Nonenzymatic Î±-Linolenic Acid Derivatives from the Sea: Macroalgae

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Non-Enzymatic #-Linolenic Acid Derivatives from the Sea: Macroalgae as Novel Sources of Phytoprostanes Mariana Barbosa, Jacinta Collado-González, Paula B. Andrade, Federico Ferreres, Patrícia Valentão, Jean-Marie Galano, Thierry Durand, and Angel Gil-Izquierdo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01904 • Publication Date (Web): 30 Jun 2015 Downloaded from http://pubs.acs.org on July 6, 2015

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Journal of Agricultural and Food Chemistry

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Non-Enzymatic α-Linolenic Acid Derivatives from the Sea: Macroalgae as Novel

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Sources of Phytoprostanes

3 4

Mariana Barbosa†, Jacinta Collado-González‡, Paula B. Andrade†, Federico Ferreres‡,

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Patrícia Valentão†, Jean-Marie Galano§, Thierry Durand§, Ángel Gil-Izquierdo‡,*

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†

REQUIMTE/LAQV, Laboratório de Farmacognosia, Departamento de Química,

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Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, nº 228,

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4050-313 Porto, Portugal

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‡

Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of

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Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus

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University Espinardo, Murcia, Spain

13

§

14

of Montpellier - ENSCM, Faculty of Pharmacy, Montpellier, France

Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 - CNRS – University

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*Corresponding author:

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Tel: +34 968396363; Fax: +34 968396213; E-mail: [email protected]

18 19

1 ACS Paragon Plus Environment


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ABSTRACT

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Phytoprostanes, autoxidation products of α-linolenic acid, have been studied in several

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plant species, but information regarding the natural occurrence of this large family of

23

biologically active oxidized lipids in macroalgae is still scarce. In this work, free

24

phytoprostane composition

25

Chlorophyta, Phaeophyta and Rhodophyta was determined through a recently validated

26

UHPLC-QqQ-MS/MS method. The phytoprostane profiles varied greatly among all

27

samples, being F1t-phytoprostanes and L1-phytoprostanes the predominant and the

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minor classes, respectively. No correlation between the amounts of α-linolenic acid in

29

algae material and phytoprostane content was found. Therefore, we hypothesize that the

30

observed variability could be species-specific or result from interspecific interactions.

31

This study provides new insight about the occurrence of phytoprostanes in macroalgae

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and opens doors for future exploitation of these marine photosynthetic organisms as

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sources of potentially bioactive oxylipins, encouraging their incorporation in food

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products, nutraceutical and pharmaceutical preparations for human health.

of twenty-four

macroalgae species

belonging

to

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KEYWORDS: α-Linolenic acid, GC-MS, Macroalgae, Oxidized lipids, Phytoprostanes,

37

UHPLC-QqQ-MS/MS.

38


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INTRODUCTION

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Membrane lipids are major targets of free radical attack. Phytoprostanes are the

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resulting products of the autoxidation of α-linolenic acid (C18:3 ω3) (1, Figure 1), one

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of the most abundant polyunsaturated fatty acid (PUFA) in terrestrial higher plant

43

membranes.1

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phytoprostanes) can be generated depending on the position where the hydrogen

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abstraction occurs and the oxygen atoms are inserted in the PUFA backbone.2 G1-

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phytoprostanes are then precursors of different classes of cyclic phytoprostanes, named

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in analogy with the prostaglandin nomenclature system as A1, B1, D1, E1, F1, dJ1, and

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L1-phytoprostanes, with the latter being the regioisomer of B1-phytoprostane.3,

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large family of regio- and stereoisomeric prostaglandin-like compounds has been found

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to occur constitutively in plants; however, reactive oxygen species (ROS) generated

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under the influence of both biotic and abiotic factors interfere with cellular redox

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balance and lead to enhanced formation of phytoprostanes, inducing, for instance, the

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biosynthesis of secondary metabolites, the expression of genes involved in

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detoxification processes and the regulation of oxidative stress-related mitogen-activated

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protein kinase (MAPK)-dependent signaling pathway.1,

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can function not only as defense signals, but also as endogenous mediators capable of

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preventing cellular damage.5

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Previous works have already reported the presence of different classes of

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phytoprostanes in vegetable oils, particularly in linseed and soybean oils, as well as in

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aqueous pollen extracts.8, 9 Nevertheless, information regarding the natural occurrence

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of this novel family of biologically active oxidized lipids in macroalgae is still scarce.

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As far as we know, only Ritter et al.10 have recently described the accumulation of

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cyclic C18 A1-phytoprostanes in the brown macroalgae Ectocarpus siliculosus

Two

regioisomeric

series

(16-G1-phytoprostanes

5-7

and

9-G1-

4

This

Therefore, these oxylipins



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(Dillwyn) Lyngbye to which copper stress was induced, supporting the occurrence of

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ROS-mediated lipid peroxidation processes.10

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Macroalgae, commonly known as seaweeds, are an abundant and heterogeneous group

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of marine photosynthetic organisms widely used as food in direct human consumption.

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Besides their key-role in the food sector, macroalgae are also employed in other

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industrial branches, such as textiles, paints, cosmetics, and more recently as renewable,

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sustainable and eco-friendly alternative sources for bioethanol production.11

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Regardless of the large algal biodiversity that remains unexplored, macroalgae have

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historically been an important group of organisms for marine drug development.12 In

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fact, researchers have shown that macroalgae are rich sources of nutraceuticals with

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numerous and remarkable biological activities.13

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Macroalgae are usually grouped into Chlorophyta (green algae), Phaeophyta (brown

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algae) and Rhodophyta (red algae), according to the presence of specific pigments.

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These marine organisms are rich sources of bioactive compounds from both primary

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and secondary metabolism, among which fatty acids are highlighted.14,

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marine algae present low lipid content (1-5% of dry matter), PUFA account for almost

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half of the lipid fraction.15 Several studies have shown that despite PUFA profiles are

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highly variable between and within algal groups, linoleic (C18:2 ω6), linolenic (C18:3

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ω3), arachidonic (C20:4 ω6), eicosapentaenoic (C20:5 ω3) and docosahexaenoic (C22:6

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ω3) acids are predominant in macroalgae.16-20

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Marine ecosystem is characterized by broad fluctuations of environmental conditions

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that can be strong stress inducers in macroalgae populations, including extreme

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temperatures, rapid salinity and nutrient changes, dessication, intense sunlight, among

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others.21

15

Although


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Taken together, these observations suggest that macroalgae can be valuable sources of

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phytoprostanes and potentially used as biomarkers of oxidative stress and/or present

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important biological effects.9, 22 Due to the fact that macroalgae are an integral part of

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the Asian food and recently became a popular addition to some Western diets, our goal

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was to assess the occurrence of free phytoprostanes, readily bioavailable and absorbed

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by the human body, in twenty-four species of macroalgae belonging to Chlorophyta,

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Phaeophyta and Rhodophyta. The reason for this characterization was based on the fact

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that humans do not possess esterases in the gastrointestinal tract, rendering impossible

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to absorb the esterified form of phytoprostanes.23

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A fast, selective and robust ultra-high performance liquid chromatography coupled to

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triple-quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) method, previously

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validated by Collado-González et al.24, was employed. Moreover, all macroalgae

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species selected for this work were characterized for their composition in α-linolenic

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acid (1) by gas chromatography-mass spectrometry (GC-MS) after alkaline hydrolysis

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and derivatization.

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MATERIALS AND METHODS

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Standards and reagents. Ten phytoprostanes standards (9-F1t-phytoprostane

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(2), 9-epi-9-F1t-phytoprostane (3), ent-16-F1t-phytoprostane (4), ent-16-epi-16-F1t-

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phytoprostane (5), 9-D1t-phytoprostane (6), 9-epi-9-D1t-phytoprostane (7), 16-B1-

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phytoprostane (8), ent-16-B1-phytoprostane (9), 9-L1-phytoprostane (10) and ent-9-L1-

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phytoprostane (11)) (Figure 1) were synthesized according to our previous procedures.

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25-27

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The d4-15-F2t-isoprostane (8-isoPGF2α-d4) (containing four deuterium atoms at

positions 3,3ʹ,4, and 4ʹ) was purchased from Cayman Chemicals (Ann Arbor, MI, US).



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Authentic standards of fatty acids methyl esters (FAME) for GC-MS analysis were

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obtained from Supelco (Bellefonte, PA, US).

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Two types of solid-phase extraction (SPE) cartridges were used in this study:

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Chromabond C18 columns (1000 mg/6 mL) were obtained from Macherey-Nagel

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(Düren, Germany) and Strata X-AW (500 mg/3 mL) from Phenomenex (Torrance, CA,

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US).

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(hydroxymethyl)-methane (BIS-TRIS), chloroform, isooctane and boron trifluoride

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(BF3) were purchased from Sigma-Aldrich (St. Louis, MO, US). Methanol was acquired

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from VWR (Fontenay-sous-Bois, France), acetonitrile was obtained from Merck

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(Darmstadt, Germany) and n-hexane was purchased from Panreac (Barcelona, Spain).

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All LC-MS grade solvents were obtained from J.T. Baker (Phillipsburg, NJ, US). Water

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was treated in a Milli-Q water purification system from (Millipore, Bedford, MA, US).

Butylated

hydroxyanisole

(BHA),

bis-(2-hydroxyethyl)-amino-tris-

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Macroalgae samples. Macroalgae samples consisted of three Chlorophyta, five

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Rhodophyta and sixteen Phaeophyta species collected between 2010 and 2013 (Table

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1). With the exceptions of G. vermiculophylla, S. latissima and Ulva sp., which were

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cultivated in integrated multi-trophic aquaculture (IMTA) systems and provided by

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CIIMAR/CIMAR – Centre for Marine and Environmental Research, all samples were

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randomly collected during low tide periods from different places of the west coast of

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Portugal, each sample corresponding to a mixture of three to four individuals in the

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same stage of development. In order to prevent sample alterations, after collection they

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were immediately placed on ice and transported to the laboratory in insulated, sealed ice

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boxes, to protect them from heat, air, and light exposure. Macroalgae were then

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carefully and quickly washed with NaCl aqueous solution (3.5%) to remove epiphytes

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and encrusting material, at room temperature, without exposure to direct light, and kept


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at –20 ºC, prior to lyophilization. The dried material was powdered (< 910 μm) and kept

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in the dark, in a desiccator, until it was subjected to extraction. No alteration (color,

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smell, humidity) of the samples was noticed during storage.

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α-Linolenic acid and extract derivatization for GC-MS analysis. The

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extraction of α-linolenic acid was performed as previously described, with slight

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modifications.28 Briefly, 0.25 g of the dried macroalgae were extracted with 25 mL of

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chloroform:methanol (2:1), under magnetic stirring at 500 rpm, for 10 min, at 40 °C.

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The extraction procedure was repeated five times and the resulting extracts were pooled

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and concentrated to dryness under reduced pressure (40 °C). The residue was then

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hydrolyzed with 1 mL of KOH methanolic solution (11 g/L), at 90 °C, for 10 min. The

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free fatty acids originally present and those resulting from the alkaline hydrolysis were

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derivatized to their methyl esters with 1 mL of BF3 methanolic solution (10%), at 90 °C,

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for 10 min. FAME were purified with 2×10 mL of isooctane and anhydrous sodium

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sulphate was added to assure the total absence of water. The resulting extract was

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evaporated to dryness under a stream of nitrogen and dissolved in 200 μL of isooctane.

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Each macroalgae species was assayed in triplicate.

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GC-MS analysis of α-linolenic acid. GC-MS analysis was performed following

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a previously established method.28 Derivatized extracts (1 μL) were analyzed using a

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Varian CP-3800 gas chromatograph (Walnut Creek, CA, US) equipped with a Varian

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Saturn 4000 mass selective detector (Walnut Creek, CA, US) and a Saturn GC/MS

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workstation software version 6.8. The column used was a 30 x 0.25 mm, i.d., 0.25 µm,

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VF-5 ms (Varian, Walnut Creek, CA, US). The injector port was heated to 250 °C.

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Injections were performed in split mode, with a ratio of 1/40. The carrier gas was



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Helium C-60 (Gasin, Portugal), at a constant flow of 1 mL/min. The oven temperature

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was set at 40 °C for 1 min, then increased 5 °C/min to 250 °C, 3 °C/min to 300 °C and

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held for 15 min. All mass spectra were acquired in EI mode. Ionization was maintained

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off during the first 4 min, to avoid solvent overloading. The Ion Trap detector was set as

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follows: transfer line, manifold and trap temperatures were respectively 280, 50 and 180

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°C. The mass ranged from m/z 50 to 600, with a scan rate of 6 scan/s. The emission

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current was 50 μA, and the electron multiplier was set in relative mode to auto tune

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procedure. The maximum ionization time was 25,000 μs, with an ionization storage

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level of m/z 35. The analysis was performed in Full Scan mode. Identification of α-

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linolenic acid was achieved by comparison of its retention index and mass spectra with

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those from pure standard injected under the same conditions, and from NIST 05 MS

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Library Database. The amount of methyl ester of α-linolenic acid present in the samples

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was achieved from the calibration curve of the respective standard prepared in

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isooctane.

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Phytoprostane extraction. Each pulverized macroalgae sample (1 g) was

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crushed in a mortar and pestle with 5 mL methanol (0.1% BHA). The sample extracts

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were centrifuged at 2,000 g during 10 min and the supernatants underwent SPE using a

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Chromabond C18 column. Briefly, 10 mL of n-hexane were added to 1 mL of the

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filtered sample and then rediluted in 2 mL of methanol and 2 mL of BIS-TRIS buffer.24

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The emulsion of each macroalgae sample was applied to a previously conditioned and

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equilibrated Strata-X-AW cartridge. After loading the column with the emulsion, 2 mL

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of n-hexane, followed by 2 mL of Milli-Q water, 2 mL of methanol:Milli-Q water (1:3)

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and 2 mL of acetonitrile were applied for removing undesired compounds. Target

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compounds were eluted with 1 mL of methanol and dried under nitrogen stream. The


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dried residue of each macroalgae sample was reconstituted with 200 µL of a mixture of

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A/B solvents (90:10, v/v), solvent A being Milli-Q water/0.01% acetic acid and solvent

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B methanol/0.01% acetic acid. Reconstituted extracts were sonicated, filtered through a

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0.45 µm filter (Millipore, Bedford, MA, US) and further injected and analyzed in an

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UHPLC-QqQ-MS/MS apparatus.

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UHPLC-QqQ-MS/MS analysis of free phytoprostanes. Separation of

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phytoprostanes present in macroalgae samples was performed using an UHPLC coupled

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to 6460 QqQ-MS/MS (Agilent Technologies, Waldbronn, Germany), as previously

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described.24 The column used was a 50 x 2.1 mm i.d., 1.7 µm, BEH C18 (Waters,

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Milford, MA, US). The column temperature was 6 ºC. The mobile phases employed

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were solvent A (Milli-Q water/acetic acid (99.99:0.01, v/v)) and solvent B

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(methanol/acetic acid (99.99:0.01, v/v)). The elution was performed at a flow rate of 0.2

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mL/min using the following gradient profile: 60% B at 0 min, 62% B at 2 min, 62.5% B

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at 4 min, reaching 65% B at 8 min, and returning to the initial conditions at 8.01 min.

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The MS analysis was applied in the multiple reaction monitoring (MRM) negative ESI

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mode. ESI conditions and ion optics were as previously described.24 Data acquisition

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and processing were performed using the MassHunter software version B.04.00

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(Agilent Technologies, Waldbronn, Germany). The quantitation of phytoprostanes

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detected in macroalgae was performed using authentic standards of compounds 2, 3, 8

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and 10. The synthetic isoprostane d4-15-F2t-isoprostane (8-isoPGF2α-d4) was used as

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internal standard.

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Statistical Analysis. All the analytical determinations were performed in

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triplicate and the mean values were reported. All values obtained were compared using



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analysis of variance (one-way ANOVA, post hoc Tukey) and unpaired t-test. Pearson

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correlations were calculated according to GraphPad Prism 6 Software, Inc. (San Diego,

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CA, US). One-way ANOVA, unpaired t-test and principal component analysis (PCA)

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were performed using IBM SPSS Statistic for windows Version 22.0 (Armonk, NY,

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US). Differences at p < 0.05 were considered statistically significant.

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RESULTS AND DISCUSSION

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The field of phytoprostanes was opened in 2,000, and currently is still in its infancy. As

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far as we know, our work is the first report of different naturally occurring classes of

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free phytoprostanes in twenty-four macroalgae species belonging to Chlorophyta,

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Phaeophyta and Rhodophyta collected along the west coast of Portugal and from IMTA

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systems. Likewise, this is the first time a correlation between α-linolenic acid levels and

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its autoxidation products was ever tried in algae material.

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Occurrence of α-linolenic acid in macroalgae. Although the fatty acid profiles

226

of some of the target species in this work have already been characterized, intra-specific

227

variability is common in macroalgae coming from different geographical locations

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and/or exposure to diverse abiotic factors, resulting in different PUFA profiles.18 In this

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work we were able to identify and quantitate α-linolenic acid (1) in the twenty-four

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selected macroalgae species (Table 2). The content of this compound ranged between

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ca. 0.7 and 5.6 g/kg of dry algae, C. tamariscifolia and U. lactuca presenting,

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respectively, the lowest and the highest amounts. Indeed, previous studies have reported

233

that members of the order Ulvales presented compound 1 as the characteristic PUFA.17,

234

18

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importance of assessing the occurrence of its autoxidation products, the phytoprostanes.

The presence of compound 1 in all the analyzed species enables and emphasizes the


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Occurrence of free phytoprostanes in macroalgae. The analysis of

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phytoprostanes in natural matrices is extremely challenging, requiring highly sensitive

238

and specific tools for their profiling and characterization.29 Moreover, the great diversity

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granted by the presence of racemic mixtures of phytoprostanes increases the complexity

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of these analyses. In this work, a fast, accurate and robust UHPLC-QqQ-MS/MS

241

method previously developed by Collado-González et al.24 for quantitative and

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qualitative determination of free phytoprostanes in foodstuffs was employed.

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Among the ten available phytoprostane standards, only three were detected in the

244

analyzed macroalgae samples (Table 2). Neither 16-series of F1t-phytoprostanes nor 9-

245

series of D1t-phytoprostanes were detected in the studied species. Phytoprostane identity

246

was confirmed according to their molecular masses, the precursor ions (m/z 327.2 and

247

m/z 307.2), the characteristic MS/MS fragmentation product ions and the corresponding

248

retention times. The mass spectrometric information of the phytoprostanes detected in

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one macroalgae sample (C. tomentosum) is summarized in Figure 2. Compounds 2 and

250

3 (Figure 2A) showed the same transition from the precursor ion at m/z [M-H]− 327.2 to

251

the product ion at m/z 171.2 (Figure 2B and 2C). Thus, their identification was only

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possible by the comparison of their retention times: compound 2 eluted at 1.75 min,

253

while compound 3 eluted at 1.93 min (Figure 2A and 2B). Contrary to prostaglandins,

254

phytoprostanes are non-enzymatically formed as regio- and stereoisomeric mixtures.3

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The analytical conditions employed in this study did not allow the enantiomers

256

separation. Therefore, compounds 8 and 10 (Figure 2A) were identified and quantitated

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according to their specific transitions from the precursor ion m/z [M-H]− 307.2 to the

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product ions m/z [M-H]– 235.2 and m/z [M-H]– 185.2, respectively (Figure 2D and 2E).

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The qualitative profile of phytoprostanes found in the analyzed macroalgae samples

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showed great variability among the three phyla (Chlorophyta, Phaeophyta and



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Rhodophyta) and even between species belonging to the same genus, i.e., compounds 2

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and 3 were detected in F. spiralis, but in F. serratus and F. guiryi none of the studied

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phytoprostanes was identified.

264

In the three studied green macroalgae (C. tomentosum, U. lactuca and Ulva sp.), as well

265

as in two of the red (S. coronopifolius and G. vermiculophylla) and in eight of the

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brown species (C. spongiosus, F. spiralis, L. ochroleuca, P. pavonica, S. latissima, S.

267

polyschides, S. vulgare and S. scoparium), compounds 2 and 3 were identified. Also,

268

compound 8 was detected in four of the brown species (P. pavonica, S. latissima, S.

269

vulgare and S. scoparium). The brown macroalgae B. bifurcata contained only

270

compound 8. None of the screened phytoprostanes were detected in ten macroalgae

271

species (A. armata, C. tamariscifolia, C. usneoides, F. guiryi, F. serratus, Gigartina sp.,

272

H. filicina, P. canaliculata, P. cartilagineum and S. muticum). The species C.

273

spongiosus and C. tomentosum exhibited the higher diversity in phytoprostanes.

274

The determination of phytoprostane levels in macroalgae is of extreme importance,

275

stimulating the exploitation and characterization of new natural dietary sources of these

276

compounds.

277

The amount of phytoprostanes found in the analyzed samples is shown in Table 2. F1t-

278

phytoprostanes were the dominant class determined in this study, while L1-

279

phytoprostanes were only detected in two macroalgae species and at very low levels.

280

Concerning to each phytoprostane class, both compounds 2 and 3 were found in higher

281

concentration in the brown macroalgae S. latissima (ca. 701 and 668 ng/100 g of dry

282

algae, respectively); F. spiralis exhibited the lowest amount of these phytoprostanes (ca.

283

19 and 17 ng/100 g of dry algae, respectively). The sample with the lowest content of

284

compound 8 was S. vulgare (ca. 4 ng/100 g of dry algae), while C. tomentosum was

285

found to have the highest amount (ca. 14 ng/100 g of dry algae). C. spongiosus and C.


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tomentosum were the only samples that presented compound 10 (ca. 5 and 6 ng/100 g of

287

dry algae, respectively).

288

As observed with the qualitative profile, the quantitation of total phytoprostanes in the

289

studied macroalgae species revealed significant variability. The total phytoprostanes

290

content ranged between ca. 6 and 1,381 ng/100 g of dry algae. The macroalgae species

291

showing the highest amount of phytoprostanes (S. latissima) was from IMTA systems.

292

However, no conclusions can be drawn regarding the advantages of IMTA for obtaining

293

higher yields of phytoprostanes, as no marine counterpart of this species was analyzed.

294

To the best of our knowledge, no information has been published before on the content

295

of naturally occurring free phytoprostanes in macroalgae. Therefore, the results obtained

296

can only be compared with those of previous work performed on plant material and

297

other foodstuffs. Karg et al.9 found that either D1 or F1t-phytoprostanes were the

298

dominant classes in vegetable oils, while B1 and L1-phytoprostanes, including their

299

enantiomers, were the minor components.9 More recently, Collado-González et al.24

300

reported that F1t-phytoprostanes were the main class in the green table olive

301

“Manzanilla de Sevilla”.24 Likewise, in macroalgae, F1t-phytoprostanes were the main

302

phytoprostanes detected. This class of compounds was also found to occur in leaves,

303

flowers and roots of taxonomically distinct plant species, ranging from 43 to 1,380 ng/g

304

of dry weight.30 Although we are aware structural differentiation in macroalgae is likely

305

to be accompanied by variation in chemical composition, the present work used full

306

algae individuals in the same stage of development, rendering impossible to conclude

307

about the presence and distribution of phytoprostanes in different morphological parts

308

of the selected species.

309

Although it was theoretically expected to find both 9- and 16-series of F1-

310

phytoprostanes in equal amounts, we were not able to assure this hypothetical relation



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in the studied macroalgae. In fact, previous studies only reported that 9- and 16-series

312

(previously referred as types II and I, respectively) of A1, B1 and E1-phytoprostanes

313

were of equal abundance in different plant tissues.9 Moreover, Imbusch et al.

314

that due to the isomeric complexity of F1-phytoprostanes, identification and

315

quantification of this class of compounds is technically difficult. Therefore, studies

316

reporting the presence of F1-phytoprostanes focused essentially on the differentiation

317

between free and esterified isomers.9, 30-32 The analytical methodology employed in this

318

work was able to differentiate 9-F1-phytoprostane and 16-F1-phytoprostane.24 Likewise,

319

the occurrence of these regioisomers was not of equal proportions: in refined sunflower

320

oil the 9-F1-phytoprostane was found in 44.48 ng/mL while the 16-F1-phytoprostane

321

was found in 24.40 ng/mL of; in 0.8º and 0.4º extra virgin oil 9-F1-phytoprostane were

322

not detected whereas 16-F1-phytoprostane was found in 2.13 ng/mL and 3.70 ng/mL,

323

respectively.24 The reason for these differences is still unclear and more studies are

324

needed to clarify the underlying response mechanisms of individual free phytoprostanes

325

in natural products.

31

stated

326 327

Statistical analysis. Principal Component Analysis (PCA) was performed to

328

determine possible distribution patterns of the identified and quantitated phytoprostanes

329

(2, 3, 8 and 10) in all of the analyzed macroalgae samples. PCA of normalized

330

phytoprostane dataset explained 92.9% of total variations, PC1 accounting for 64.0% of

331

the variance and PC2 for 28.9%. Five groups were distinguished (Figure 3A). One

332

group (G1) includes C. spongiosus and C. tomentosum, the only two macroalgae species

333

in which compound 10 was identified. G2 contains G. vermiculophylla, S.

334

coronopifolius, U. lactuca and Ulva sp., which presented similar amounts of both

335

compounds 2 and 3, while G3 comprises the three macroalgae species that clearly stood


Page 15 of 36


336

out for their high levels in these exact phytoprostanes (S. latissima, S. scoparium and S.

337

polyschides). The low amounts of compounds 2 and 3 in F. spiralis and L. ochroleuca

338

led to the inclusion of these two species in another group (G4), together with A. armata,

339

C. tamariscifolia, C. usneoides, F. guiryi, F. serratus, Gigartina sp., H. filicina, P.

340

canaliculata, P. cartilagineum and S. muticum, in which none of the available

341

phytoprostanes was identified. Finally, B. bifurcata, P. pavonica and S. vulgare were

342

grouped together (G5) due to their similar amounts of compound 8 (Figure 3). The total

343

amount of phytoprostanes is visibly influenced by the levels of both compounds 2 and

344

3, confirming that F1t-phytoprostane is the dominant phytoprostane class in the analyzed

345

samples (Figure 3B).

346

Pearson correlations were calculated in order to establish a potential relationship

347

between the presence of α-linolenic acid (1) in algae material and phytoprostane

348

composition. Our results showed a lack of correlation between the amount of compound

349

1 and total phytoprostane content (r=-0.318). However, the possibility of enzymatic

350

oxidation of compound 1 cannot be ignored. Although this compound is prone to

351

undergo autoxidation reactions, it can also be released from membrane lipids and

352

metabolized by the enzymatic action of lipoxygenases (LOX).33 In fact, several studies

353

have already reported the presence of diverse structurally unique oxylipins from

354

enzymatic routes in macroalgae.34-39 Also recently, Barden et al.40 conducted a clinical

355

trial with healthy volunteers whose diet was supplemented with linseed oil. They

356

detected high levels of phytoprostanes, but they were not able to assess whether

357

phytoprostanes increased due to enhanced concentration of compound 1 or by direct

358

intake from linseed oil.40

359

Altogether, these observations suggest that the large variations observed in

360

phytoprostane composition can be partially explained by intrinsic factors (e.g.,



Page 16 of 36

361

physiological variations within algae organs) and/or extrinsic factors (e.g., geographical

362

origin or area of cultivation, seasonal and environmental variations, time of harvest,

363

water temperature, salinity levels, and processing methods).

364 365

At present, the interest in phytoprostanes comprises two general areas: as biomarkers of

366

oxidative stress in plant-derived foodstuffs and as bioactive mediators with potential

367

benefits in human health. As example, Collado-González et al.41 have recently reported

368

that some phytoprostanes could be considered as early candidate biomarkers of water

369

stress in olive tree.41 Concerning to their biological potential, studies have been shown

370

that certain phytoprostane classes were active in various experimental models. 9, 22, 42, 43

371

However, further studies are required using different natural dietary sources of

372

phytoprostanes to evaluate the real effect of these oxidized lipids on human health.

373

In this regard, our study represents a first approach in the assessment of different

374

naturally occurring classes of free phytoprostanes in macroalgae. The rational

375

exploitation of photosynthetic marine organisms as valuable sources of prominent

376

oxidized lipids is encouraged for future application as substitutes of chemically

377

synthesized oxylipins and as pharmaceuticals and/or nutraceuticals, providing a

378

complementary treatment for chronic diseases, including neurodegenerative and

379

inflammation-related pathologies.

380 381

ABBREVIATIONS USED

382

BHA,

383

(hydroxymethyl)-methane); IMTA, integrated multi-trophic aquaculture; MAPK,

384

mitogen-activated protein kinase; MRM, multiple reaction monitoring; PCA, principal

385

component analysis; ROS, reactive oxygen species; SPE, solid-phase extraction;

butylated

hydroxyanisole;

BIS-TRIS,

bis-(2-Hydroxyethyl)-amino-tris-


Page 17 of 36


386

UHPLC-QqQ-MS/MS,

387

quadrupole-mass spectrometry.

ultra-high

performance

liquid

chromatography-triple-

388 389

FUNDING

390

This work received financial support from the European Union (FEDER funds through

391

COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through

392

project UID/QUI/50006/2013. The work also received financial support from CYTED

393

Programme (Ref. 112RT0460) CORNUCOPIA Thematic Network and projects

394

AGL2011-23690, AGL2013-45922-C2-1-R and AGL2013-45922-C2-2-R (CICYT). To

395

all financing sources the authors are greatly indebted. M. Barbosa and J. Collado-

396

Gonzalez are indebted to FCT and MINECO (Ministerio de Economia y Competitividad

397

Español) for their grants, SFRH/BD/95861/2013 and BES-2011-048401, respectively.

398



399

REFERENCES

400

1.

401 402

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acid B1- and L1-phytoprostanes protect immature neurons from oxidant injury

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types I and II. J. Org. Chem. 2004, 69, 2498-2503.

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M. W.; Hamberg, M. Biologically active oxylipins from seaweeds.

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Jiang, Z.-D.; Gerwick, W. H. Novel oxylipins from the temperate red alga

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cymatherols A− C, polycyclic oxylipins from the marine brown alga Cymathere

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Barden, A. E.; Croft, K. D.; Durand, T.; Guy, A.; Mueller, M. J.; Mori, T. A.

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Flaxseed oil supplementation increases plasma F1-phytoprostanes in healthy

524

men. J. nutr. 2009, 139, 1890-1895.

525

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Durand, T.; Guy, A.; Galano, J.-M.; Ferreres, F.; Torrecillas, A.; Gil-Izquierdo,

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A. Water deficit during pit hardening enhances phytoprostanes content, a plant

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biomarker of oxidative stress, in extra virgin olive oil. J Agric Food Chem. 2015,

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63, 3784-3792.

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Gutermuth, J.; Bewersdorff, M.; Traidl-Hoffmann, C.; Ring, J.; Mueller, M. J.;

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Behrendt, H.; Jakob, T. Immunomodulatory effects of aqueous birch pollen

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extracts and phytoprostanes on primary immune responses in vivo. J. Allergy

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Clin. Immunol. 2007, 120, 293-299.

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43.

Barden, A.; Mas, E.; Henry, P.; Durand, T.; Galano, J.-M.; Roberts, L. J.; Croft,

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K. D.; Mori, T. A. The effects of oxidation products of arachidonic acid and n3

536

fatty acids on vascular and platelet function. Free Radical Res. 2011, 45, 469-

537

476.

538



Page 24 of 36

539

FIGURE CAPTIONS

540

Figure 1. Chemical structures of α-linolenic acid (1) and of the studied phytoprostanes:

541

9-F1t-phytoprostane (2), 9-epi-9-F1t-phytoprostane (3), ent-16-F1t-phytoprostane (4),

542

ent-16-epi-16-F1t-phytoprostane (5), 9-D1t-phytoprostane (6), 9-epi-9-D1t-phytoprostane

543

(7), 16-B1-phytoprostane (8), ent-16-B1-phytoprostane (9), 9-L1-phytoprostane (10) and

544

ent-9-L1-phytoprostane (11).

545 546

Figure 2. (A) Representative UHPLC-QqQ-MS/MS chromatogram of detected

547

phytoprostanes (C. tomentosum) and MRM transitions for quantitation of (B) 9-F1t-

548

phytoprostane (2), (C) 9-epi-9-F1t-phytoprostane (3), (D) 16-B1-phytoprostane (8) and

549

(E) 9-L1-phytoprostane (10).

550 551

Figure 3. Projection of macroalgae (A) (variables: A. armata (AA), B. bifurcata (BB),

552

C. spongiosus (CS), C. tomentosum (Ctom), C. tamariscifolia (CT), C. usneoides (CU),

553

F. guiryi (FG), F. serratus (Fser), F. spiralis (Fspi), Gigartina sp. (Gsp), G.

554

vermiculophylla (GV), H. filicina (HL), L. ochroleuca (LO), P. pavonica (PP), P.

555

canaliculata (PC), P. cartilagineum (Pcart), S. latissima (SL), S. polyschides (SP), S.

556

muticum (SM), S. vulgare (SV), S. coronopfolius (SC), S. scoparium (SS), U. lactuca

557

(UL) and Ulva sp. (Usp) and loadings (B) by phytoprostane composition (variables: 2,

558

3, 8 and 10) into the plane composed by the principal components PC1 and PC2

559

containing 92.9% of the total variance.


Page 25 of 36


Table 1. Characterization of Macroalgae Samples. Phylum Chlorophyta

Species

Origin

Location

Date of collection

Codium tomentosum Stackhouse

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

April 2011

Ulva lactuca Linnaeus

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

August 2011

Ulva sp.

IMTA

N 41°27’11.20, W 8°46’28.29"

July 2012

Asparagopsis armata Harvey

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

June 2010

Gigartina sp.

Praia da Amorosa

N 41°38’51.44, W 8°49’32.53"

December 2013

Gracilaria vermiculophylla (Ohmi) Papenfuss

IMTA

N 41°27’11.20, W 8°46’28.29"

July 2012

Plocamium cartilagineum (Linnaeus) P. S. Dixo

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

July 2012

Sphaerococcus coronopifolius Stackhouse

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

June 2010

Bifurcaria bifurcata R. Ross

Praia da Amorosa

N 41°38’51.44, W 8°49’32.53"

December 2013

Cladostephus spongiosus (Hudson) C. Agardh

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

June 2010

Cystoseira tamariscifolia (Hudson) Papenfuss

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

July 2012

Cystoseira usneoides (Linnaeus) M. Roberts

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

August 2011

Fucus guiryi G. I. Zardi, K. R. Nicastro, E. S. Serrão & G. A. Pearson

Praia da Amorosa

N 41°38’51.44, W 8°49’32.53"

December 2013

Rhodophyta

Phaeophyta



Page 26 of 36

Table 1. Continue. Phylum Phaeophyta

Macroalgae species

Origin

Location

Date of collection

Fucus serratus Linnaeus

Praia da Amorosa

N 41°38’51.44, W 8°49’32.53"

December 2013

Fucus spiralis Linnaeus

Praia da Amorosa

N 41°38’51.44, W 8°49’32.53"

December 2013

Halopteris filicina (Grateloup) Kützing

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

July 2012

Laminaria ochroleuca Bachelot de la Pylaie

Praia da Amorosa

N 41°38’51.44, W 8°49’32.53"

December 2013

Padina pavonica (Linnaeus) Thivy

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

July 2012

Pelvetia canaliculata (Linnaeus) Decaisne & Thuret

Praia do Norte

N 41°41’49.75, W 8°51’3.52"

December 2013

Saccharina latissima (Linnaeus) C. E. Lane, C. Mayes, Druehl & G. W.

IMTA

N 41°27’11.20, W 8°46’28.29"

December 2013

Saccorhiza polyschides (Lightfoot) Batters

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

September 2012

Sargassum muticum (Yendo) Fensholt

Praia da Amorosa

N 41°38’51.44, W 8°49’32.53"

December 2013

Sargassum vulgare C. Agardh

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

August 2011

Stypocaulon scoparium (Linnaeus) Kützing

Praia do Quebrado

N 39°22’0.91, W 9°22’25.86"

June 2010

Saunders


Page 27 of 36


Table 2. α-Linolenic Acid and Phytoprostanes Contents in the Analyzed Macroalgae Species. Phytoprostanes* α-Linolenic acid (g/kg dry algae) 0.96 (0.00)d,e

Compound 2 (ng/100g dry algae) n.d.




Total (ng/100g dry algae) -

B. bifurcata

3.57 (0.04)e,f

n.d.

n.d.

5.68 (1.09)d

n.d.

5.68 (1.09)

C. spongiosus

4.09 (2.77)i

75.25 (3.71)d,e

38.05 (6.57)d

10.53 (2.32)b,c

4.67 (1.17)a

128.49 (6.78)

C. tomentosum

3.77 (0.28)i

22.67 (3.38)e

32.49 (4.45)d

14.42 (2.10)a

6.36 (0.25)a

75.94 (9.22)

C. tamariscifolia

0.69 (0.01)b

n.d.

n.d.

n.d.

n.d.

-

C. usneoides

2.62 (0.05)f

n.d.

n.d.

n.d.

n.d.

-

F. guiryi

5.08 (0.06)b

n.d.

n.d.

n.d.

n.d.

-

F. serratus

4.23 (0.06)c,d

n.d.

n.d.

n.d.

n.d.

-

F. spiralis

3.98 (0.14)d,e

18.61 (3.04)e

17.27 (1.32)d

n.d.

n.d.

35.87 (2.18)

Gigartina sp.

1.66 (0.02)h

n.d.

n.d.

n.d.

n.d.

-

G. vermiculophylla

1.79 (0.03)h

54.46 (6.31)d,e

43.49 (2.14)d

n.d.

n.d.

97.96 (4.91)

H. filicina

3.43 (0.08)f

n.d.

n.d.

n.d.

n.d.

-

L. ochroleuca

3.90 (0.13)d,e

36.42 (6.23)d,e

21.07 (5.54)d

n.d.

n.d.

57.48 (11.69)

P. pavonica

5.03 (0.08)a

26.33 (4.15)d,e

28.91 (3.60)d

6.39 (0.52)d

n.d.

61.36 (7.03)

P. canaliculata

4.40 (0.10)c

n.d.

n.d.

n.d.

n.d.

-

P. cartilagineum

2.63 (0.01)g

n.d.

n.d.

n.d.

n.d.

-

S. latissima

2.01 (0.07)h

700.94 (53.14)a

667.62 (51.34)a

12.33 (0.55)a,b

n.d.

1,380.90 (103.83)

Species A. armata



Page 28 of 36

Table 2. Continue. Phytoprostanes* α-Linolenic acid (g/kg dry algae) 1.10 (0.03)i

Compound 2 (ng/100g dry algae) 413.35 (50.88)b

Compound 3 (ng/100g dry algae) 305.97 (39.56)b



Total (ng/100g dry algae) 719.62 (24.22)

S. muticum

3.94 (0.22)d,e

n.d.

n.d.

n.d.

n.d.

-

S. vulgare

3.62 (0.12)e,f

99.94 (17.63)d

39.54 (9.62)d

4.00 (0.86)d

n.d.

143.48 (26.92)

S. coronopifolius

0.78 (0.01)i

38.10 (4.91)d,e

36.80 (2.44)d

n.d.

n.d.

74.90 (4.93)

S. scoparium

0.80 (0.01)i

334.28 (44.67)c

170.23 (15.97)c

7.43 (0.37)c,d

n.d.

511.94 (52.69)

U. lactuca

5.11 (0.01)b

74.95 (6.10)d,e

55.17 (4.04)d

n.d.

n.d.

130.13 (2.14)

Ulva sp.

3.49 (0.30)f

88.28 (23.82)d,e

64.98 (6.72)d

n.d.

n.d.

153.25 (25.44)

Species S. polyschides

* 9-F1t-phytoprostane (2), 9-epi-9-F1t-phytoprostane (3), 16-B1-phytoprostane (8) and L1-phytoprostane (10). Results are expressed as mean (standard deviation) of three determinations. Not detected (n.d.). Different superscript letters in the same column indicate significant differences (p

Nonenzymatic Î±-Linolenic Acid Derivatives from the Sea: Macroalgae

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