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Profiling phlorotannins from Fucus spp. of the Northern Portuguese


coastline: chemical approach by HPLC-DAD-ESI/MSn and UPLC-ESI-
QTOF/MS

Article  in  Algal Research · November 2017


DOI: 10.1016/j.algal.2017.11.025

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Algal Research 29 (2018) 113–120

Contents lists available at ScienceDirect

Algal Research
journal homepage: www.elsevier.com/locate/algal

Profiling phlorotannins from Fucus spp. of the Northern Portuguese T


coastline: Chemical approach by HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/
MS
Graciliana Lopesa, Mariana Barbosaa, Fernando Vallejob, Ángel Gil-Izquierdob,
⁎ ⁎
Paula B. Andradea, , Patrícia Valentãoa, David M. Pereiraa, Federico Ferreresb,
a
REQUIMTE/LAQV, Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-
313 Porto, Portugal
b
Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus University
Espinardo, Murcia, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Several attempts have been made in recent years to fully characterize phlorotannin-rich extracts. Nevertheless,
Phlorotannins this remains a challenging quest, not only because of seaweed complex chemical composition, but also due to the
Fucales diverse structural assemblage within phlorotannin molecules (i.e., type of linkage, modification site, and number
Fucus spp. of additional hydroxyl groups). In this work, 22 phlorotannins were tentatively identified and their presence
HPLC-DAD-ESI/MSn
confirmed, in purified extracts obtained from four Fucus species, by HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/
UPLC-ESI-QTOF/MS
MS analyses. The characterized phlorotannins exhibited molecular weights ranging from 370 to 746 Da and
relatively low degree of polymerization (3–6 phloroglucinol units). Isomers of fucophlorethol, dioxinodehy-
droeckol, difucophlorethol, fucodiphlorethol, bisfucophlorethol, fucofuroeckol, trifucophlorethol, fucotriphlor-
ethol, tetrafucophlorethol, and of fucotetraphlorethol were identified. Among the analysed phlorotannin pur-
ified extracts, only the ones wild-sourced and from aquaculture-grown Fucus vesiculosus exhibited five and six-
ringed phloroglucinol oligomers in their composition. The remaining extracts from Fucus guiryi, Fucus serratus
and Fucus spiralis were richer in both trimers and tetramers. The variability observed for the overall phlorotannin
composition points to the influence of species-specific and/or external factors. As far as we know, mass spectra
confirming the presence and degree of polymerization of phlorotannins in Portuguese-sourced brown macro-
algae, specifically F. guiryi and F. serratus, as well as of aquaculture-grown F. vesiculosus, are not found in lit-
erature. The information gained in this work ascertains the role of advanced analytical tools in facilitating the
use of this valuable natural resource for the development of macroalgal-based products, opening doors for future
application of phlorotannin-rich extracts in the commercial areas related to nutraceuticals, pharmaceuticals, and
cosmetics.

1. Introduction pivotal role as primary metabolites with transitional secondary biolo-


gical functions in brown macroalgae [3].
An extremely variable and often complex molecular assemblage Over the last decade, studies on the biological activities of phlor-
from the polymerization of phloroglucinol (1,3,5-trihydroxybenzene) otannins have increased exponentially, and a growing commercial in-
gives origin to an almost inexhaustible family of naturally occurring terest into their potential application in a range of therapeutics has
polyphenolic entities: phlorotannins. These compounds exhibit a wide arisen [4,5]. Nevertheless, some obstacles have hampered the devel-
range of molecular weights (126 Da–650 kDa) and are biosynthesized opment of phlorotannin-based products (e.g., difficulties in separation
exclusively by brown seaweeds (Ochrophyta), via the acetate-malonate and purification steps, and subsequent characterization due to their
pathway [1]. The amount and the degree of polymerization of phlor- similar polarity and polymeric structure) [6]. Six different classes of
otannins are highly variable among seaweed species, and deeply af- phlorotannins (phlorethols, fuhalols, fucols, fucophlorethols, eckols and
fected by surrounding factors (e.g., UV radiation) [2], reinforcing their carmalols) can be established according to the nature of the structural


Corresponding authors.
E-mail addresses: pandrade@ff.up.pt (P.B. Andrade), federico@cebas.csic.es (F. Ferreres).

https://doi.org/10.1016/j.algal.2017.11.025
Received 23 May 2017; Received in revised form 7 November 2017; Accepted 18 November 2017
2211-9264/ © 2017 Elsevier B.V. All rights reserved.
G. Lopes et al. Algal Research 29 (2018) 113–120

linkages between phloroglucinol units, as well as to the number and Table 1


distribution of hydroxyl (OH) groups [4]; however, within each class, it Sample characterization.
is still possible to have both structural and conformational isomers,
Sample Species Origin Collection date
increasing the complexity and variability of these molecules [7]. In a
general way, phlorethols and fuhalols are characterized by the presence Fg F. guiryi Praia da Amorosa Dec 2013
of aryl-ether linkages between phloroglucinol units; however, fuhalols (N 41°41′49.75, W 8°51′3.52)
Fser F. serratus Praia da Amorosa Dec 2013
exhibit a regular sequence of para- and ortho- ether bonds, as well as
(N 41°41′49.75, W 8°51′3.52)
additional OH groups in every third ring, and one or more OH groups in Fspi F. spiralis Praia Norte Dec 2013
the whole molecule. Fucols, on the other hand, consist of phlor- (N 41°38′51.44, W 8°49′32.53)
oglucinol monomers linked by aryl-aryl bonds, while fucophlorethols Fves-w F. vesiculosus Praia Norte May 2015
present a mixture of ether and phenyl linkages between the basic unit. (N 41°38′51.44, W 8°49′32.53)
Fves-a F. vesiculosus IMTA Jul 2016
The generally low molecular weight of eckols and the presence of a
(N 40°36′45.27, W 8°40′26.88)
phenoxyl substitution at C4 are key-structural motifs that differentiate
eckols from carmalols, both characterized by the presence of dibenzo-
dioxin linkages [4,8]. 2.2. Sampling, phlorotannin extraction and quantification
Phlorotannins have been conventionally determined on crude ex-
tracts as the total amount of phenolic compounds, using non-specific Brown macroalgae (Ochrophyta) of the order Fucales used in this
spectrophotometric-based assays like Folin-Ciocalteu [9]. Another work were randomly collected during low-tide periods from different
quantitative colorimetric method, employing 2,4-dimethox- rocky shores of Northern Portugal (Table 1): F. guiryi (Fg), F. serratus
ybenzaldehyde (DMBA) that reacts specifically with 1,3- and 1,3,5- (Fser), F. spiralis (Fspi), and F. vesiculosus (Fves-w). Of the selected spe-
substituted phenols to form a coloured product, has also been employed cies, F. vesiculosus was also grown in an integrated multi-trophic
with good repeatability and high precision [8,10]. Although these aquaculture (IMTA) system (Fves-a), and supplied by ALGAPlus (Ílhavo,
methods are quite simple to use, giving a general estimation of the Portugal). The collected samples, consisting of, at least, five individuals
amount of phlorotannins in the extract, they provide no information on in the same stage of development, were washed to remove epiphytes,
the qualitative phlorotannin profile. Chromatographic techniques arise freeze-dried in a Virtis SP Scientific Sentry 2.0 apparatus (Gardiner, NY,
then as a powerful tool for the analysis of these algal constituents, and a USA) and ground to a fine powder (particle size ≤ 910 μm).
few recent studies employing advanced liquid chromatography-mass The extracts were prepared with approximately 1 g of powdered
spectrometry (LC-MS) methods have achieved, at least in part, the lyophilized material, using 10 mL of methanol:water (1:1, v/v), under
characterization of phlorotannins [6,7,9,11–16]. More than 150 the following conditions: 1 h of sonication, 24 h maceration at room
phlorotannins were identified in several brown seaweed species [8], but temperature, followed by 1 h of sonication. Afterwards, the extracts
the noteworthy structural heterogeneity and complexity of these com- were centrifuged (10,000 rpm, 10 min) and the methanol present in
pounds makes their profiling an almost unlimited field of research. each supernatant was removed in a Savant™ SPD121P SpeedVac™
The aim of this work was to establish the phlorotannin composition Concentrator (Thermo Scientific, Alcobendas, Spain). The remaining
of four Fucus species (Fucus guiryi G.I. Zardi, K.R. Nicastro, E.S. Serrão & aqueous mixture was loaded onto a Sep-Pak C18 Plus Short Cartridge
G.A. Pearson, Fucus serratus Linnaeus, Fucus spiralis Linnaeus, and Fucus (360 mg sorbent per cartridge, 55–105 μm particle size, 50/pk
vesiculosus Linnaeus) widely represented in the Northern Portuguese [WAT020515]) (Waters, Milford, MA, USA), which had been pre-con-
coastline. Some studies have previously characterized phlorotannins ditioned with methanol followed by water, and then washed with
found in Fucus species, such as F. vesiculosus [9,12,13,15,17], F. spiralis water. Phlorotannins were eluted with methanol and the solvent was
[6,7,9,11] and F. serratus [13], from different regions of the globe. To evaporated under reduced pressure until complete dryness. The re-
the best of our knowledge, this is the first study confirming the presence sulting phlorotannin-rich fraction was resuspended in a mixture of
and degree of polymerization of phlorotannins in F. guiryi. Moreover, as methanol:water (1:1, v/v) and filtered through a 0.45 μm pore size
macroalgal cultivation in integrated multi-trophic aquaculture (IMTA) membrane (Millipore) before analysis.
systems has become more widespread, there is a need to expand the The phlorotannin content of the Fucus spp. extracts was spectro-
knowledge on this material [18,19]. Therefore, F. vesiculosus grown in photometrically determined by the specific reaction between DMBA
IMTA was also analysed. High-performance liquid chromatography- and 1,3- and 1,3,5-substituted phenols to form a coloured product, as
diode array detection coupled to tandem electrospray ionization mass before [11]. The amount of phlorotannins in each extract was de-
spectrometry (HPLC-DAD-ESI/MSn) and ultra-performance liquid termined from a standard calibration curve (y = 0.0233x + 0.0125;
chromatography-electrospray ionization coupled to quadrupole time- r2 = 0.9995) with serial dilutions of phloroglucinol (2.3–75 μg/mL)
of-flight high-definition mass spectrometry (UPLC-ESI-QTOF/MS) were and expressed as mean ± SD (mg phloroglucinol equivalents (PGE)/kg
employed to provide evidence of the presence of phlorotannins, their dry algae) of 3 independent experiments performed in duplicate.
varying degree of polymerization and tentative identification in pur-
ified extracts of Fucus spp.
2.3. HPLC-DAD-ESI/MSn qualitative analyses

2. Materials and methods Chromatographic analyses were performed in an Agilent HPLC 1200
series equipped with a diode array and mass detectors in series (Agilent
2.1. Standards and reagents Technologies, Waldbronn, Germany), as previously described [20]. The
HPLC consisted of a binary pump (model G1376A), an autosampler
All solvents were of HPLC-grade. Formic acid, phloroglucinol (model G1377A) refrigerated at 4 °C (G1330B), a degasser (model
(≥ 99.0%) and 2,4-dimethoxybenzaldehyde (DMBA) were purchased G1379B), and a diode array detector (model G1315D). The HPLC
from Sigma-Aldrich (Steinheim, Germany). Methanol, acetonitrile, system was controlled by ChemStation software (Agilent, v. B.01.03-
glacial acetic acid and hydrochloric acid were obtained from Merck SR2). The mass detector was a Bruker ion trap spectrometer (model
(Darmstadt, Germany). Water was treated in a Milli-Q water purifica- HCT Ultra) equipped with an electrospray ionization interface and was
tion system (Millipore, Bedford, MA, USA). controlled by LCMSD software (Agilent, v. 6.1). Briefly, extract (20 μL)
elution was carried out at a flow rate of 0.8 mL/min, on a Kinetex
column (5 μm, C18, 100 Å, 150 × 4.6 mm; Phenomenex, Macclesfield,

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G. Lopes et al. Algal Research 29 (2018) 113–120

UK), with formic acid (1%) in water (A) and acetonitrile (B), starting phlorotannin amount (mg PGE/Kg dry algae) was ordered as follows:
with 1% B and installing a gradient to obtain 10% B at 12 min, 30% B at Fves-a (9.55 ± 0.20) < Fspi (16.72 ± 0.22) < Fves-w (32.76 ±
25 min, 50% B at 27 min, 50% B at 28 min, 1% B at 29 min, and 1% B 0.72) < Fg (181.70 ± 1.46) < Fser (264.73 ± 2.78). All samples
at 33 min. The ionization conditions were adjusted at 350 °C and 4.0 kV were screened for their phlorotannin composition by HPLC-DAD-ESI/
for capillary temperature and voltage, respectively. The nebulizer MSn and UPLC-ESI-QTOF/MS. The peaks were not abundant in the UV
pressure and flow rate of nitrogen were 65.0 psi and 11 L/min, re- chromatograms recorded at 280 nm (data not shown), and could not be
spectively. The full scan mass covered the range from m/z 100 up to m/ related to the MS ions corresponding to phlorotannins. Thus, the
z 1200. Collision-induced fragmentation experiments were performed Extracted Ion Chromatogram (EIC), together with the analysis of the MS
in the ion trap using helium as the collision gas, with voltage ramping fragmentation (Ion Trap) of the deprotonated molecular ions ([M-H]−),
cycles from 0.3 up to 2 V. Mass spectrometry data were acquired in the as well as their exact molecular mass (QTOF), were used as an attempt
negative ionization mode. to identify simple phloroglucinol polymers (fucols/phlorethols/fu-
cophlorethols), polymers with additional OH groups (fuhalols/hydro-
2.4. UPLC-ESI-QTOF/MS analyses xyfuhalols) and derivatives presenting dibenzodioxin moieties (eckols/
carmalols). The tentative identification of phlorotannin structures was
Exact mass determination was carried out in an Agilent 1290 based in the loss of phloroglucinol units (126/125/124 amu) or their
Infinity LC system coupled to 6550 Accurate-Mass QTOF (Agilent derivatives (Table 2 footnote), and considering that the CeC linkages
Technologies, Waldbronn, Germany) with an electrospray interface (Jet between phloroglucinol units, characteristic of fucols, are of difficult
Stream Technology). Samples (1 μL) were injected onto a reversed fragmentation, when compared to CeOeC bonds, characteristic of
phase Luna Omega column (1.6 μm, PS C18, 100 Å, 50 × 2.1 mm; phlorethols. Apart from the loss of neutral fragments, fragments cor-
Phenomenex, Macclesfield, UK) with SecurityGuard ULTRA Cartridges responding to the loss of neutral fragments with one or two protons less
of the same material operating at 30 °C and a flow rate of 0.5 mL/min. were also observed. For instance, fragmentation of phloroglucinol may
The mobile phases used were acidified water (0.1% formic acid) (A) present a deprotonated molecular ion with less 1 amu or 2 amu than the
and acidified acetonitrile (0.1% formic acid) (B). Compounds were se- parent compound (126 amu), which corresponds to the loss of one or
parated using the following gradient conditions: elution started with two hydrogen atoms (125 amu and 124 amu, respectively).
1% B to obtain 10% B at 8 min, 30% B at 14 min, 50% B at 16 min, and
1% B at 20 min. The optimal conditions for the electrospray interface 3.2. Phlorotannin profile of Fucus spp.
were as follows: gas temperature 280 °C, drying gas 11 L/min, nebulizer
pressure 45 psi, sheath gas temperature 400 °C, sheath gas flow 12 L/ At present, only a small number of phlorotannins have been isolated
min. The MS system was operated in negative ion mode with the mass and chemically characterized from the four macroalgae species ana-
range set at m/z 50–1100 in full scan resolution mode. Other analytical lysed herein. As far as we know, mass spectra confirming the presence
conditions were as previously described [21]. and degree of polymerization of phlorotannins in Portuguese-sourced
brown macroalgae, specifically F. guiryi and F. serratus, as well as of
3. Results and discussion aquaculture-grown F. vesiculosus, are not found in literature. The 22
phlorotannin compounds detected in this work were then tentatively
3.1. General considerations identified using mass spectrometry, as well as literature data
[8,11,12,23–26]. The analytical methodology employed herein had
The presence of phlorotannins is widely acknowledged in brown already been successfully used by us [11], allowing an adequate re-
seaweeds, but a rather limited characterization of such complex poly- solution of phlorotannins with between 3 and 8 phloroglucinol units
meric structures has been accomplished [16]. The strong biological (PGU). Furthermore, any polymer with mass lower than 1200 could be
potential already pointed for the use of phlorotannins as therapeutic detected herein and the gradient used allows the elution of compounds
agents makes the characterization of phlorotannin-rich extracts of great with several degrees of polymerization. Preliminary experiences, not
importance for both discovery and product development [3,4]. Due to included in the manuscript, were performed with softer gradients at the
the generally high complexity of the extracts, advanced mass spectro- end to search for other polymers; nevertheless, as these were not ob-
metry-methods constitute attractive options for phlorotannin analysis. served, we used a gradient with a fast increase of the organic phase at
Although recent works have successfully employed UPLC-tandem the end for cleaning.
quadrupole detector (TQD)-MS analysis to investigate the isomeric The identified polyphenols exhibited distinct molecular weights
complexity of phlorotannins present in different algal extracts, the full (370–746 Da), and relatively low degree of polymerization (3–6 PGU)
characterization of their structures was not achieved and it is highly (Table 2). Though there are seaweed species with monomeric and di-
improbable that it will be in a near future [7,13]. meric phlorotannins [27], it seems they are not commonly found in
To properly profile phlorotannins, several protocol steps must be Fucus spp. extracts. The variability observed for the overall phlor-
considered. Suitable sample preparation (e.g., the choice of the best otannin composition among the analysed algal extracts (Fig. S1) points
extractant) is crucial in the analysis of natural matrices, and primary also to the influence of species-specific factors (e.g., thallus age, me-
purification of the crude extract is also beneficial to target compounds tabolic activity, and physiological variations within algal organs) and/
and to improve chromatographic resolution [22]. The procedure em- or extrinsic factors (origin, time of harvest, and general surrounding
ployed herein (Fig. 1) for obtaining phlorotannin-rich fractions from conditions). For instance, in a previous work undertaken by our group
the different Fucus species benefited from the purification of the extract [11], phlorotannins of higher degree of polymerization were tentatively
by using a C18 Sep-Pak cartridge. This step allowed the retention of identified in a Fucus species. This can be explained, at least in part, by
phlorotannins and removal of other co-extracted compounds, such as the geographical location of the Fucus spiralis used in that work: a
polysaccharides, with which phlorotannins are usually associated [6]. southern location, with generally higher temperatures and light ex-
In fact, preliminary MS analyses were conducted in the SPE washing posure, can be determinant for the profile of secondary metabolites.
fractions and no phlorotannins were detected. These fractions were Besides phlorotannin amount, these abiotic factors can also be re-
then discarded, and methanol eluates were considered of suitable purity sponsible for the production of phlorotannins with higher degree of
for MS profiling analysis. polymerization, explaining the differences observed between seaweed
The five samples of Fucus under study were quantified for their total species from southern and northern origin [11]. Of the Fucus spp. stu-
phlorotannin content through the specific DMBA colorimetric method, died so far, a study of Heffernan and co-workers [13] with F. serratus
using their monomeric unit (phloroglucinol) as standard. Total and F. vesiculosus harvested off the Irish coast described the presence of

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G. Lopes et al. Algal Research 29 (2018) 113–120

Fig. 1. Schematic representation of the general procedure for the obtainment and profiling of purified extracts from Fucus spp. By HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/MS analyses.

phlorotannins with 3 and up to 12 PGU [13]. However, the majority of was detected, more than two of these oligomers were found in the re-
the polyphenols found in Fucus spp. were of low molecular weight, maining extracts (Fig. S1). Excepting compound 12, the remaining
which is in accordance with our findings for species of this genus. Other tetramers (5–11) presented deprotonated molecular ions at m/z 497
studies [6,7,9,12,15,17] are also in agreement with this and it seems (Table 2). In the MS2 and MS3 fragmentation of both compounds 5 and
that the abundance of low molecular weight phlorotannins can be 6 the loss of a single phloroglucinol unit indicates the presence of only
characteristic of Fucus spp. one aryl-ether bond, likely corresponding to difucophlorethol isomers
In this section, the main features of the different phlorotannin (Table 2). As far as we are aware, to date, these phlorotannin oligomers
classes identified in the extracts from Fucus spp. will be discussed. were not described in any Fucus species; however, the difucophlorethol
A isomer had already been isolated from other brown seaweeds of the
3.2.1. Phlorotannin trimers order Fucales (Himanthalia elongata (Linnaeus) S.F. Gray and Cystophora
Apart from Fves-a, trimers of phloroglucinol (1–4) were found in all retroflexa (Labillardière) J. Agardh) [24,25]. In their MS fragmentation
the other purified extracts (Fig. S1). Compounds 1 and 2 have the same (MS2 and MS3), compounds 7–9 exhibited losses of one and two
[M-H]− at m/z 373, but slightly different fragmentation patterns, phloroglucinol units, the ion characterizing the fucol moiety (m/z 229
suggesting that they would be isomers (Table 2). The MS2 fragmenta- in MS3) being observed, differing from each other by their base peaks
tion of compound 1, detected only in Fves-w (Fig. S1A), exhibited the and the relative abundance of their product ions (Table 2). Based on the
loss of a single unit of phloroglucinol (− 126 amu) and of a derivative MS data collected (Table 2), two phloroglucinol units linked by an aryl-
with water (124 + 18 amu) to give rise to the ions at m/z 247 and 231, ether bond (diphlorethol) may be present, leading to the tentative
respectively. Important losses of 18 and 44 amu were also detected, the identification of compounds 7–9 as fucodiphlorethol isomers. As it
latter probably generated by the combined elimination of an ethylene happened with 7–9, the loss of a phloroglucinol unit (−124 amu) was
group and water, a consequence of the internal cleavage of benzene detected in the MS2 of compound 11, originating the base peak at m/z
ring structures [12]. Likewise, in the MS2 fragmentation of compound 373 that is fragmented in MS3 with loss of 140 amu (124 + 16). No
2, detected in both Fser and Fspi extracts (Fig. S1K and M), the loss of other loss of phloroglucinol was noticed, leading to the putative iden-
only one phloroglucinol unit was observed (Table 2). In MS3 and MS4 tification of compound 11 as a fucodiphlorethol isomer. These tetra-
(data not shown) of compounds 1 and 2 no additional losses of phlor- mers of phloroglucinol isomers have already been identified by our
oglucinol or derivative were observed, the ion at m/z 229 in MS3 does group [11] in purified extracts from F. spiralis and Cystoseira tamar-
not undergo further fragmentation into phloroglucinol, suggesting the iscifolia (Hudson) Papenfuss, as well as by Wang et al. [12] in Sephadex
presence of a fucol moiety (two units of phloroglucinol linked by an subfractions of the ethyl acetate fraction from F. vesiculosus. This par-
aryl-aryl bond). Compounds 1 and 2 were then tentatively identified as ticular phlorotannin assemblage have been associated to a number of
fucophlorethol isomers, as they are formed by phloroglucinol units interesting properties (e.g., free radical scavenging [28] and anti-al-
linked through both ether and phenyl bonds. Either one of them may, in lergic [29]), making their presence in these extracts of great relevance
fact, correspond to fucophlorethol A (Fig. 2), previously isolated from F. for further biological studies.
vesiculosus by Parys et al. [23], and described as a strong radical sca- Despite sharing the same molecular ion of the previously identified
venger with chemopreventive potential [23]. tetramers (m/z 497), the main MS2 fragments observed for compound
The trimers 3 and 4, detected in the purified extracts of Fspi and Fg, 10 result from the loss of two phloroglucinol molecules and a methyl
respectively (Fig. S1N and H), presented deprotonated molecular ions group (262 amu = 124 + 124 + 14), to give rise to the base peak,
with less 4 amu than the others (m/z 369), consistent with three-ringed which did not originate other phloroglucinol units in MS3. As so,
phloroglucinol containing dibenzo[1,4]-dioxin structural elements, compound 10 should correspond to two fucol moieties linked by an
such as dioxinodehydroeckol derivatives (Fig. 2). Dioxinodehydroeckol, aryl-ether bond, tentatively labelled as bisfucophlorethol (Fig. 2). Bis-
also known as eckostolonol, is a common phlorotannin reported in the fucophlorethols, together with several other phlorotannin oligomers,
composition of several seaweed species belonging to the orders Fucales have been found by Glombitza and Hauperich [26] in the composition
and Laminariales [11]. of Cystophora torulosa (R. Brown ex Turner) J. Agardh (Fucales).
The tentative identification of the tetramer 12, detected only in the
3.2.2. Phlorotannin tetramers purified extract from Fg (Fig. S1J), proved to be a challenging task
Phlorotannins composed of four phloroglucinol units (5–12) were mainly because of its atypical molecular formula (C24H14O12). Its de-
generally the most representative of the analysed extracts (Fig. S1, protonated molecular ion (m/z 493) is 4 amu lower than those of the
Table 2). With the exception of Fves-w, in which only one tetramer (7) previous tetramers. Based on the MS data and published literature [8],

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G. Lopes et al.

Table 2
Retention times (Rt), molecular formula and mass spectrometric data of molecular ions and main observed fragments of phlorotannins in F. vesiculosus wild (1, 7, 14–19, 21, 22), F. vesiculosus aquaculture (5, 7, 13, 20, 22), F. guiryi (4–6, 8, 10–12),
F. serratus (2, 5, 9, 11) and F. spiralis (2, 3, 7, 10) extracts.a

Compoundb Phlorotannin oligomer Rt (min) Molecular formula MS1 [M-H]−, m/z MS2 [M-H]−, m/z (%, − lossc) MS3 [(M-H) → base peak]−, m/z (%, − lossc)

1 Trimer 3.9 C18H14O9 373.0567 355(100, −18), 329 (40, − 44), 247(30, −126), 231(60, −142) 229 (30, − 126), 215(100, −140)
2 Trimer 6.6 C18H14O9 373.0560 247(100, −126), 233(70, − 140) 229(100, − 18)
3 Trimer 14.5 C18H10O9 369.0249 238(100) 195(100, − 43), 167(60), 112(50, −126)
4 Trimer 18.6 C18H10O9 369.0246 351(50, − 18), 325(100, − 44), 307(40, − 62) 307(100, − 18), 297(60, −28), 281(45, − 44)
5 Tetramer 3.2 C24H18O12 497.0715 479(100, −18), 353(10, − 144) 331(70) 461(100, − 18), 435(40, −44), 353(20, − 126)
6 Tetramer 4.6 C24H18O12 497.0719 479(100, −18), 461(35, − 36), 355 (50, −142) 420(35), 353(80, − 126), 337(100, − 142)
7 Tetramer 5.2 C24H18O12 497.0717 479(100, −18), 371(5, −126), 353(25, − 144), 339(30, − 158)d 339(100, − 140), 229(5, −250)
8 Tetramer 6.7 C24H18O12 497.0713 479(10, − 18), 355(100, − 142), 311(50) 311(100, − 44), 229(10, −126)
9 Tetramer 8.2 C24H18O12 497.0715 355(100, −142), 235(40, − 262) 269(40), 229(100, − 126)
10 Tetramer 8.5 C24H18O12 497.0716 235(100, −262) 207(75, − 28), 191(100, −44)
11 Tetramer 9.4 C24H18O12 497.0715 479(25, − 18), 373(100, − 124), 265(50) 233(50, − 140), 139(100)
12 Tetramer 17.4 C24H14O12 493.0405 475(100, −18), 367(15, − 126) 431(60, − 44), 405(100)
13 Pentamer 4.2 C30H22O15 621.0899 603(100, −18), 577(35, − 44), 559(40), 497(5, − 124), 477(5, 585(100, − 18), 559(50), 477(25, −126)
− 144)
14 Pentamer 4.7 C30H22O15 621.0883 603(100, −18), 495(10, − 126) 585(100, − 18), 463(35, −140), 477(25, − 126), 459(60, − 144)
15 Pentamer 6.7 C30H22O15 621.0902 603(100, −18), 495(5, −126), 461(39, − 160), 355(45, − 266) 461(60, − 142), 355(100, − 248)e

117
16 Pentamer 7.4 C30H22O15 621.0887 603(100, −18), 479(10, − 142), 461(5, − 160), 353(20, − 268) 479(70, − 124)f, 335(100, −268)
17 Pentamer 7.8 C30H22O15 621.0885 603(100, −18), 479(50, − 142), 461(60, −160)g, 353(30, −268) 461(60, − 142)g, 353(100, − 250), 335(25, − 268)
18 Pentamer 8.2 C30H22O15 621.0901 603(100, −18), 463(5, −158), 339(10, − 282)h 477(40, − 126), 463(50, −140), 339(100, − 264)h
19 Pentamer 8.5 C30H22O15 621.0900 603(100, −18)i, 479(5, − 142), 337(25, − 284), 229(10) 479(50, − 124), 339(100, − 264), 229(60)
20 Hexamer 6.4 C36H26O18 745.1040 727(100, −18), 601(10, − 144) 709(100, − 18), 602(20, −125), 585(20, − 142)
21 Hexamer 9.9 C36H26O18 745.1050 727(100, −18), 601(35, − 144), 461(5, − 284), 335(12, − 410), 601(35, − 126), 583(90, −144), 479(50, − 248), 353(60, − 374),
229(5) 229(20)
22 Hexamer 10.3 C36H26O18 745.1048 727(100, −18), 601(25, − 144), 479(10, −286), 353(12, − 392), 601(45, − 126), 583(60, −144), 461(35, − 266), 335(100, −392),
229(5) 229(45)

a
Ions in bold comprise the loss of phloroglucinol moieties.
b
Peak identity as in Fig. S1.
c
62: 44 + 18; 140: 124 + 16; 142: 126 + 16/124 + 18; 140: 124 + 16; 142: 124 + 18; 144: 126 + 18; 158: 124 + 16 + 18; 160: 126 + 16 + 18/124 + 18 + 18; 170: 126 + 44; 248: 124 + 124; 250: 126 + 124; 262: 124 + 124 + 14;
264: 124 + 124 + 16; 266: 124 + 124 + 18; 268: 126 + 126 + 16/124 + 126 + 18; 282: 124 + 124 + 18 + 16; 284: 124 + 124 + 18 + 18; 286: 124 + 126 + 18 + 18; 374: 124 + 124 + 126; 376: 126 + 126 + 124; 392:
124 + 124 + 126 + 18; 410: 126 + 126 + 126 + 16 + 16.
d
7 MS3(497 → 339): 230; MS4(497 → 479 → 229): 229(100); 201(25).
e
15 MS4(355): 229(100, − 126).
f
16 MS4(479): 339(100, − 140), 230(40).
g
17 MS3(461): 230; MS4(461 → 230): 229.
h
18 MS3(339): 230; MS4(621 → 603 → 463): 339(100); MS4(621 → 603 → 339): 230(100).
i
19 MS3(621 → 603): 479(50, − 124), 337(100, − 266), 229(65); MS4(621 → 603 → 337): 229(100).
Algal Research 29 (2018) 113–120
G. Lopes et al. Algal Research 29 (2018) 113–120

Fig. 2. Proposed fragmentation patterns of the structures tentatively identified in phlorotannin purified extracts from Fucus spp.: 1, m/z 373; 4, m/z 369; 6, 9 and 10, m/z 497; 12, m/z
493; 13 and 15, m/z 621; 20 and 22, m/z 745.

it is possible that the chemical structure of compound 12 resembles that [124 + 124 + 126 + 18 amu]). Additionally, this ion does not un-
of a fucofuroeckol containing an additional OH group in its backbone dergo further fragmentation in MS4, pointing to a fucol moiety (two
(Fig. 2). units of phloroglucinol linked by an aryl-aryl bond), as observed in the
fragmentation of other compounds described above. In some cases, like
3.2.3. Phlorotannin pentamers that of compound 18, it was possible to observe the sequential loss of
Among the analysed phlorotannin purified extracts, the ones from phloroglucinol units: MS4 (621 → 603 → 463): 339; MS4 (621 →
both wild-sourced and aquaculture-grown F. vesiculosus (Fves-w and 603 → 339): 230 (Fig. 3). Pentamers 15–19 were then tentatively
Fves-a, respectively) were the only exhibiting five-ringed phloroglucinol identified as isomers of fucotriphlorethol (Fig. 2). The work previously
oligomers (Fig. S1C and F). In all the pentamers (13–19), as well as in conducted by our group [11] had already identified a fucotriphlorethol
most of the phlorotannins detected in this work, the base peak in MS2 derivative in the seaweed species Cystoseira usneoides (Linnaeus) M.
results from the loss of water from the deprotonated molecular ion Roberts, belonging to Fucales. Still, of relevance, was the isolation of
(Table 2). Both MS2 and MS3 fragmentation of compounds 13 and 14 fucotriphlorethol A from F. vesiculosus extracts, suggesting that one of
are similar, with losses of a single phloroglucinol unit (Table 2) being the 15–19 pentamers detected herein, in the same seaweed species,
observed, with no further loss of phloroglucinol or phloroglucinol de- may, in fact, correspond to that isomer [23].
rivative in MS4 (data not shown). The chemical features of compounds
13 and 14 are therefore consistent with the ones of trifucophlorethol 3.2.4. Phlorotannin hexamers
isomers (Fig. 2). As far as we know, this is the first tentative identifi- The extracts of F. vesiculosus (Fves-w and Fves-a) were the only ones
cation of this fucophlorethol-type phlorotannins in Fucales. exhibiting phlorotannin hexamers (2–22) (Fig. S1D and G). The three
All the remaining pentamers (15–19), detected only in Fves-w (Fig. hexamers had the same deprotonated molecular ion at m/z 745, but
S1C), had similar MS2 fragmentation patterns, characterized by losses different fragmentation patterns (Table 2). In the MS3 of compound 20
of a phloroglucinol (−126 amu) or phloroglucinol derivative (−142/ detected in Fves-a (Fig. S1G), the loss of water and the fragmentation of
146/158/160 amu), as well as by the simultaneous loss of two units of a phloroglucinol unit (− 125 amu) indicates the presence of a single
phloroglucinol combined with oxygen and/or water (− 266/268/282/ ether bond in its structure (Fig. 2). The loss of 125 amu was observed
284 amu) (Table 2). The same losses were detected in the MS3 frag- only for this compound (Table 2). A similar fragmentation pattern was
mentation. In the MS2 of compound 19, in the MS3 of compounds 17 recently reported for phorethols and fucophlorethols extracted from
and 18, as well as in the MS4 of 15–19, an ion at m/z 229/230 was Sargassum fusiforme (Harvey) Setchell (Fucales) [16]. Although the MS
found (Table 2). As for several other phlorotannins detected herein, this fragmentations provided raw data to ensure the identity of compound
ion may be due to the loss of three phloroglucinol units linked to each 20, it can be included within the fucophlorethol-type, and possibly la-
other through aryl-ether bonds (229 = 621–392 belled as tetrafucophlorethol.

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G. Lopes et al. Algal Research 29 (2018) 113–120

Fig. 3. Mass spectra analysis of the pentamer 18 detected in wild-sourced F. vesiculosus (Fves-w).

Compounds 21 and 22 showed a MS fragmentation similar to that of Acknowledgments


the pentamers referred above (15–19), though with an additional
phloroglucinol unit. Besides the ion [(M-H)-18]− as base peak, their This work received financial support from National Funds (FCT/MEC,
MS2 fragmentation exhibited the ions at m/z 601(−144), 461/ Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência)
479(−284/286), 335/353(− 410/392) and the radical fucol at m/z through project UID/QUI/50006/2013, co-financed by European Union
229, the corresponding ions being also found in the MS3. These data (FEDER under the Partnership Agreement PT2020), from Norte Portugal
suggest the presence of four phloroglucinol units linked by aryl-ether Regional Operational Programme (NORTE 2020), under the PORTUGAL
bonds, and of a fucol moiety (ion observed at m/z 229), leading to the 2020 Partnership Agreement, through the European Regional
tentative identification of compounds 21 and 22 as fucotetraphorethol Development Fund (ERDF) (project NORTE-01-0145-FEDER-000024), and
isomers (Fig. 2), already isolated from H. elongata and C. retroflexa from Programa de Cooperación Interreg V-A España–Portugal (POCTEP)
(Fucales) [24,25]. 2014–2020 (project 0377_IBERPHENOL_6_E). To all financing sources the
authors are greatly indebted. To all financing sources the authors are
4. Concluding remarks greatly indebted. Mariana Barbosa (SFRH/BD/95861/2013) thank FCT/
MEC for the grant.
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