Identification of Phlorotannins in the Brown Algae, Saccharina latissima and Ascophyllum nodosum by Ultra-High-Performance Liquid Chromatography Coupled to High-Resolution Tandem Mass Spectrometry

Abstract

Phlorotannins are bioactive polyphenols in brown macroalgae that make these algae interesting as healthy food. Specific phlorotannins are, however, seldom identified, and extracts from different species are often only analysed for total phenolic content (TPC). In this study, our focus was to identify phlorotannin molecules from Saccharina latissima and Ascophyllum nodosum (a species rich in these compounds) using ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS2). Water and ethanol (30 and 80% v/v) were used at solid:liquid ratios, extraction times and temperatures, proposed to result in high TPC in extracts from other species. The S. latissima extracts, however, did not allow phlorotannin detection by either UHPLC-UV/Vis or UHPLC-HRMS2, despite a TPC response by the Folin–Ciocalteu assay, pinpointing a problem with interference by non-phenolic compounds. Purification by solid phase extraction (SPE) led to purer, more concentrated fractions and identification of four phlorotannin species in A. nodosum and one in S. latissima by UHPLC-HRMS2, using extracts in ethanol 80% v/v at a solid:liquid ratio of 1:10 for 20 h at 25 °C with an added 10 h at 65 °C incubation of remaining solids. The phlorotannin with the formula C12H10O7 (corresponding to bifuhalol) is the first identified in S. latissima.

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

“…[82][83][84] Overall, the presented data indicate that brown algae contain more phlorethol-like phlorotannins (Table 4), supporting the reports by Visser et al and Ford et al and reinforced by mass spectrometry analyses previously reported. 41,85,86 Furthermore, the phlorotannin 13 C NMR characterisation can facilitate an estimate of the number of protons per aromatic phlorotannin ring in the seaweed samples. This estimate is made to improve the accuracy of the total polyphenolic content quantification using 1 H qNMR.…”

“…Additionally, other studies have found indications that seaweeds belonging to the Fucaceace species contain phlorotannins consisting of 2-16 phloroglucinol units. 41,85,87,88 Montero et al (2016) found that for the brown algae Sargassum muticum, the degree of polymerisation of the phlorotannins ranged from 2 to 10 for samples collected in Norway. 89 Taking these studies and the indications of the relative intensity ratios measured into account, it can be assumed that phlorotannins with polymerisation degrees ≥ 4 make up a large part of the phlorotannin matrix of the alga in this study.…”

“…[22][23][24][25][26][27][28][29][30][31][32][33][34][35] Various polyphenols have been identified in macroalgae, with phlorotannins being the predominant polyphenol group in the class of Phaeophyta (brown algae). 12,[36][37][38][39][40][41][42] Phlorotannins, which are exclusive to brown algae, are oligomers of phloroglucinol and are separated into different subgroups depending on the linkage of the phloroglucinol units. These linkages can be either phenyl linkages (C-C), ether linkages (C-O-C), or both (Figure 1).…”

Comprehensive reviews on phenolic compounds from Phaeophyceae as potential therapeutic agent

“…Using HPLC-MS-TOF and a modified UHPLC-QQQ-MS technique, several phlorotannin derivatives, Eckol, Fuhalol, and phloroglucinol from Silvetia compressa and Sargassum fusiforme were discovered [35,36]. UHPLC-HRMS2 was used to analyze phlorotannin derivatives from brown seaweeds such as F. spiralis, Ascophyllum nodosum, and Saccharina latissima, which yielded phlorotannin-rich extracts [37,38]. These approaches could be used to characterize and discover biologically important phlorotannin molecules.…”

Evaluation on Intestinal Permeability of Phlorotannins Using Caco-2 Cell Monolayers

“…14,15 The A. nodosum is one of the representative seaweed species rich in phlorotannins, 16 and compounds belonging to the subclasses of phlorethols and fucols have been mainly identified. 17 In the above reports, findings included the discovery that several phlorotannin-metabolites attributed to both unconjugated and conjugated metabolites in urine and plasma samples and indicated that ingested phlorotannins may be metabolized and absorbed predominantly in the large intestine. 14,15 However, findings on the bioavailability of phlorotannins are limited to these two studies, and so far there are no reports analyzing the permeability and metabolism of isolated compounds using the evaluation system of the small intestine model, unlike the research field of terrestrial polyphenols.…”