Extraction, Enrichment, and LC-MS n-Based Characterization of Phlorotannins and Related Phenolics from the Brown Seaweed, Ascophyllum nodosum

Abstract

Phenolic components from the edible brown seaweed, Ascophyllum nodosum, have been associated with considerable antioxidant activity but also bioactivities related to human health. This study aims to select and identify the main phlorotannin components from this seaweed which have been previously associated with potential health benefits. Methods to enrich phenolic components then further select phlorotannin components from ethanolic extracts of Ascophyllum nodosum were applied. The composition and phenolic diversity of these extracts were defined using data dependent liquid chromatography mass spectroscopic (LC-MSⁿ) techniques. A series of phlorotannin oligomers with apparent degree of polymerization (DP) from 10 to 31 were enriched by solid phase extraction and could be selected by fractionation on Sephadex LH-20. Evidence was also obtained for the presence of dibenzodioxin linked phlorotannins as well as sulphated phlorotannins and phenolic acids. As well as diversity in molecular size, there was evidence for potential isomers at each DP. MS² fragmentation analyses strongly suggested that the phlorotannins contained ether linked phloroglucinol units and were most likely fucophlorethols and MS³ data suggested that the isomers may result from branching within the chain. Therefore, application of these LC-MSⁿ techniques provided further information on the structural diversity of the phlorotannins from Ascophyllum, which could be correlated against their reported bioactivities and could be further applied to phlorotannins from different seaweed species.

Keywords: Ascophyllum; LC-MSn; diversity; health benefits; isomers; phenolics; phlorotannins; seaweed.

Figure 1

Profiles of the fractions from the SPE procedure. (A) UV profiles, (B) MS spectra from 12–21 min for each sample. Blue bracket in (A) represents area for MS spectra in (B). Arrows show 124 amu differences between peaks. FSD = full scale deflection.

Figure 2

Possible nonaphorethol structure.

Figure 3

Structures of difucol, diphlorethol and trifucotetraphlorethol. These structures are discussed in the text. Fragmentation at the positions noted with blue arrows would produce THB containing fragments with neutral losses of 142 amu = THB, 266 amu = PG-THB, 390 amu = 2PG-THB and 514 amu = 3PG-THB respectively from right to left.

Figure 4

Profiles of the fractions from the Sephadex LH-20 procedure. (A) UV profiles, (B) MS spectra from 12–21 min for each sample. FSD = full scale deflection.

Figure 5

(A) MS properties of phlorotannin peaks in negative and positive mode. The top panel shows the UV spectra of the SPE bound sample from 10–21 min, the next shows the MS spectra in negative mode from 10–21 min and the third panel shows the MS spectra in positive mode from 10–21 min. The fourth panel shows the MS spectrum over 10–21 min in negative mode and the last panel shows the same in positive mode. FSD = full scale deflection. The peaks denoted in bold green were examined further (Figure 5B). (B). MS spectra of selected phlorotannin peaks in negative and positive mode. Examples of negative and positive MS spectra of specific peaks labelled in bold in Figure 5A. Green arrows denote m/z signals discussed in the text.

Figure 6

The phlorotannin peaks yield doubly charged MS signals in negative mode. The top panel (A) shows the MS spectra across 12–21 min of the SPE bound sample and the second panel shows the MS spectra of the main UV peak. The third panel (B) shows the zoom spectra across the m/z 1116 peak.

Figure 7

Relative abundance of phlorotannin oligomers in the SPE bound fraction.

Figure 8

MS² and MS³ data of major phlorotannin isomers at m/z 1116.6. (A) The top panel shows the MS profile between RT = 21–21 min, the second panel shows the profile for base peak at m/z 1116.6 and the bottom two panels show the MS spectra at the two peaks at 17.75 min and 16.35 min respectively. (B) The top panel shows the MS³ fragments from the main MS² peak (m/z 1098) derived from the major m/z 1116.6 peak at RT = 17.75. The bottom panel shows the MS³ fragments from the main MS² peak (m/z 1098) derived from the minor m/z 1116.6 peak at RT = 16.35.