceae, Fucus vesiculosus (qNMR: 1.1%; TPC: 4.1%; PGE, DW) and Ascophyllum nodosum (qNMR: 0.9%; TPC: 2.0%; PGE, DW), the TPC results were found to be up to three times higher than the qNMR results. The 13 C NMR characterisation showed the highest phlorotannin polymerisation degree for F. vesiculosus. CONCLUSION: The TPC assay provided similar polyphenolic amounts to the selective qNMR method for sublittoral species. For eulittoral growing species, the TPC method showed amounts up to three times higher than the qNMR method-most likely illustrating the lack of selectivity in the TPC assay.

Quantification of Polyphenols in Seaweeds: A Case Study of Ulva intestinalis.

In this case study, we explored quantitative 1H NMR (qNMR), HPLC-DAD, and the Folin-Ciocalteu assay (TPC) as methods of quantifying the total phenolic content of a green macroalga, Ulva intestinalis, after optimized accelerated solvent extraction. Tentative qualitative data was also acquired after multiple steps of purification. The observed polyphenolic profile was complex with low individual concentrations. The qNMR method yielded 5.5% (DW) polyphenols in the crude extract, whereas HPLC-DAD and TPC assay yielded 1.1% (DW) and 0.4% (DW) respectively, using gallic acid as the reference in all methods. Based on the LC-MS observations of extracts and fractions, an average molar mass of 330 g/mol and an average of 4 aromatic hydrogens in each spin system was chosen for optimized qNMR calculations. Compared to the parallel numbers using gallic acid as the standard (170 g/mol, 2 aromatic H), the optimized parameters resulted in a similar qNMR result (5.3%, DW). The different results for the different methods highlight the difficulties with total polyphenolic quantification. All of the methods contain assumptions and uncertainties, and for complex samples with lower concentrations, this will be of special importance. Thus, further optimization of the extraction, identification, and quantification of polyphenols in marine algae must be researched.