Effect of Food Preparations on In Vitro Bioactivities and Chemical Components of Fucus vesiculosus

Abstract

Fucus vesiculosus is a brown macroalgae used in food and generally considered safe to be consumed, according to EU Directive (EC 258/97). The aim of this study is to analyze the effect of food preparation on F.vesiculosus of different origins on what concerns its chemical constituents and final bioactivities. The aqueous extract of the seaweeds were obtained at different temperatures, similar to food preparation and then purified by SPE. The compound identification was carried out by Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS/MS) and algae extracts microstructure were observed by Scanning Electron Microscopy (SEM). The activities were determined by using antioxidant activity, inhibition of acetylcholinesterase (AChE) and 3-hidroxi-3-methyl-glutaril-CoA (HMG-CoA) reductase (HMGR) together with Caco-2 cells line simulating the intestinal barrier. The activity of AChE and the HMGR were inhibited by the extracts giving IC50 values of 15.0 ± 0.1 µg/mL and 4.2 ± 0.1 µg/mL, respectively and 45% of the cholesterol permeation inhibition. The main compounds identified were phlorotannins and peptides derivatives. The mode of preparation significantly influenced the final bioactivities. Moreover, the in vitro results suggest that the preparation of F. vesiculosus as a soup could have hypercholesterolemia lowering effect.

Full text

foods
Article
Effect of Food Preparations on In Vitro Bioactivities
and Chemical Components of Fucus vesiculosus
Rebeca André1, Laura Guedes 1, Ricardo Melo 2, Lia Ascensão3, Rita Pacheco 1,4,
Pedro D. Vaz 5and Maria Luísa Serralheiro 1,6, *
1BioISI—Biosystems & Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa,
1749-016 Lisboa, Portugal; rebeca.esperanca@gmail.com (R.A.); lrguedes@fc.ul.pt (L.G.);
rpacheco@deq.isel.ipl.pt (R.P.)
2Marine and Environmental Sciences Centre (MARE), Faculdade de Ciências, Universidade de Lisboa,
1749-016 Lisboa, Portugal; ramelo@fc.ul.pt
3Centre for Environmental and Marine Studies (CESAM) Faculdade de Ciências, Universidade de Lisboa,
1749-016 Lisboa, Portugal; lmpsousa@fc.ul.pt
4Department of Chemical Engineering, ISEL—Instituto Superior de Engenharia de Lisboa,
Rua Conselheiro Emídio Navarro, 1, 1959-007 Lisboa, Portugal
5Champalimaud Foundation, Champalimaud Centre for the Unknown, 1400-038 Lisboa, Portugal;
pedro.vaz@fundacaochampalimaud.pt
6Department of Chemistry and Biochemistry, Faculdade de Ciências, Universidade de Lisboa,
Campo Grande, C8 bldg, 1749-016 Lisboa, Portugal
*Correspondence: mlserralheiro@fc.ul.pt; Tel.: +351-21-750-0935
Received: 15 June 2020; Accepted: 15 July 2020; Published: 18 July 2020


Abstract:
Fucus vesiculosus is a brown macroalgae used in food and generally considered safe to be
consumed, according to EU Directive (EC 258/97). The aim of this study is to analyze the effect of
food preparation on F.vesiculosus of different origins on what concerns its chemical constituents and
final bioactivities. The aqueous extract of the seaweeds were obtained at different temperatures,
similar to food preparation and then purified by SPE. The compound identification was carried
out by Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS/MS) and algae
extracts microstructure were observed by Scanning Electron Microscopy (SEM). The activities
were determined by using antioxidant activity, inhibition of acetylcholinesterase (AChE) and
3-hidroxi-3-methyl-glutaril-CoA (HMG-CoA) reductase (HMGR) together with Caco-2 cells line
simulating the intestinal barrier. The activity of AChE and the HMGR were inhibited by the extracts
giving IC
50
values of 15.0
±
0.1
µ
g/mL and 4.2
±
0.1
µ
g/mL, respectively and 45% of the cholesterol
permeation inhibition. The main compounds identified were phlorotannins and peptides derivatives.
The mode of preparation significantly influenced the final bioactivities. Moreover, the
in vitro
results
suggest that the preparation of F. vesiculosus as a soup could have hypercholesterolemia lowering effect.
Keywords:
Fucus vesiculosus; phlorotannins; peptides; acetylcholinesterase; HMG-CoA reductase;
cholesterol; LC-HRMS/MS
1. Introduction
Macroalgae have been used for centuries as a source of food, fuel, feed, and agricultural
fertilizers [
1
]. The consumption of seaweeds has been growing in the past years because people are
looking for a more environmentally friendly and healthier lifestyle, being aware of the influence of diet
on health and well-being [2,3]. The global seaweed industry produces approximately 12 million tons
per annum in volume of which 85 percent comprises food products [4].
Foods 2020,9, 955; doi:10.3390/foods9070955 www.mdpi.com/journal/foods

Foods 2020,9, 955 2 of 20
Fucus vesiculosus L. (Fucaceae), commonly known as bladderwrack, is a brown macroalgae used
as “sea-vegetable” to make tea or in cooked dishes, soups, or sprinkled in salads, despite having
a strong iodine and very salty taste [
5
–
7
]. In traditional medicine this seaweed is administered
orally for different conditions such as weight loss, prevention of atherosclerosis, viscous blood
and hypercholesterolemia, mineral deficit, complaints, arthritis, arthrosis, and as an adjuvant for
menopause [
8
–
11
]. Traditionally it is also administered topically for obesity and arthritis [
8
]. F.
vesiculosus grows in different environmental conditions from saline lagoons to rocky shores and it
is widespread along the coastlines worldwide [
5
]. Its chemical composition depends on the harvest
season, geographic location, and environmental factor such as substrate firmness, exposure to ice
and waves, salinity, wave force, light, or competition between macroalgae [
12
,
13
]. However, it is
essentially constituted by polyphenolic compounds, proteins, minerals, iodine, vitamins, fatty acids,
and non-digestible polysaccharides [7,12].
Phlorotannins, the phenolic compounds high in concentration in brown algae, are highly
hydrophilic, have a molecular size from 234 to more than 100,000 Da [
14
] and are composed of oligomers
of phloroglucinol (1,3,5-Trihydroxybenzene) [
15
]. According to different studies, phlorotannins
reaches 15% of F. vesiculosus dry weight and are characterized by low toxicity and high antioxidant
activity [
16
,
17
]. Phlorotannins have also been described as having
in vitro
anti-cancer, anti-diabetic,
anti-lipidemic, and anti-hypertensive activities, among others [
6
,
18
–
20
] and therefore have been used
as food additives [3].
Recent studies have found that protein hydrolysates from macroalgae extracts contain
peptides showing various
in vitro
activities, such as antimicrobial, antihypertensive, anticoagulant,
and anticancer activity. Some studies have also shown the presence of peptides capable of lowering
plasma cholesterol levels [
18
,
21
] and with antioxidant activity [
22
]. The bioactive peptides found are
usually composed of hydrophobic amino acid residues (aromatic or branched side chain) like histidine,
proline, tyrosine, and tryptophan, which were also associated with high antioxidant activity [2].
The development of seaweed-derived ingredients for cosmetic, pharmaceutical, and food
industries is a growing research area [
23
]. Considering that F. vesiculosus is an edible alga, it will be
studied using different aqueous extracts prepared under distinct conditions similar to food preparations.
The antioxidant activity of the extracts is evaluated, and one of their main components, phlorotannins,
has been reported as having antioxidant activity [
17
]. Antioxidants may neutralize the excessive
formation of reactive species and free radicals inside the cell, contributing to the prevention of cell
damage in different diseases [
24
]. The capacity of the extracts to inhibit acetylcholinesterase (AChE)
(EC 3.1.1.7) enzyme is also studied. The enzyme AChE is located in the synaptic gaps and neuromuscular
junctions and its reversible inhibition is used as an approach to treat gastrointestinal disorders and
Alzheimer’s disease [
25
]. Different studies with phlorotannins derivatives have demonstrated that this
type of compounds has in vitro inhibitory potential against AChE [26,27].
Also, the capacity of the extracts to reduce hypercholesterolemia, one of the major risks to
cardiovascular diseases and the principal cause of mortality in Europe [
28
], is evaluated by inhibition of
3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) (E.C.1.1.1.34), the enzyme that regulates
the biosynthesis of intracellular cholesterol; also the effect on the
in vitro
cholesterol cell permeability
in the intestinal lining is studied. There are still no studies with aqueous extracts of F. vesiculosus, but it
has already been demonstrated the Seapolynol
™
, a commercially available Ecklonia cava extract and the
purified phlorotanin dieckol show inhibitory capacity for HMGR in vitro [20].
In addition, the microstructure of the different extracts is observed by scanning electron microscopy
(SEM) and a comparative analysis of the Liquid-Chromatography-High Resolution Mass Spectrometry
(LC-HRMS/MS) chromatograms of the extracts is done in an attempt to provide a scientific explanation
for the different results of the bioactivities found in the F. vesiculosus extracts from different origins
(ocean, Tagus, and commercial capsules) and under the effect of different food preparations.
The aim of the present study is to evaluate the effect of the seaweed origin and the cooking
processes on the final antioxidant activity, digestion improvement, and cholesterol biosynthesis

Foods 2020,9, 955 3 of 20
and intestinal permeability, as information on the effect of phlorotannins on these items is scarce.
To accomplish these objectives different algae origin, drying processes, and different aqueous extraction
methods are performed, and the described bioactivities are evaluated using the extracts obtained.
2. Materials and Methods
2.1. Chemicals
All chemicals were of analytical grade. Water, methanol (MeOH), formic acid, and acetonitrile
LC-MS grade Optima were purchased from Fisher Scientific (Hampton, USA). Roswell Park
Memorial Institute (RPMI) medium, HBSS (Hanks balanced salt solution), glutamine, Pen-Strep
(penicillin and streptomycin mixture), and FBS (fetal bovine serum) were bought from Lonza
(Verviers, Belgium). Tris(hydroxymethyl)aminomethane (Tris), sodium chloride, and sodium carbonate
were obtained from Merck kGaA (Darmstadt, Germany). Magnesium chloride hexahydrate was
obtained from PanReac (Barcelona, Spain). Acetylcholinesterase (AChE), acetylcholine iodide (AChI),
5-5
0
-Dithiobis (2-nitrobenzoic acid) (DTNB), HMGR assay kit, 2,2-Diphenyl-1-picrylhydrazyl (DPPH),
Folin-Ciocalteu’s phenol reagent, and phloroglucinol
≥
99.0% ( HPLC - High Performance Liquid
Chromatography) were bought from Sigma-Aldrich (Barcelona, Spain).
2.2. Algae Material
Samples of the brown seaweed F. vesiculosus Linnaeus from three different sources were studied:
(1) Harvested during low tide in the Tagus estuary (Lisbon, 15 February, 2018), named here as Tagus
samples; (2) commercially available whole dried algae (imported by Am
é
rico Duarte Paix
ã
o Lda
and commercialized by Celeiro diet™, Lisbon, Portugal, Lot number 03ALG2731901) from the North
Atlantic Ocean, denominated here as ocean samples; and (3) capsules sold in health food stores
as food supplement (sold by Dalipharma
™
, Lot number D325), named here as capsule samples,
label indicates that the capsules contain midrib of F.vesiculosus (83%), tricalcium phosphate, lactose,
and magnesium stearate.
Fresh Tagus fronds were washed with distilled water; the fertile receptacle regions were removed,
and the fronds were sectioned into small pieces before being lyophilized, and then finely pulverized,
and stored at −20 ◦C until analysis. In parallel, other Tagus fronds were dried also in the oven at 180 ◦C.
2.3. Aqueous Extract Preparation
Aqueous extracts were prepared with two extraction conditions: (1) 24 h of incubation with water
at room temperature (25 ◦C); (2) boiling (decoction) for 30 min.
The extract preparation with water at room temperature was carried out; 5 g of the algae powder
was mixed with 100 mL of distilled water, incubated in a platform shaker for 24 h at 200 rpm and at
room temperature. Decoction was prepared using 5 g of the algae powder boiled for 30 min in 100 mL
of distilled water, similar to a soup preparation. Both extracts were centrifuged at 5000
×
gfor 10 min at
4
◦
C. The supernatants were lyophilized and then stored at
−
20
◦
C until analysis. Both extractions
conditions were done with two replicates.
2.4. SPE Purification
The extracts from Tagus and the oceans samples, extracted as decoctions, were purified as described
in [
29
], with some modifications. The extracts were resuspended with water to a concentration of
30 mg/mL and loaded into a Sep-Pak C18 Plus Short Cartridge (360 mg sorbent per cartridge, 55–105
µ
m
particle size, 50/pk [WAT020515]) (Waters, Milford, MA, USA), which had been pre-conditioned with
methanol followed by water, and then washed with water. Phlorotannins were eluted with methanol and
the solvent was evaporated under compressed air. The resulting purified fraction rich in phlorotannins
was resuspended in water and centrifuged in an Eppendorf 5415D equipment, DE (Marshall Scientific,
Hampton, NH, United States) at 3500
×
gfor 10 min. This SPE fraction (eluted with methanol) was

Foods 2020,9, 955 4 of 20
used for LC-HRMS/MS analysis, and for the biological activities under study. The purification process
resulted in a concentration factor of 4.8 and 11.4 for the Tagus and ocean sample, respectively.
2.5. Chemical Analysis by HPLC-DAD and LC-HRMS/MS
The high-performance liquid chromatographic (HPLC) analysis was carried out in an Elite
LaChrom
®
VWR Hitachi liquid chromatograph (Tokyo, Japan) equipped with a Column oven L-2300
and Diode array detector L-2455 (VWR, USA). A column LiChroCART
®
250-4 LiChrospher
®
100 RP-18
(5
µ
m) was used. The extracts in study were analyzed injecting 25
µ
L with an auto injector and using a
gradient composed of 0.05% trifluoroacetic acid (solution A), and acetonitrile (solution B) as follows:
0 min, 100% A; 30 min, 70% A, 30% B; 40 min, 20% A, 80% B; 45 min, 20% A, 80% B; 50 min 70% A,
30% B; 52 min, 100% A; 55 min 100% A. The flow rate was 0.8 mL/min and the detection were carried
out between 200 and 600 nm using a diode-array detector (DAD). The chromatograms were extracted
by representing the highest intensity regardless of the wavelength measured. The quantification of the
cholesterol for the permeation studies was performed as described in [30].
The chromatographic analysis to identify the compounds of the extracts was carried out
by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) using an
Elute OLE UHPLC system interfaced with a quadrupole time-of-flight (QqToF) Impact II mass
spectrometer equipped with an electrospray source (ESI) (Bruker DaltoniK GmbH, Bremen, German).
Chromatography separation was carried out on an Intensity Solo 2 1.8
µ
m C18 100
×
2.1 mm column
(Bruker Daltonics, Bremen, Germany). The mobile phase, the elution conditions, and data acquisition
by mass spectrometry were done as described in [
31
]. The column and the sampler were maintained at
35
◦
C and 10
◦
C, respectively. The mass analysis was carried out in ESI negative and positive mode,
the optimized parameters being:
−
3.5 kV and +4.0 kV; end plate offset, 500 V, nebulizer gas (N
2
)
2.0 bars; dry gas (N
2
), 8 Lmin-1; dry heater, 200
◦
C; collision cell energy was set to 5.0 eV. The internal
calibration was performed with 250 mL H
2
O, 50 mL iPrOH, 750
µ
L acetic acid, 250
µ
L formic acid,
and 0.5 mL 1N NaOH solution on HPC mode. The acquired data were processed by DataAnalysis 4.1
software (Bruker Daltonik GmbH, Bremen, Germany). This identification was carried out by taking
into account the suggestions from the DataAnalysis
®
program version 4.4 from BRUKER and the
Metlin database. Putative structures for the identified compounds were drawn by MassFrag from
Bruker (Bremen, Germany) taking into account the MS/MS fragmentation.
2.6. Total Phenol Content Quantification
Total phenolic compound content was determined spectrophotometrically as described in [
32
].
The absorbance was measured at 760 nm in triplicates. The standard curve (y =0.007 x +0.0061;
R
2
=0.9958) was performed with serial phloroglucinol solutions (0.2–100 ug/mL). The concentration of
the total phenolic compounds was calculated as micrograms of phloroglucinol equivalents (PGE) per
milligrams of dry extract as the mean of three replicates.
2.7. Determination of Antioxidant Activity
Antioxidant activity was measured by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method as described
in [
30
]. To calculate the percentage of antioxidant activity, the following expression was used:
AA (%) =[(A control −A sample)/A control] ×100
In which AA (%) corresponds to the percentage of antioxidant activity, A control refers to the
absorbance of the control sample containing water, and A sample to the absorbance of the different
extract solution.
2.8. Acetylcholinesterase Activity
Acetylcholinesterase enzymatic activity was measured using the method described in [30].

Foods 2020,9, 955 5 of 20
2.9. HMG-CoA Reductase Activity
The inhibition of the enzymatic activity of HMGR was determined by measuring the decrease in
NADPH (Reduced for of Nicotinamide Adenine Dinucleotide Phosphate) absorbance in the presence
of HMG-CoA substrate using the HMG-CoA reductase assay kit (Sigma-Aldrich, Barcelona, Spain)
following the manufacturer’s indications and the process described in [32].
2.10. Permeation Studies
Caco-2 cells (ATCC#HTB37), a human colorectal adenocarcinoma epithelial cell line, were cultured
in RPMI medium supplemented with 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM
L-glutamine, in T25 cell culture flasks at 37
◦
C in an atmosphere with 5% CO
2
. The culture medium
was changed every 48–72 h. The transport and metabolism experiments and the calculations were
performed as described in [
33
]. For these studies, 0.25 mg/mL of ocean sample extract, the methanol
fraction containing the phlorotannins purified by Solid Phase Extraction (SPE), and 5 mM of cholesterol
were used.
2.11. SEM Observations
The microstructure of the different extracts was investigated by SEM as described in [34].
2.12. Statistical Analysis
The results were expressed as average
±
standard deviation and the analysis of variance (ANOVA)
was performed with p=0.05, using the software Microsoft®Excel 2016 (Microsoft Office 365).
3. Results
3.1. Chromatographic Characterization and Phenolic Content Determination According to Several Origins and
Different Extraction Processes
To evaluate the biological activity of F. vesiculosus, that can be used as food in soups or salads
or acquired in capsules as food supplement, extracts obtained from Fucus of different origins and
prepared by several extraction methods were studied. The comparison between all the methods of
extraction and sources of algae was carried out analyzing the chromatographic profile of the compounds
present in the different extracts before and after SPE purification and determining their total phenolic
content. Since algae composition is affected by environmental factors, different sources of F. vesiculosus
were studied. Beyond the different sources of F. vesiculosus, studies were performed using several
conditions of aqueous extractions: extraction with water at 25
◦
C (room temperature) for 24 h and
extraction with water at 100
◦
C for 30 min to simulate a boiling cooking process, followed by a SPE
purification. The samples from the ocean were bought already dry, samples from the Tagus were dried
by lyophilization and in the oven at 180
◦
C, and the capsules consisting mainly of midrib part of the
algae were already dry.
3.1.1. Phenolic Profile Characterization by RP-HPLC-DAD
The different extracts were analyzed by Reverse Phase-High Performance Liquid Chromatography
– Diode Array Detector (RP-HPLC-DAD). Figure 1shows the chromatograms of the different extracts,
all injected in the concentration of 10 mg/mL. As observed from Figure 1a, F. vesiculosus extracted with
water at 100
◦
C gave different chromatographic profiles according to the alga origin. The chromatogram
of ocean sample presented nine peaks, the Tagus sample—eight, the Tagus oven-dried—six, and the
capsules only presented five peaks. The extract of the ocean samples was the one with higher number
and higher intensity of compounds, mainly the peaks with retention time of 8 and 10 min.

Foods 2020,9, 955 6 of 20
Foods 2020, 9, x 6 of 20
Figure 1. RP-HPLC-DAD of aqueous extract (100 µg/mL) from F. vesiculosus ( ) Ocean samples;(
)Tagus samples; ( )capsules; ( )Tagus samples oven-dried. (a): Ocean, Tagus, capsules, and Tagus
oven-dried extracted at 100 °C, 30 min; (b): ocean, Tagus, capsules, and Tagus oven-dried extracted
at 25 °C, 24h; (c): ocean and Tagus samples extracted at 100 °C and purified by SPE.
The capsules (Figure 1a,b) have almost no phenolic compounds or other type of biological
material that can absorb under the Ultraviolet-Visible (UV-Vis) – light.
The chromatographic profile of the different algae extracts with water at room temperature
(Figure 1b) is very similar to the results of the extraction with hot water (Figure 1a). In both situations
the ocean sample showed the highest number of compounds (9) together with the highest intensities.
Comparing both temperatures of extraction for the ocean samples, the peaks at 8 and 10 min have
higher intensities, (268001 mUA and 170,626 mUA at 100 °C and 252,534 mUA and 162,551 mUA at
25 °C, respectively) that is an increase of 6 and 5% of area, respectively, when the extraction is carried
out with boiling water. Therefore, each temperature of extraction leads to similar profiles, but the
extraction at 100 °C produces higher intensities of compounds.
3.1.2. Separation of Bioactive Metabolites by Solid Phase Extraction (SPE)
The chromatographic analyses indicated that there were a few compounds in the extracts that
were able to be absorbed in the UV-VIS region. Compounds, such as polysaccharides, that have high
solubility in water as well as other bioactive compounds co-extracted with phlorotannins, lead to an
extract with compounds that are not absorbed in the UV-Vis region. This may be preventing the
visualization of phenolic compounds in the chromatographic process. Knowing that phenolic
compounds are absorbed in this region of the spectrum, a purification of the matrix extracted with
water was accomplished by using SPE methodology, C18 Sep-Pak cartridge (WatersTM, Milford, MA,
USA) with the ocean and Tagus samples boiled at 100 °C, as their chromatographic profile showed a
higher number of compounds than the capsules or the dried-in-the-oven sample. The objective of this
purification was to separate the phlorotannins and other bioactive compounds from non-phenolic
metabolites [29]. The fraction containing the bioactive molecules was obtained in the methanol
elution step. This process was carried out with the Tagus samples and the ocean samples, extracted
at 100 °C, since these extracts presented higher concentration of compounds. The chromatographic
analysis carried out by RP-HPLC-DAD is shown in Figure 1c. This purification process produced an
increase in the compounds with retention time 8 min of approx. 9 and 11 times, for the ocean and
Tagus extract respectively, while the increase in compounds with retention time 10 min was of
Figure 1.
RP-HPLC-DAD of aqueous extract (100
µ
g/mL) from F. vesiculosus (
Foods 2020, 9, x 6 of 20
Figure 1. RP-HPLC-DAD of aqueous extract (100 µg/mL) from F. vesiculosus ( ) Ocean samples;(
)Tagus samples; ( )capsules; ( )Tagus samples oven-dried. (a): Ocean, Tagus, capsules, and Tagus
oven-dried extracted at 100 °C, 30 min; (b): ocean, Tagus, capsules, and Tagus oven-dried extracted
at 25 °C, 24h; (c): ocean and Tagus samples extracted at 100 °C and purified by SPE.
The capsules (Figure 1a,b) have almost no phenolic compounds or other type of biological
material that can absorb under the Ultraviolet-Visible (UV-Vis) – light.
The chromatographic profile of the different algae extracts with water at room temperature
(Figure 1b) is very similar to the results of the extraction with hot water (Figure 1a). In both situations
the ocean sample showed the highest number of compounds (9) together with the highest intensities.
Comparing both temperatures of extraction for the ocean samples, the peaks at 8 and 10 min have
higher intensities, (268001 mUA and 170,626 mUA at 100 °C and 252,534 mUA and 162,551 mUA at
25 °C, respectively) that is an increase of 6 and 5% of area, respectively, when the extraction is carried
out with boiling water. Therefore, each temperature of extraction leads to similar profiles, but the
extraction at 100 °C produces higher intensities of compounds.
3.1.2. Separation of Bioactive Metabolites by Solid Phase Extraction (SPE)
The chromatographic analyses indicated that there were a few compounds in the extracts that
were able to be absorbed in the UV-VIS region. Compounds, such as polysaccharides, that have high
solubility in water as well as other bioactive compounds co-extracted with phlorotannins, lead to an
extract with compounds that are not absorbed in the UV-Vis region. This may be preventing the
visualization of phenolic compounds in the chromatographic process. Knowing that phenolic
compounds are absorbed in this region of the spectrum, a purification of the matrix extracted with
water was accomplished by using SPE methodology, C18 Sep-Pak cartridge (WatersTM, Milford, MA,
USA) with the ocean and Tagus samples boiled at 100 °C, as their chromatographic profile showed a
higher number of compounds than the capsules or the dried-in-the-oven sample. The objective of this
purification was to separate the phlorotannins and other bioactive compounds from non-phenolic
metabolites [29]. The fraction containing the bioactive molecules was obtained in the methanol
elution step. This process was carried out with the Tagus samples and the ocean samples, extracted
at 100 °C, since these extracts presented higher concentration of compounds. The chromatographic
analysis carried out by RP-HPLC-DAD is shown in Figure 1c. This purification process produced an
increase in the compounds with retention time 8 min of approx. 9 and 11 times, for the ocean and
Tagus extract respectively, while the increase in compounds with retention time 10 min was of
) Ocean samples; (
Foods 2020, 9, x 6 of 20
Figure 1. RP-HPLC-DAD of aqueous extract (100 µg/mL) from F. vesiculosus ( ) Ocean samples;(
)Tagus samples; ( )capsules; ( )Tagus samples oven-dried. (a): Ocean, Tagus, capsules, and Tagus
oven-dried extracted at 100 °C, 30 min; (b): ocean, Tagus, capsules, and Tagus oven-dried extracted
at 25 °C, 24h; (c): ocean and Tagus samples extracted at 100 °C and purified by SPE.
The capsules (Figure 1a,b) have almost no phenolic compounds or other type of biological
material that can absorb under the Ultraviolet-Visible (UV-Vis) – light.
The chromatographic profile of the different algae extracts with water at room temperature
(Figure 1b) is very similar to the results of the extraction with hot water (Figure 1a). In both situations
the ocean sample showed the highest number of compounds (9) together with the highest intensities.
Comparing both temperatures of extraction for the ocean samples, the peaks at 8 and 10 min have
higher intensities, (268001 mUA and 170,626 mUA at 100 °C and 252,534 mUA and 162,551 mUA at
25 °C, respectively) that is an increase of 6 and 5% of area, respectively, when the extraction is carried
out with boiling water. Therefore, each temperature of extraction leads to similar profiles, but the
extraction at 100 °C produces higher intensities of compounds.
3.1.2. Separation of Bioactive Metabolites by Solid Phase Extraction (SPE)
The chromatographic analyses indicated that there were a few compounds in the extracts that
were able to be absorbed in the UV-VIS region. Compounds, such as polysaccharides, that have high
solubility in water as well as other bioactive compounds co-extracted with phlorotannins, lead to an
extract with compounds that are not absorbed in the UV-Vis region. This may be preventing the
visualization of phenolic compounds in the chromatographic process. Knowing that phenolic
compounds are absorbed in this region of the spectrum, a purification of the matrix extracted with
water was accomplished by using SPE methodology, C18 Sep-Pak cartridge (WatersTM, Milford, MA,
USA) with the ocean and Tagus samples boiled at 100 °C, as their chromatographic profile showed a
higher number of compounds than the capsules or the dried-in-the-oven sample. The objective of this
purification was to separate the phlorotannins and other bioactive compounds from non-phenolic
metabolites [29]. The fraction containing the bioactive molecules was obtained in the methanol
elution step. This process was carried out with the Tagus samples and the ocean samples, extracted
at 100 °C, since these extracts presented higher concentration of compounds. The chromatographic
analysis carried out by RP-HPLC-DAD is shown in Figure 1c. This purification process produced an
increase in the compounds with retention time 8 min of approx. 9 and 11 times, for the ocean and
Tagus extract respectively, while the increase in compounds with retention time 10 min was of
)
Tagus samples; (
Foods 2020, 9, x 6 of 20
Figure 1. RP-HPLC-DAD of aqueous extract (100 µg/mL) from F. vesiculosus ( ) Ocean samples;(
)Tagus samples; ( )capsules; ( )Tagus samples oven-dried. (a): Ocean, Tagus, capsules, and Tagus
oven-dried extracted at 100 °C, 30 min; (b): ocean, Tagus, capsules, and Tagus oven-dried extracted
at 25 °C, 24h; (c): ocean and Tagus samples extracted at 100 °C and purified by SPE.
The capsules (Figure 1a,b) have almost no phenolic compounds or other type of biological
material that can absorb under the Ultraviolet-Visible (UV-Vis) – light.
The chromatographic profile of the different algae extracts with water at room temperature
(Figure 1b) is very similar to the results of the extraction with hot water (Figure 1a). In both situations
the ocean sample showed the highest number of compounds (9) together with the highest intensities.
Comparing both temperatures of extraction for the ocean samples, the peaks at 8 and 10 min have
higher intensities, (268001 mUA and 170,626 mUA at 100 °C and 252,534 mUA and 162,551 mUA at
25 °C, respectively) that is an increase of 6 and 5% of area, respectively, when the extraction is carried
out with boiling water. Therefore, each temperature of extraction leads to similar profiles, but the
extraction at 100 °C produces higher intensities of compounds.
3.1.2. Separation of Bioactive Metabolites by Solid Phase Extraction (SPE)
The chromatographic analyses indicated that there were a few compounds in the extracts that
were able to be absorbed in the UV-VIS region. Compounds, such as polysaccharides, that have high
solubility in water as well as other bioactive compounds co-extracted with phlorotannins, lead to an
extract with compounds that are not absorbed in the UV-Vis region. This may be preventing the
visualization of phenolic compounds in the chromatographic process. Knowing that phenolic
compounds are absorbed in this region of the spectrum, a purification of the matrix extracted with
water was accomplished by using SPE methodology, C18 Sep-Pak cartridge (WatersTM, Milford, MA,
USA) with the ocean and Tagus samples boiled at 100 °C, as their chromatographic profile showed a
higher number of compounds than the capsules or the dried-in-the-oven sample. The objective of this
purification was to separate the phlorotannins and other bioactive compounds from non-phenolic
metabolites [29]. The fraction containing the bioactive molecules was obtained in the methanol
elution step. This process was carried out with the Tagus samples and the ocean samples, extracted
at 100 °C, since these extracts presented higher concentration of compounds. The chromatographic
analysis carried out by RP-HPLC-DAD is shown in Figure 1c. This purification process produced an
increase in the compounds with retention time 8 min of approx. 9 and 11 times, for the ocean and
Tagus extract respectively, while the increase in compounds with retention time 10 min was of
) capsules; (
Foods 2020, 9, x 6 of 20
Figure 1. RP-HPLC-DAD of aqueous extract (100 µg/mL) from F. vesiculosus ( ) Ocean samples;(
)Tagus samples; ( )capsules; ( )Tagus samples oven-dried. (a): Ocean, Tagus, capsules, and Tagus
oven-dried extracted at 100 °C, 30 min; (b): ocean, Tagus, capsules, and Tagus oven-dried extracted
at 25 °C, 24h; (c): ocean and Tagus samples extracted at 100 °C and purified by SPE.
The capsules (Figure 1a,b) have almost no phenolic compounds or other type of biological
material that can absorb under the Ultraviolet-Visible (UV-Vis) – light.
The chromatographic profile of the different algae extracts with water at room temperature
(Figure 1b) is very similar to the results of the extraction with hot water (Figure 1a). In both situations
the ocean sample showed the highest number of compounds (9) together with the highest intensities.
Comparing both temperatures of extraction for the ocean samples, the peaks at 8 and 10 min have
higher intensities, (268001 mUA and 170,626 mUA at 100 °C and 252,534 mUA and 162,551 mUA at
25 °C, respectively) that is an increase of 6 and 5% of area, respectively, when the extraction is carried
out with boiling water. Therefore, each temperature of extraction leads to similar profiles, but the
extraction at 100 °C produces higher intensities of compounds.
3.1.2. Separation of Bioactive Metabolites by Solid Phase Extraction (SPE)
The chromatographic analyses indicated that there were a few compounds in the extracts that
were able to be absorbed in the UV-VIS region. Compounds, such as polysaccharides, that have high
solubility in water as well as other bioactive compounds co-extracted with phlorotannins, lead to an
extract with compounds that are not absorbed in the UV-Vis region. This may be preventing the
visualization of phenolic compounds in the chromatographic process. Knowing that phenolic
compounds are absorbed in this region of the spectrum, a purification of the matrix extracted with
water was accomplished by using SPE methodology, C18 Sep-Pak cartridge (WatersTM, Milford, MA,
USA) with the ocean and Tagus samples boiled at 100 °C, as their chromatographic profile showed a
higher number of compounds than the capsules or the dried-in-the-oven sample. The objective of this
purification was to separate the phlorotannins and other bioactive compounds from non-phenolic
metabolites [29]. The fraction containing the bioactive molecules was obtained in the methanol
elution step. This process was carried out with the Tagus samples and the ocean samples, extracted
at 100 °C, since these extracts presented higher concentration of compounds. The chromatographic
analysis carried out by RP-HPLC-DAD is shown in Figure 1c. This purification process produced an
increase in the compounds with retention time 8 min of approx. 9 and 11 times, for the ocean and
Tagus extract respectively, while the increase in compounds with retention time 10 min was of
) Tagus samples oven-dried. (
a
): Ocean, Tagus, capsules, and Tagus
oven-dried extracted at 100
◦
C, 30 min; (
b
): ocean, Tagus, capsules, and Tagus oven-dried extracted at
25 ◦C, 24 h; (c): ocean and Tagus samples extracted at 100 ◦C and purified by SPE.
The capsules (Figure 1a,b) have almost no phenolic compounds or other type of biological material
that can absorb under the Ultraviolet-Visible (UV-Vis) – light.
The chromatographic profile of the different algae extracts with water at room temperature
(Figure 1b) is very similar to the results of the extraction with hot water (Figure 1a). In both situations
the ocean sample showed the highest number of compounds (9) together with the highest intensities.
Comparing both temperatures of extraction for the ocean samples, the peaks at 8 and 10 min have
higher intensities, (268001 mUA and 170,626 mUA at 100
◦
C and 252,534 mUA and 162,551 mUA at
25
◦
C, respectively) that is an increase of 6 and 5% of area, respectively, when the extraction is carried
out with boiling water. Therefore, each temperature of extraction leads to similar profiles, but the
extraction at 100 ◦C produces higher intensities of compounds.
3.1.2. Separation of Bioactive Metabolites by Solid Phase Extraction (SPE)
The chromatographic analyses indicated that there were a few compounds in the extracts that
were able to be absorbed in the UV-VIS region. Compounds, such as polysaccharides, that have high
solubility in water as well as other bioactive compounds co-extracted with phlorotannins, lead to
an extract with compounds that are not absorbed in the UV-Vis region. This may be preventing
the visualization of phenolic compounds in the chromatographic process. Knowing that phenolic
compounds are absorbed in this region of the spectrum, a purification of the matrix extracted with
water was accomplished by using SPE methodology, C18 Sep-Pak cartridge (Waters
TM
, Milford, MA,
USA) with the ocean and Tagus samples boiled at 100
◦
C, as their chromatographic profile showed
a higher number of compounds than the capsules or the dried-in-the-oven sample. The objective of
this purification was to separate the phlorotannins and other bioactive compounds from non-phenolic
metabolites [
29
]. The fraction containing the bioactive molecules was obtained in the methanol elution
step. This process was carried out with the Tagus samples and the ocean samples, extracted at 100
◦
C,
since these extracts presented higher concentration of compounds. The chromatographic analysis
carried out by RP-HPLC-DAD is shown in Figure 1c. This purification process produced an increase in
the compounds with retention time 8 min of approx. 9 and 11 times, for the ocean and Tagus extract

Foods 2020,9, 955 7 of 20
respectively, while the increase in compounds with retention time 10 min was of approx. 9 times,
for both the ocean and Tagus extract. This indicated an increase in the compounds absorbing in the
UV-Vis region with the SPE purification.
3.1.3. Total Phenolic Content
The effect of the extraction temperature on the total phenol content of ocean samples, Tagus samples,
and capsules can be observed in Figure 2. One possible explanation for the different amount of total
phenol content is the geographic location where the algae were collected, as all the procedures were
identical for both ocean and Tagus samples.
Foods 2020, 9, x 7 of 20
approx. 9 times, for both the ocean and Tagus extract. This indicated an increase in the compounds
absorbing in the UV-Vis region with the SPE purification.
3.1.3. Total Phenolic Content
The effect of the extraction temperature on the total phenol content of ocean samples, Tagus
samples, and capsules can be observed in Figure 2. One possible explanation for the different amount
of total phenol content is the geographic location where the algae were collected, as all the procedures
were identical for both ocean and Tagus samples.
Figure 2. Total phenol content of Fucus vesiculosus extracts obtained by different aqueous extraction
processes. Total phenolic content (µg of PGE/mg of extract); different superscript letters (a,b)
correspond to values in the same extraction condition that can be considered statistically different (p
≤ 0.05).( )H2O 25 °C; ( )H2O 100 °C; ( )SPE purification (H2O 100 °C).
The aqueous extracts from the ocean samples presented a higher concentration of µg of
phloroglucinol equivalents (PGE)/mg of extract. Comparing with the Tagus samples, the ocean
samples had 2–3 times more phloroglucinol equivalents, 45.6 ± 3.8 and 40.2 ± 2.1 µg PGE/mg Ext,
while the Tagus samples present 15.0 ± 1.2 and 17.1 ± 2.1 µg PGE/mg Ext, with extraction at 25 °C,
and 100 °C, respectively (Figure 2). For the ocean samples, with the SPE purification, the total phenol
content increased from 48.8 ± 2.4 to 322.39 ug PGE equivalents/mg of extract. While for the Tagus
samples it increased from 21.8 ± 2.5 to 104.7 ± 4.1 µg PGE equivalents/mg of extract (Figure 2).
The results showed that the process of drying in oven leads, approximately, to losses of 25–45%
on the total phenolic content 11.1 ± 1.4 and 9.2 ± 1.2 µg PGE/mg Ext with extraction at 25 °C and 100
°C, respectively. The capsules F. vesiculosus have lower concentration of PGE equivalents 6.1 ± 0.7 and
0.7 ± 0.3 µg PGE/mg Ext with extraction at 25 °C, and 100 °C, respectively (Figure 2). These results
agree with the expected, as the capsules, used as a food supplement, are essentially constituted by
fibers.
3.1.4. SEM Observations of F. vesiculosus Particles Obtained by Different Drying Methods and
Extraction Processes
In an attempt to see if the different drying and extraction processes could be reflected into
different microscopic changes, SEM analysis was carried out. The analysis was performed with the
Tagus samples dried by lyophilization or in the oven before and after the various water extraction
processes (Figure 3) and with the aqueous extracts of the ocean algae and capsules obtained at 100 °C
(Figure 4).
Figure 2.
Total phenol content of Fucus vesiculosus extracts obtained by different aqueous extraction
processes. Total phenolic content (
µ
g of PGE/mg of extract); different superscript letters (
a
,
b
) correspond
to values in the same extraction condition that can be considered statistically different (p
≤
0.05). (
Foods 2020, 9, x 7 of 20
approx. 9 times, for both the ocean and Tagus extract. This indicated an increase in the compounds
absorbing in the UV-Vis region with the SPE purification.
3.1.3. Total Phenolic Content
The effect of the extraction temperature on the total phenol content of ocean samples, Tagus
samples, and capsules can be observed in Figure 2. One possible explanation for the different amount
of total phenol content is the geographic location where the algae were collected, as all the procedures
were identical for both ocean and Tagus samples.
Figure 2. Total phenol content of Fucus vesiculosus extracts obtained by different aqueous extraction
processes. Total phenolic content (µg of PGE/mg of extract); different superscript letters (a,b)
correspond to values in the same extraction condition that can be considered statistically different (p
≤ 0.05).( )H2O 25 °C; ( )H2O 100 °C; ( )SPE purification (H2O 100 °C).
The aqueous extracts from the ocean samples presented a higher concentration of µg of
phloroglucinol equivalents (PGE)/mg of extract. Comparing with the Tagus samples, the ocean
samples had 2–3 times more phloroglucinol equivalents, 45.6 ± 3.8 and 40.2 ± 2.1 µg PGE/mg Ext,
while the Tagus samples present 15.0 ± 1.2 and 17.1 ± 2.1 µg PGE/mg Ext, with extraction at 25 °C,
and 100 °C, respectively (Figure 2). For the ocean samples, with the SPE purification, the total phenol
content increased from 48.8 ± 2.4 to 322.39 ug PGE equivalents/mg of extract. While for the Tagus
samples it increased from 21.8 ± 2.5 to 104.7 ± 4.1 µg PGE equivalents/mg of extract (Figure 2).
The results showed that the process of drying in oven leads, approximately, to losses of 25–45%
on the total phenolic content 11.1 ± 1.4 and 9.2 ± 1.2 µg PGE/mg Ext with extraction at 25 °C and 100
°C, respectively. The capsules F. vesiculosus have lower concentration of PGE equivalents 6.1 ± 0.7 and
0.7 ± 0.3 µg PGE/mg Ext with extraction at 25 °C, and 100 °C, respectively (Figure 2). These results
agree with the expected, as the capsules, used as a food supplement, are essentially constituted by
fibers.
3.1.4. SEM Observations of F. vesiculosus Particles Obtained by Different Drying Methods and
Extraction Processes
In an attempt to see if the different drying and extraction processes could be reflected into
different microscopic changes, SEM analysis was carried out. The analysis was performed with the
Tagus samples dried by lyophilization or in the oven before and after the various water extraction
processes (Figure 3) and with the aqueous extracts of the ocean algae and capsules obtained at 100 °C
(Figure 4).
)
H2O 25 ◦C; (
Foods 2020, 9, x 10 of 20
approximately 60 ug/mL, value similar to those obtained in infusions from medicinal plants [30].
Capsules, as well as the Tagus samples dried in the oven showed a very low antioxidant activity,
Figure 5.
Figure 5. Percentage of antioxidant activity of F. vesiculosus extracts (100 µg/mL of extract). Different
superscript letters (a–c) correspond to values in the same extraction condition that can be considered
statistically different (p ≤ 0.05).( )H2O 25 °C; ( )H2O 100 °C; ( )SPE purification
(H2O 100 °C).
3.2.2. Acetylcholinesterase Activity
In this study, only the extracts with higher antioxidant activity were included, but the
comparisons were carried out without the purification of the extracts by SPE. The results obtained,
summarized in Table 1, showed that the AChE inhibitory activity at 1 mg/mL of the ocean samples
was approximately 20 times higher than that presented by the Tagus samples. After the SPE
purification the capacity to inhibit the AChE was higher for both extracts compared to the initial
aqueous extracts under study. The purified ocean samples extract presented an IC50 of 14.97 µg/mL,
while the IC50 of the purified Tagus extract is almost 56 times higher (840.85 µg/mL), meaning lower
activity (Table 1). Comparing with other type of aqueous extracts like those obtained from medicinal
plants, these aqueous extracts are much more active than most of the extracts containing also phenolic
compounds [25,30]. Nevertheless, both extracts are far less potent than the standard galantamine that
has an IC50 of 0.14 µg/mL [35], which means that when consumed it will not affect strongly the
intestinal motility as the drug might do [37].
Table 1. Enzyme inhibitory activity of acetylcholinesterase and HMGR of the ocean and Tagus
samples aqueous extracts (100 °C, 30 min) and after SPE purified extract.
Extract
Acetylcholinesterase
Inhibition HMGR * Inhibition Inhibition of Cholesterol
Permeation (%)
1000 µg/mL IC50 (µg/mL) 10 µg/mL IC50
(µg/mL)
Ocean samples 89.96% ±
0.54 a
210.86 ±
22.11 a
27.74% ±
1.60 a - -
Ocean samples–SPE
fraction - 14.97 ± 0.13 b 86.41% ±
3.20 b 4.16 ± 0.11 45.3 ± 4.4
Tagus samples 4.199% ±
0.36 b - 1.07% ± 0.65
c- -
Tagus samples–SPE
fraction
60.44% ±
1.21c
849.80 ±
10.24 c
52.95% ±
3.17 d - -
* 3-hidroxi-3-methyl-glutaril-CoA reductase; Superscript letters (a–d) correspond to values in the same
column that can be considered statistically different (p = 0.05)
3.2.3. HMG-CoA Reductase Inhibitory Activity
) H2O 100 ◦C; (
Foods 2020, 9, x 10 of 20
approximately 60 ug/mL, value similar to those obtained in infusions from medicinal plants [30].
Capsules, as well as the Tagus samples dried in the oven showed a very low antioxidant activity,
Figure 5.
Figure 5. Percentage of antioxidant activity of F. vesiculosus extracts (100 µg/mL of extract). Different
superscript letters (a–c) correspond to values in the same extraction condition that can be considered
statistically different (p ≤ 0.05).( )H2O 25 °C; ( )H2O 100 °C; ( )SPE purification
(H2O 100 °C).
3.2.2. Acetylcholinesterase Activity
In this study, only the extracts with higher antioxidant activity were included, but the
comparisons were carried out without the purification of the extracts by SPE. The results obtained,
summarized in Table 1, showed that the AChE inhibitory activity at 1 mg/mL of the ocean samples
was approximately 20 times higher than that presented by the Tagus samples. After the SPE
purification the capacity to inhibit the AChE was higher for both extracts compared to the initial
aqueous extracts under study. The purified ocean samples extract presented an IC50 of 14.97 µg/mL,
while the IC50 of the purified Tagus extract is almost 56 times higher (840.85 µg/mL), meaning lower
activity (Table 1). Comparing with other type of aqueous extracts like those obtained from medicinal
plants, these aqueous extracts are much more active than most of the extracts containing also phenolic
compounds [25,30]. Nevertheless, both extracts are far less potent than the standard galantamine that
has an IC50 of 0.14 µg/mL [35], which means that when consumed it will not affect strongly the
intestinal motility as the drug might do [37].
Table 1. Enzyme inhibitory activity of acetylcholinesterase and HMGR of the ocean and Tagus
samples aqueous extracts (100 °C, 30 min) and after SPE purified extract.
Extract
Acetylcholinesterase
Inhibition HMGR * Inhibition Inhibition of Cholesterol
Permeation (%)
1000 µg/mL IC50 (µg/mL) 10 µg/mL IC50
(µg/mL)
Ocean samples 89.96% ±
0.54 a
210.86 ±
22.11 a
27.74% ±
1.60 a - -
Ocean samples–SPE
fraction - 14.97 ± 0.13 b 86.41% ±
3.20 b 4.16 ± 0.11 45.3 ± 4.4
Tagus samples 4.199% ±
0.36 b - 1.07% ± 0.65
c- -
Tagus samples–SPE
fraction
60.44% ±
1.21c
849.80 ±
10.24 c
52.95% ±
3.17 d - -
* 3-hidroxi-3-methyl-glutaril-CoA reductase; Superscript letters (a–d) correspond to values in the same
column that can be considered statistically different (p = 0.05)
3.2.3. HMG-CoA Reductase Inhibitory Activity
) SPE purification (H2O 100 ◦C).
The aqueous extracts from the ocean samples presented a higher concentration of
µ
g of
phloroglucinol equivalents (PGE)/mg of extract. Comparing with the Tagus samples, the ocean
samples had 2–3 times more phloroglucinol equivalents, 45.6
±
3.8 and 40.2
±
2.1
µ
g PGE/mg Ext,
while the Tagus samples present 15.0
±
1.2 and 17.1
±
2.1
µ
g PGE/mg Ext, with extraction at 25
◦
C,
and 100
◦
C, respectively (Figure 2). For the ocean samples, with the SPE purification, the total phenol
content increased from 48.8
±
2.4 to 322.39 ug PGE equivalents/mg of extract. While for the Tagus
samples it increased from 21.8 ±2.5 to 104.7 ±4.1 µg PGE equivalents/mg of extract (Figure 2).
The results showed that the process of drying in oven leads, approximately, to losses of 25–45% on
the total phenolic content 11.1
±
1.4 and 9.2
±
1.2
µ
g PGE/mg Ext with extraction at 25
◦
C and 100
◦
C,
respectively. The capsules F. vesiculosus have lower concentration of PGE equivalents 6.1
±
0.7 and
0.7
±
0.3
µ
g PGE/mg Ext with extraction at 25
◦
C, and 100
◦
C, respectively (Figure 2). These results agree
with the expected, as the capsules, used as a food supplement, are essentially constituted by fibers.
3.1.4. SEM Observations of F. vesiculosus Particles Obtained by Different Drying Methods and
Extraction Processes
In an attempt to see if the different drying and extraction processes could be reflected into different
microscopic changes, SEM analysis was carried out. The analysis was performed with the Tagus
samples dried by lyophilization or in the oven before and after the various water extraction processes
(Figure 3) and with the aqueous extracts of the ocean algae and capsules obtained at 100
◦
C (Figure 4).

Foods 2020,9, 955 8 of 20
Foods 2020, 9, x 8 of 20
Figure 3. Scanning electron micrographs of the dried particles of Tagus algae obtained by different
drying and extraction processes. (a), Lyophilized algae. (b), Dried in the oven (180 °C, 30 min). (c),
Lyophilized and extracted at 25 °C. (d), Dried in the oven and extracted at 25 °C. (e), Lyophilized and
extracted at 100 °C. (f), Dried in the oven and extracted at 100 °C. Scale bars = 5 µm.
The observations show that the microstructure of the two samples of dried algae, lyophilized,
and dried in the oven, were different (Figure 3a,b), as well as the respective extracts obtained at 25
°C (Figure 3d) and 100 °C (Figure 3e,f). In both cases, the extracts of the algae lyophilized or dried in
the oven exhibit groups of cubic and parallelepiped-shaped crystals, but in the aqueous extracts of
the lyophilized algae they seem to be embedded in an amorphous matrix (Figure 3c,d).
Figure 3.
Scanning electron micrographs of the dried particles of Tagus algae obtained by different
drying and extraction processes. (
a
), Lyophilized algae. (
b
), Dried in the oven (180
◦
C, 30 min).
(
c
), Lyophilized and extracted at 25
◦
C. (
d
), Dried in the oven and extracted at 25
◦
C. (
e
), Lyophilized
and extracted at 100 ◦C. (f), Dried in the oven and extracted at 100 ◦C. Scale bars =5µm.
The observations show that the microstructure of the two samples of dried algae, lyophilized,
and dried in the oven, were different (Figure 3a,b), as well as the respective extracts obtained at 25
◦
C
(Figure 3c,d) and 100
◦
C (Figure 3e,f). In both cases, the extracts of the algae lyophilized or dried in the
oven exhibit groups of cubic and parallelepiped-shaped crystals, but in the aqueous extracts of the
lyophilized algae they seem to be embedded in an amorphous matrix (Figure 3c,d).

Foods 2020,9, 955 9 of 20
Foods 2020, 9, x 9 of 20
Figure 4. Scanning electron micrographs of the dried particles from the ocean algae and capsules
extracts obtained at 100 °C. (a,b), Ocean samples extract microstructure. (c,d), Capsules extract
microstructure. Scale bars = 10 µm (a,c); 5 µm (b,d).
The water extract obtained from the ocean samples at 100 °C, seem to be essentially constituted
by large aggregates of cubic and parallelepiped-shaped crystals of diverse sizes (Figure 4a,b), while
the capsules extract obtained at the same conditions appeared as a fibrous framework formed by
rosette aggregates made by irregular long-shaped particles of different sizes (Figure 4c,d).
In the current study it was observed that the different drying processes as well as the several
extraction methods, although giving identical brownish powder, showed different microstructures.
3.2. Biological Activities
Biological activities of the extracts obtained at 100 °C without and with SPE purification were
studied by determining its antioxidant activity, measured as scavenging radical capacity,
acetylcholinesterase inhibitory activity and the capacity for reducing the cholesterol biosynthesis, as
well as the inhibition of cholesterol intestinal permeation. The antioxidant activity was also evaluated
for the two temperatures of extraction used.
3.2.1. Free Radical Scavenging Capacity
The antioxidant activity for the different extracts studied was determined using the DPPH
scavenging activity. Antioxidant activity reflects the ability to eliminate free radicals that have the
capacity to damage cellular constituents, such as proteins, DNA (Deoxyribonucleic acid), and lipids
[34]. In this study all extracts were used at a concentration of 0.1 mg/mL.
Analyzing the effect of the temperature extraction on the antioxidant activity (Figure 4), the extraction
at 100 °C leads to a higher activity, except for the capsules. The ocean samples extracted at 100 °C and
purified by SPE was the extract with a higher antioxidant activity (76.54% ± 0.17) followed by the
Tagus samples extracted with the same conditions (41.98% ± 0.55). The results present in Figure 4
demonstrate that although the antioxidant activity even in the best situation is lower than the
standard like butylated hydroxytoluene (BHT), whose IC50 is 15.7 ug/mL [35,36], it would be
Figure 4.
Scanning electron micrographs of the dried particles from the ocean algae and capsules
extracts obtained at 100
◦
C. (
a
,
b
), Ocean samples extract microstructure. (
c
,
d
), Capsules extract
microstructure. Scale bars =10 µm (a,c); 5 µm (b,d).
The water extract obtained from the ocean samples at 100
◦
C, seem to be essentially constituted
by large aggregates of cubic and parallelepiped-shaped crystals of diverse sizes (Figure 4a,b), while the
capsules extract obtained at the same conditions appeared as a fibrous framework formed by rosette
aggregates made by irregular long-shaped particles of different sizes (Figure 4c,d).
In the current study it was observed that the different drying processes as well as the several
extraction methods, although giving identical brownish powder, showed different microstructures.
3.2. Biological Activities
Biological activities of the extracts obtained at 100
◦
C without and with SPE purification
were studied by determining its antioxidant activity, measured as scavenging radical capacity,
acetylcholinesterase inhibitory activity and the capacity for reducing the cholesterol biosynthesis,
as well as the inhibition of cholesterol intestinal permeation. The antioxidant activity was also evaluated
for the two temperatures of extraction used.
3.2.1. Free Radical Scavenging Capacity
The antioxidant activity for the different extracts studied was determined using the DPPH
scavenging activity. Antioxidant activity reflects the ability to eliminate free radicals that have the
capacity to damage cellular constituents, such as proteins, DNA (Deoxyribonucleic acid), and lipids [
34
].
In this study all extracts were used at a concentration of 0.1 mg/mL.

Foods 2020,9, 955 10 of 20
Analyzing the effect of the temperature extraction on the antioxidant activity (Figure 4),
the extraction at 100
◦
C leads to a higher activity, except for the capsules. The ocean samples extracted
at 100
◦
C and purified by SPE was the extract with a higher antioxidant activity (76.54%
±
0.17) followed
by the Tagus samples extracted with the same conditions (41.98%
±
0.55). The results present in Figure 4
demonstrate that although the antioxidant activity even in the best situation is lower than the standard
like butylated hydroxytoluene (BHT), whose IC
50
is 15.7 ug/mL [
35
,
36
], it would be approximately
60 ug/mL, value similar to those obtained in infusions from medicinal plants [
30
]. Capsules, as well as
the Tagus samples dried in the oven showed a very low antioxidant activity, Figure 5.
Foods 2020, 9, x 10 of 20
approximately 60 ug/mL, value similar to those obtained in infusions from medicinal plants [30].
Capsules, as well as the Tagus samples dried in the oven showed a very low antioxidant activity,
Figure 5.
Figure 5. Percentage of antioxidant activity of F. vesiculosus extracts (100 µg/mL of extract). Different
superscript letters (a–c) correspond to values in the same extraction condition that can be considered
statistically different (p ≤ 0.05).( )H2O 25 °C; ( )H2O 100 °C; ( )SPE purification (H2O 100 °C).
3.2.2. Acetylcholinesterase Activity
In this study, only the extracts with higher antioxidant activity were included, but the
comparisons were carried out without the purification of the extracts by SPE. The results obtained,
summarized in Table 1, showed that the AChE inhibitory activity at 1 mg/mL of the ocean samples
was approximately 20 times higher than that presented by the Tagus samples. After the SPE
purification the capacity to inhibit the AChE was higher for both extracts compared to the initial
aqueous extracts under study. The purified ocean samples extract presented an IC50 of 14.97 µg/mL,
while the IC50 of the purified Tagus extract is almost 56 times higher (840.85 µg/mL), meaning lower
activity (Table 1). Comparing with other type of aqueous extracts like those obtained from medicinal
plants, these aqueous extracts are much more active than most of the extracts containing also phenolic
compounds [25,30]. Nevertheless, both extracts are far less potent than the standard galantamine that
has an IC50 of 0.14 µg/mL [35], which means that when consumed it will not affect strongly the
intestinal motility as the drug might do [37].
Table 1. Enzyme inhibitory activity of acetylcholinesterase and HMGR of the ocean and Tagus
samples aqueous extracts (100 °C, 30 min) and after SPE purified extract.
Extract
Acetylcholinesterase
Inhibition HMGR * Inhibition Inhibition of Cholesterol
Permeation (%)
1000 µg/mL IC50 (µg/mL) 10 µg/mL IC50
(µg/mL)
Ocean samples 89.96% ±
0.54 a
210.86 ±
22.11 a
27.74% ±
1.60 a - -
Ocean samples–SPE
fraction - 14.97 ± 0.13 b 86.41% ±
3.20 b 4.16 ± 0.11 45.3 ± 4.4
Tagus samples 4.199% ±
0.36 b - 1.07% ± 0.65
c - -
Tagus samples–SPE
fraction
60.44% ±
1.21c
849.80 ±
10.24 c
52.95% ±
3.17 d - -
* 3-hidroxi-3-methyl-glutaril-CoA reductase; Superscript letters (a–d) correspond to values in the same
column that can be considered statistically different (p = 0.05)
3.2.3. HMG-CoA Reductase Inhibitory Activity
Figure 5.
Percentage of antioxidant activity of F. vesiculosus extracts (100
µ
g/mL of extract). Different
superscript letters (
a
–
c
) correspond to values in the same extraction condition that can be considered
statistically different (p≤0.05). (
Foods 2020, 9, x 7 of 20
approx. 9 times, for both the ocean and Tagus extract. This indicated an increase in the compounds
absorbing in the UV-Vis region with the SPE purification.
3.1.3. Total Phenolic Content
The effect of the extraction temperature on the total phenol content of ocean samples, Tagus
samples, and capsules can be observed in Figure 2. One possible explanation for the different amount
of total phenol content is the geographic location where the algae were collected, as all the procedures
were identical for both ocean and Tagus samples.
Figure 2. Total phenol content of Fucus vesiculosus extracts obtained by different aqueous extraction
processes. Total phenolic content (µg of PGE/mg of extract); different superscript letters (a,b)
correspond to values in the same extraction condition that can be considered statistically different (p
≤ 0.05).( )H2O 25 °C; ( )H2O 100 °C; ( )SPE purification (H2O 100 °C).
The aqueous extracts from the ocean samples presented a higher concentration of µg of
phloroglucinol equivalents (PGE)/mg of extract. Comparing with the Tagus samples, the ocean
samples had 2–3 times more phloroglucinol equivalents, 45.6 ± 3.8 and 40.2 ± 2.1 µg PGE/mg Ext,
while the Tagus samples present 15.0 ± 1.2 and 17.1 ± 2.1 µg PGE/mg Ext, with extraction at 25 °C,
and 100 °C, respectively (Figure 2). For the ocean samples, with the SPE purification, the total phenol
content increased from 48.8 ± 2.4 to 322.39 ug PGE equivalents/mg of extract. While for the Tagus
samples it increased from 21.8 ± 2.5 to 104.7 ± 4.1 µg PGE equivalents/mg of extract (Figure 2).
The results showed that the process of drying in oven leads, approximately, to losses of 25–45%
on the total phenolic content 11.1 ± 1.4 and 9.2 ± 1.2 µg PGE/mg Ext with extraction at 25 °C and 100
°C, respectively. The capsules F. vesiculosus have lower concentration of PGE equivalents 6.1 ± 0.7 and
0.7 ± 0.3 µg PGE/mg Ext with extraction at 25 °C, and 100 °C, respectively (Figure 2). These results
agree with the expected, as the capsules, used as a food supplement, are essentially constituted by
fibers.
3.1.4. SEM Observations of F. vesiculosus Particles Obtained by Different Drying Methods and
Extraction Processes
In an attempt to see if the different drying and extraction processes could be reflected into
different microscopic changes, SEM analysis was carried out. The analysis was performed with the
Tagus samples dried by lyophilization or in the oven before and after the various water extraction
processes (Figure 3) and with the aqueous extracts of the ocean algae and capsules obtained at 100 °C
(Figure 4).
) H2O 25 ◦C; (
Foods 2020, 9, x 10 of 20
approximately 60 ug/mL, value similar to those obtained in infusions from medicinal plants [30].
Capsules, as well as the Tagus samples dried in the oven showed a very low antioxidant activity,
Figure 5.
Figure 5. Percentage of antioxidant activity of F. vesiculosus extracts (100 µg/mL of extract). Different
superscript letters (a–c) correspond to values in the same extraction condition that can be considered
statistically different (p ≤ 0.05).( )H2O 25 °C; ( )H2O 100 °C; ( )SPE purification
(H2O 100 °C).
3.2.2. Acetylcholinesterase Activity
In this study, only the extracts with higher antioxidant activity were included, but the
comparisons were carried out without the purification of the extracts by SPE. The results obtained,
summarized in Table 1, showed that the AChE inhibitory activity at 1 mg/mL of the ocean samples
was approximately 20 times higher than that presented by the Tagus samples. After the SPE
purification the capacity to inhibit the AChE was higher for both extracts compared to the initial
aqueous extracts under study. The purified ocean samples extract presented an IC50 of 14.97 µg/mL,
while the IC50 of the purified Tagus extract is almost 56 times higher (840.85 µg/mL), meaning lower
activity (Table 1). Comparing with other type of aqueous extracts like those obtained from medicinal
plants, these aqueous extracts are much more active than most of the extracts containing also phenolic
compounds [25,30]. Nevertheless, both extracts are far less potent than the standard galantamine that
has an IC50 of 0.14 µg/mL [35], which means that when consumed it will not affect strongly the
intestinal motility as the drug might do [37].
Table 1. Enzyme inhibitory activity of acetylcholinesterase and HMGR of the ocean and Tagus
samples aqueous extracts (100 °C, 30 min) and after SPE purified extract.
Extract
Acetylcholinesterase
Inhibition HMGR * Inhibition Inhibition of Cholesterol
Permeation (%)
1000 µg/mL IC50 (µg/mL) 10 µg/mL IC50
(µg/mL)
Ocean samples 89.96% ±
0.54 a
210.86 ±
22.11 a
27.74% ±
1.60 a - -
Ocean samples–SPE
fraction - 14.97 ± 0.13 b 86.41% ±
3.20 b 4.16 ± 0.11 45.3 ± 4.4
Tagus samples 4.199% ±
0.36 b - 1.07% ± 0.65
c- -
Tagus samples–SPE
fraction
60.44% ±
1.21c
849.80 ±
10.24 c
52.95% ±
3.17 d - -
* 3-hidroxi-3-methyl-glutaril-CoA reductase; Superscript letters (a–d) correspond to values in the same
column that can be considered statistically different (p = 0.05)
3.2.3. HMG-CoA Reductase Inhibitory Activity
) H2O 100 ◦C; (
Foods 2020, 9, x 10 of 20
approximately 60 ug/mL, value similar to those obtained in infusions from medicinal plants [30].
Capsules, as well as the Tagus samples dried in the oven showed a very low antioxidant activity,
Figure 5.
Figure 5. Percentage of antioxidant activity of F. vesiculosus extracts (100 µg/mL of extract). Different
superscript letters (a–c) correspond to values in the same extraction condition that can be considered
statistically different (p ≤ 0.05).( )H2O 25 °C; ( )H2O 100 °C; ( )SPE purification
(H2O 100 °C).
3.2.2. Acetylcholinesterase Activity
In this study, only the extracts with higher antioxidant activity were included, but the
comparisons were carried out without the purification of the extracts by SPE. The results obtained,
summarized in Table 1, showed that the AChE inhibitory activity at 1 mg/mL of the ocean samples
was approximately 20 times higher than that presented by the Tagus samples. After the SPE
purification the capacity to inhibit the AChE was higher for both extracts compared to the initial
aqueous extracts under study. The purified ocean samples extract presented an IC50 of 14.97 µg/mL,
while the IC50 of the purified Tagus extract is almost 56 times higher (840.85 µg/mL), meaning lower
activity (Table 1). Comparing with other type of aqueous extracts like those obtained from medicinal
plants, these aqueous extracts are much more active than most of the extracts containing also phenolic
compounds [25,30]. Nevertheless, both extracts are far less potent than the standard galantamine that
has an IC50 of 0.14 µg/mL [35], which means that when consumed it will not affect strongly the
intestinal motility as the drug might do [37].
Table 1. Enzyme inhibitory activity of acetylcholinesterase and HMGR of the ocean and Tagus
samples aqueous extracts (100 °C, 30 min) and after SPE purified extract.
Extract
Acetylcholinesterase
Inhibition HMGR * Inhibition Inhibition of Cholesterol
Permeation (%)
1000 µg/mL IC50 (µg/mL) 10 µg/mL IC50
(µg/mL)
Ocean samples 89.96% ±
0.54 a
210.86 ±
22.11 a
27.74% ±
1.60 a - -
Ocean samples–SPE
fraction - 14.97 ± 0.13 b 86.41% ±
3.20 b 4.16 ± 0.11 45.3 ± 4.4
Tagus samples 4.199% ±
0.36 b - 1.07% ± 0.65
c- -
Tagus samples–SPE
fraction
60.44% ±
1.21c
849.80 ±
10.24 c
52.95% ±
3.17 d - -
* 3-hidroxi-3-methyl-glutaril-CoA reductase; Superscript letters (a–d) correspond to values in the same
column that can be considered statistically different (p = 0.05)
3.2.3. HMG-CoA Reductase Inhibitory Activity
) SPE purification (H2O 100 ◦C).
3.2.2. Acetylcholinesterase Activity
In this study, only the extracts with higher antioxidant activity were included, but the comparisons
were carried out without the purification of the extracts by SPE. The results obtained, summarized in
Table 1, showed that the AChE inhibitory activity at 1 mg/mL of the ocean samples was approximately
20 times higher than that presented by the Tagus samples. After the SPE purification the capacity
to inhibit the AChE was higher for both extracts compared to the initial aqueous extracts under
study. The purified ocean samples extract presented an IC
50
of 14.97
µ
g/mL, while the IC
50
of the
purified Tagus extract is almost 56 times higher (840.85
µ
g/mL), meaning lower activity (Table 1).
Comparing with other type of aqueous extracts like those obtained from medicinal plants, these aqueous
extracts are much more active than most of the extracts containing also phenolic compounds [
25
,
30
].
Nevertheless, both extracts are far less potent than the standard galantamine that has an IC
50
of
0.14
µ
g/mL [
35
], which means that when consumed it will not affect strongly the intestinal motility as
the drug might do [37].
Table 1.
Enzyme inhibitory activity of acetylcholinesterase and HMGR of the ocean and Tagus samples
aqueous extracts (100 ◦C, 30 min) and after SPE purified extract.
Extract
Acetylcholinesterase Inhibition HMGR * Inhibition Inhibition of Cholesterol
Permeation (%)
1000 µg/mL IC50 (µg/mL) 10 µg/mL IC50 (µg/mL)
Ocean samples 89.96% ±0.54 a210.86 ±22.11 a27.74% ±1.60 a- -
Ocean samples–SPE
fraction -14.97 ±0.13 b86.41% ±3.20 b4.16 ±0.11 45.3 ±4.4
Tagus samples 4.199% ±0.36 b- 1.07% ±0.65 c- -
Tagus samples–SPE
fraction 60.44% ±1.21c849.80 ±10.24 c52.95% ±3.17 d- -
* 3-hidroxi-3-methyl-glutaril-CoA reductase; Superscript letters
(a–d)
correspond to values in the same column that
can be considered statistically different (p=0.05).

Foods 2020,9, 955 11 of 20
3.2.3. HMG-CoA Reductase Inhibitory Activity
For the study of the HMGR inhibition, the high activity before and after the SPE purification was
again obtained with the ocean samples, Table 1. The aqueous extract of the ocean samples, at 1 mg/mL,
has a percentage of inhibition approximately 27 times higher than the Tagus samples. With the
purification of SPE, the ability to inhibit HMGR for ocean samples increased 3–4 times, and for Tagus
samples it increased 50 times compared to the respective aqueous extracts initially studied, meaning
that there are compounds in these aqueous preparations with the capacity to inhibit this enzyme.
After the SPE purification, the IC
50
value for the purified ocean samples extract is approximately
4.16
µ
g/mL. The IC
50
of simvastatin (0.198
±
0.015
µ
g/mL) is almost 20 times lower compared to the
value determined for the alga water extract, that in this case is a mixture of several compounds, while
simvastatin is a pure compound used as a drug.
3.2.4. Cholesterol Permeation
Once the ocean samples extract purified by SPE demonstrated a positive effect inhibiting the
rate-limiting enzyme of the cholesterol biosynthesis as previously demonstrated, a study to evaluate
the effect of this extract on cholesterol permeation through the intestine was performed. The intestinal
barrier was simulated using Caco-2 cells, that have the capacity to differentiate into a monolayer
of polarized cells with morphological and functional characteristics of enterocytes of the small
intestine [
33
,
38
]. F. vesiculosus is one of the 22 species of seaweed that is considered safe to be consumed
as vegetables and condiments under the European directive (EC 258/97) [
1
], which means that it is not
cytotoxic and can be used for the permeation study.
The ocean sample extract SPE purified at 0.25 mg/mL and the cholesterol at 5 mM in HBSS were
added to the apical side of the cells and a control was prepared only with cholesterol in the same
concentration. After 6 h into contact with the cells, the cholesterol on the basolateral compartment and
inside the cells was quantified by HPLC-DAD. Comparing the permeation of the cholesterol in the
presence of the extract with the cholesterol permeation in the control, a reduction of 45.3
±
4.4% in the
cholesterol permeation in the presence of the extract was observed, Table 1.
3.3. Compound Identification by LC-HRMS/MS
Based on the better values of the biological activity of the extracts after the SPE purification, a
tentative identification of the compounds in the extracts obtained from the ocean and Tagus samples was
carried out by HRMS using LC-HRMS/MS. The mass was acquired in positive and negative mode, but
only the negative mode is shown here, because identical chromatograms were obtained, the negative
mode being the one showing the higher intensity of the compounds. The chromatograms for the
ocean and Tagus samples are shown in Supplementary Material Figure S1. The mass analysis and the
intensity of the several compounds in the HRMS chromatograms (Figure S1) allowed the establishment
of the heatmap shown in Table 2. The aqueous extract from the ocean samples had a higher number of
compounds (108 compounds) than the extract of the Tagus samples (85 compounds). On the other
side, this last extract had compounds with retention time at 3.2, 6.9, and 9.8 min, compounds 39, 40,
and 43 (Table 2) in a much higher intensity than the extract of the ocean samples.
A comparison of the compounds present in both algae extracts was carried out by analyzing the
intensities of each peak in a heatmap, Table 2. There are 65 common compounds in both extracts, but
to explain the differences in these extracts, the activities of the compounds present only in the ocean
sample (compounds 5, 8, 10, 15, 18, 20–27, 29–38, 41, and 44) were identified first, Table 3.

Foods 2020,9, 955 12 of 20
Table 2.
Heatmap representing the intensity of all compounds present in the Tagus and ocean samples
after SPE purification. The green values correspond to the compounds with proposed identification
and respective compound number. Intensity: 160000; 1000.
Mass
[M −H]−
Samples
Origin
Mass
[M −H]−
Samples
Origin
Mass
[M −H]−
Samples
Origin
Nom/zOcean Tagus Nom/zOcean Tagus Nom/zOcean Tagus
1.0–1.3 min 1.9–2.0 min 268.9734
102.9572 191.0198 516.9721
1181.0722 117.0194 268.9734
146.0460 269.0874 494.9909
165.0406 203.9732 531.0673
227.0773 223.0820 536.1138
240.0080 230.9893 699.1822
2191.0201 7276.0184 3.2–3.4 min
217.0484 384.1527 164.0715
219.0457 8 455.1898 185.0454
126.9051 713.2366 232.0592
111.0091 9 497.0728 16 277.0932
1.5–1.6 min 10 875.2911 299.0754
117.0193 2.0–2.1 min 17 351.1334
140.0115 11 327.1299 375.0613
149.9627 425.0990 397.0428
181.0715 2.1–2.3 min 18 410.1695
219.9681 12 237.0078 19 555.1934
3256.0940 475.0231 855.2730
300.9335 265.9819 3.5–3.6 min
319.9066 13 125.0247 20 355.1642
322.9155 174.9560 21 497.0748
338.8896 2.4–2.6 min 387.0309
370.1380 14 246.9916 4.5–4.7 min
4403.0557 328.9980 22 355.1622
5445.0962 15 373.0572 227.1037
541.1551 680.2201 453.1301
6551.1836 536.1125 23 711.3323
248.9894
4.7–4.8 min 6.1–6.2 min 12.7–12.8 min
24 203.0824 131.0714 174.9558
25 291.0988 186.1133 43 527.2535
369.1787 785.3471
26 419.1573 6.7–6.8 min
27 621.0891 147.0452
5.0–5.1 min 40 165.0557
138.0562 206.0822
28 175.0611 119.0504
187.9041 331.1190
375.8086 353.1007
29 745.1056 619.2774
5.4–5.7 min 41 930.1220
30 324.1576 1054.6409
31 369.1787 7.8 min
405.1553 42 361.1503
427.9317 383.1323
32 437.1652 587.2592
33 467.1465 433.1622
34 739.3642 9.8–10
241.1192 394.9648
35 531.2215 395.9679
36 682.0892 10.9–11.1 min
37 806.1051 396.9627
38 869.1216 160.8416
467.1461 162.8387
5.7–5.8 min 174.9556
39 247.0823 447.1349
145.0598
269.0646

Foods 2020,9, 955 13 of 20
Table 3.
Identification proposal of the compounds detected by LC-HRMS/MS in ESI negative mode
only present in the ocean samples extract. «No» indicates compound number corresponding to Table 2.
Rt
(min)
[M −H]−
m/z
Molecular
Formula
Error
(ppm) Fragmentation (%) Name No
1.5 445.0962 C18H22 O13 3.3
314 (50%); 221 (100%); 80 (60%)
Phloroglucinol derivative 5
1.9 455.1898 C18H28 N6O8−0.5 238.0839 (96.97%); 126.0196
(37.10%); 88.0404 (23.56%) Peptide derivative ThrGluHisAla 8
1.9 875.2911 C49H48 O15 −0.2
829 (100%); 125 (7%); 179 (14%);
383 (16%) Phloroglucinol derivative (7 units) 10
2.6 373.0572 C18H14 O9−1.5 355 (37%); 141.0192 (100%);
207.0303 (20%) Phloroglucinol derivative (3 units) 15
3.2 410.1695 C17H25 N5O7−4.8
136 (45%); 109 (45%); 366 (58%);
339 (35%) Benarthin 18
3.5 355.1642 C15H24 N4O6−4.5
311 (74%); 252 (19%); 194 (24%);
96 (46%) Tetrapeptide ProProGlySer 20
3.6 497.0748 C24H18 O12 0.6
125 (45%); 141 (30%); 372 (20%);
356 (20%) Phloroglucinol derivative (4 units) 21
4.5 355.1622 C15H24 N4O60.2
311 (12%); 238 (95%); 240 (82%);
183 (3 7%); 116 (100%); 70 (8%) Tetrapeptide ProSerGlyPro 22
4.5 711.3323
C
30
H
48
N
8
O
12 −2.2 355 (100%); 129 (2%); 116 (9%);
356 (20%) Octapeptide of ProProGlySer 23
4.7 203.0824 C11H12 N2O21116.0508 (100%); 74.0251
(37,77%) Tryptophan 24
4.7 291.0982 C14H16 N2O51.5 Didn’t fragment Tryptophan derivative 25
4.7 419.1573 C19H24 N4O70 Didn’t fragment Tetrapeptide of (GluSerTrp) 26
4.7 621.0891 C30H22 O15 −2.5 125 (84%); 141(6%); 165 (6%);
485 (3%); 289 (3%); 478 (50%) Phloroglucinol derivative (5 units) 27
5.0 745.1039 C36H26 O18 −0.5 125 (29%); 141 (14%); 497
(100%); 413 (4%) Phloroglucinol derivative (6 units) 29
5.4 324.1576 C15H22 N3O5−1.9 209.0944 (45.86%); 166.0868
(34.90%); 114.0565 (100%);
N-(5-Hydrazinyl-5-oxopentyl)-3,
4,5-trimethoxybenzamide 30
5.4 369.1787 C16H26 N4O6−0.1
325.1879 (19.73%); 238.0834
(87.76%); 130.0872 (100%)
58.0303 (15.04%);
Tetrapeptide ThrProProGly 31
5.4 437.1652 C19H26 N4O83.8 392 (23%); 320 (11%); 348 (1%) Tetrapeptide GlyAspPheThr 32
5.4 467.1459
C
17
H
32
N
4
O
5
S
3−1.7 423; 319 (0.4%) Tetrapeptide LeuCysCysMet 33
5.4 739.3642
C
32
H
52
N
8
O
12 −2.9 369 (100%); 143 (1%); 238 (5%) Peptide derivative 34
5.6 531.2215 C24H32 N6O81.0 Didn’t fragment Tetrapeptide
ValAspTrpAsn 35
5.6 682.0892 C27H25 NO20 −4.4
125 (44%); 141 (27%); 134 (13%);
495 (43%) Phloroglucinol derivative 36
5.6 806.1051 C33H29 NO23 −0.8 369 (95%); 195 (21%); 761(23%);
238 (31%); 567(19%) Phloroglucinol derivative 37
5.6 869.1216 C42H30 O21 -2.4 125 (40%); 141 (9%); 137
(9%);194 (6%); 335 (14%); Phloroglucinol derivative (7 units) 38
6.7 930.1225 C39H32 NO26 −0.7 Didn’t fragment Phloroglucinol and amino acid
derivative 41
12.7 527.2535 C32 H36N2O53.1 418 (0.2%); 165 (0.8%); 130
(0.3%) Chaetoglobosin A 44
There are compounds in both alga extract with high intensity (compounds 1, 2, 3, 4, 6, 7, 9,
11–14, 16, 17, 19, 28, Table 2) as well as compounds present only in Tagus sample with high intensity,
(compounds 39, 40, 42, Table 2). These compounds were also identified, Table 4.

Foods 2020,9, 955 14 of 20
Table 4.
Identification proposal of the compounds detected by LC-HRMS/MS in ESI negative mode
present in the ocean and Tagus (river estuary) extracts and present only in the Tagus with high intensity.
«No» indicates compound number corresponding to Table 2.
Rt
(min)
Accurate
[M −H]−m/z
Molecular
Formula
(Error, ppm)
Fragmentation (%) Proposed Compound No
1.1 181.0722 C6H14 O6
(2.3) - Mannitol * 1
1.2 191.0201 C6H8O7
(2.0) - Citric acid * 2
1.5 256.0941 C10H16N3O5
(5.4)
212.10(6); 194.09(93); 166.06(7);
141.06(9); 82.03(32) Cytidine derivative 3
1.5 403.0555 C12H20 O13
(−3.7)
384.98(2); 370.13(4); 231.45(1) 219.96(21);
149.96(100); 96.96(2); 79.95(46)
Galactose-sulfate
derivative 4
1.6 551.1836 C19H36 O18
(−5.3)
506.12(10); 341.10(15); 325.11(6);
179.05(100); 161.04(17) Glycidyl compound 6
1.9 276.0187 C9H12 NO7S
(−3.0)
230.01(9); 196.06(100);
179.05(45);135.04(92) Tyrosine sulfate 7
1.9 497.0727 C24H18 O12
(−2.6)
479.05(10); 383.17(26);
331.04(27);287.05(6); 165.01(100);
137.02(26)
Phloroglucinol
derivative 9
2.0 327.1299 C12H24 O10
(−1.3) 238.08(26); 101.07(8); 88.04(100) Glycosidic derivative 11
2.1 237.0078 C10 H6O7
(−4.4)
153.05(12); 136.73(1); 123.04(13);
96.96(93)M 79.95(100)
3. ,5,7-Trihydroxy-4-
oxochromene-2-carboxylic
acid
12
2.2 125.0247 C6H6O3
(−2.6) - Phloroglucinol * 13
2.4 246.9916 C8H8O7S
(−0.4)
164.93(2); 121.02(23); 108.02(100);
80.96(76) Vanillic acid sulfate 14
3.2 277.0930 C11H18 O8
(0.7) 141.05(5); 97.06(100); 91.03(10) Phloroglucinol
glycosidic derivative 16
3.2 351.1334 C15H20N4O6
(6) 238.12(5); 181.04(3); 164.07(100) Tetrapeptide
Gly Tyr 17
3.2 555.1930 C22H36 O19
(0.1) 277.09(100); 185.04(97); 141.05(23) Phloroglucinol
glycosidic derivative 19
5.0 175.0611 C7H12 O5
(0.0) 115.04(45); 85.06(100); 59.01(13) Butanedioic derivate 28
5.7 247.0823 C10H16 O7
(0.1)
145.05(40); 127.04(5);
101.06(100);83.05(97) Glycosidic derivative 39
6.7 165.0557 C9H10 O3
(0.1)
147.04(18); 119.05(60); 91.05(5);
73.99(100) Chroman-2,5-diol 40
7.8 361.1503 C16H26 O9
(−3.3)
233.10(13); 145.05(16); 127.04(11);
88.05(100) Gibboside isomer 42
* dentification confirmed with standard analysis.
The compounds identified indicated that there are two main groups of chemical structures,
phlorotannin derivatives and small peptides, these together represent 94% of the intensities detected.
The three compounds that do not belong to this type, compounds 18, 30, and 44 represent only 6% of
the intensities determined. Of the identified compounds, 38% were phlorotannins and 50% peptide
derivatives. The bioactivities found result from the activities of this mixture. The algae from the
ocean has a higher amount of phlorotannins and peptide derivatives than the algae from the Tagus.
14 phlorotannins and 13 peptide derivatives were identified in the ocean sample that were not present
in Tagus, while in the Tagus sample only 4 phlorotannins and 3 peptide derivatives were identified.
The compounds found in higher amount in the Tagus sample, a glycosidic derivative, an iridoid,
and the third one a chroman derivative did not show a strong bioactivity. The phlorotannins detected

Foods 2020,9, 955 15 of 20
seem to be phloroglucinol units binding together originating compounds with different degrees of
polymerization with molecular weight ranging from 374 Da to 876 Da. This type of phlorotannins
are usually called fuhalols and have been detected in other brown alga [
39
]. The phloroglucinol
with higher molecular weight, 930 Da was binding an amino acid residue. A search on the web
did not provide description of this type of compound before in algae. On what concerns the small
peptides detected in the aqueous extract was composed of 8 amino acid residues, all the others had 4
binding amino acid residues, forming small polymers of mass oscillating between 356 Da and 531 Da.
The octapeptide had a molecular weight of 712 Da. In different species of brown alga other small
peptides were detected, some with 3 amino acid residues, others with 4, and most of them with higher
amino acid sequences [
40
]. In most of the small peptides detected in F. vesiculosus extract essential
amino acids are present, indicating the high nutritional value of this food preparation.
4. Discussion
In the present study the aqueous extraction at high temperatures leads to higher total phenol
content and consequently higher biological activities. These results are in accordance with previous
studies that reported that the extraction of phenolic compounds at high temperatures leads to high yields
of extraction, once the molecular movement and the solubility increase, facilitating the dissolution
of flavonoids from the plant cells and disrupting the cell membranes, facilitating the extraction
process [
41
,
42
]. The results demonstrated that the process of drying with high temperature also affect
the composition of the algae. The Tagus extract dried in the oven presented lower content of phenolic
compounds, even when extracted with water at 100
◦
C, and consequently loss of compounds that were
absorb in the UV-VIS light compared with the Tagus extract dried in the freeze dryer. The results show
that F. vesiculosus from the Atlantic Ocean is different from that collected in the Tagus River estuary,
on what concerns the bioactive compounds and, consequently, in their biological activities. The SEM
analysis also gave an indication of the different texture in the microstructure of all the extracts.
Independently of genetic characteristics, one possible explanation for the differences between
the ocean and the Tagus samples is the fact that in the ocean the algae are exposed to more stressful
conditions, such as high salinity, big wave action, and great shear stress, which lead to the synthesis of
compounds to stress protection. The analysis by LC-HRMS/MS of the F. vesiculosus extracts confirmed
that the ocean samples have higher amount of phlorotannins and peptides derivatives comparing with
the Tagus samples. Of the 14 phlorotannins identified in the ocean sample, only 3 are also found in the
Tagus sample. In relation to the peptide derivatives 11 were identified in the ocean sample but only 1
was also found in the Tagus sample, when analyzed the compounds present in both samples.
To study the enzymatic activity of the different extracts, the acetylcholinesterase (AChE) was
chosen as a model enzyme, once AChE inhibitors are used in the treatment of important diseases like
Alzheimer’s disease and severe gastrointestinal disorders [
30
,
35
]. The search for natural compounds
with cholinesterase inhibitory activity arises since synthetic cholinesterase inhibitors have adverse
side effects [
26
,
43
]. Different natural compounds, including phenolic compounds, have been shown to
be potent AChE inhibitors [
44
]. The results for the inhibition of the acetylcholinesterase can also be
explained by the composition of the ocean samples, presenting a higher amount of phenolic compounds
like phlorotannins compared to the Tagus algae and higher amount of small peptides. Previous studies
with marine algae reported that purified phlorotannins from Ecklonia stolonifera algae can prevent
the biding of the substrates like acetylcholine (Ach) and butyrylthiocholine to cholinesterase in a
non-competitive manner [
26
]. The different peptide derivatives with phenolic groups identified in the
ocean sample can also be inhibitors of the AChE enzyme. Previous studies reported marine-derived
peptides as acetylcholinesterase-inhibitors [
45
,
46
]. Phlorotannins have in their molecular structure
a phloroglucinol moiety, containing an aromatic ring. Compounds containing aromatic rings can
show affinity to the enzyme active site because of the presence of Phe residue at the entrance of the
enzyme active site [
27
,
35
]. Phlorotannins and the small peptides detected in the aqueous extracts
can act as AChE inhibitors. Some of the small peptides detected also contain benzene ring in the

Foods 2020,9, 955 16 of 20
amino acid residues. These aromatic rings may establish pi-pi interactions with the amino acid at the
entrance of the active site pocket [
35
]. In the present study the value obtained for the inhibition of
acetylcholinesterase indicates that the algae, when consumed in a meal, like a soup, may accelerate the
digestive tract, also avoiding for instance the cholesterol absorption. This can be an indirect way of
reducing the absorption of cholesterol from the diet. It is known that the polysaccharides present in
algae have the effect of sequestering cholesterol and diminishing its absorption [
12
]. With the presence
of other bioactive molecules like phlorotannins this effect may be reinforced. In the aqueous extract of
the ocean samples there were phlorotannin with high molecular weight. It can be speculated that these
compounds have difficulty in permeating the intestinal cell membranes, probably because smaller
compounds can penetrate the cell membrane and be active inside the cell. The bioactivities described
may be attributed to the small weight molecules of phlorotannins and small peptides. The peptides
found are mostly small peptides, with molecular weight between 204 and 740 Da that may permeate
the intestinal barrier or may be hydrolyzed during the digestive process.
Hypercholesterolemic levels are the main causes for the cardiovascular diseases, the leading causes
of death worldwide [
28
,
47
]. The drugs widely used to reduce the cholesterol levels act by inhibiting the
HMGR or by preventing its absorption though the intestinal barrier [
48
]. HMGR regulates the de novo
biosynthesis of the intracellular cholesterol, being the most common HMGR inhibitors those that include
lovastatin, fluvastatin, simvastatin, pravastatin, pitavastatin, rosuvastatin, and atorvastatin. Although
statins are well-known for the effective cholesterol-lowering therapy, they can cause adverse effects,
which leads to the search for natural drugs [
49
]. The
in vitro
studies here presented demonstrated
that the aqueous ocean extract of F. vesiculosus rich in phlorotannins and peptide derivatives have the
capacity to decrease the synthesis of cholesterol by inhibiting the HMGR. Although the active site of
HMGR has hydrophobic amino acid residues with which the usual enzyme inhibitors, statins, can
interact, it also has polar residues with which compounds having phenolic structure can bind [
50
].
Phlorotannins and the small peptides have more hydrophilic groups able to establish hydrogen bonds
that can attach to these hydrophilic amino acid residues in the MHGR active site.
This extract also has the potential to decrease the permeation of the cholesterol through a
simulated intestinal barrier, with a similar value to those obtained with medicinal plants aqueous
extracts containing phenolic compounds [
33
,
34
,
51
]. Cholesterol permeation may be affected because of
the competition of bioactive compounds for membrane transporters like NPC1L1, which transport
cholesterol into the intestinal cell or by increasing the efflux from inside the cells, through the ABCG5
and ABCG8 transporters [
48
]. Similar studies, with plants have already demonstrated that compounds
such as phenols and flavonoid derivatives can interfere with the cholesterol membrane transport
proteins by these types of mechanisms [
52
,
53
]. The results here reported are also in agreement
with previous
in vivo
studies that demonstrated that marine-derived peptides have the capacity to
lower the level of plasma and hepatic cholesterol [
2
] and phlorotannins like eckol and dieckol have
a hypolipidemic effect [
20
,
47
]. However, nothing was reported about phlorotannins with fuhalol
structure as those referred to in the present work.
In all the studies done in the present work the ocean sample demonstrated better activity relatively
to the Tagus sample. This can be described to the presence of a higher number of secondary metabolites,
including small peptides and phlorotannins. In fact, there was a correlation, in the present study,
between the total phenolic content and the higher activities detected.
The results here presented also indicated that the way of preparing a meal from algae is very
important. If the algae are dried in the oven before the meal preparation much of the bioactivity will
be lost. Soups seem to be a good way of preparing the algae, as it corresponds to the extract obtained
at 100
◦
C that showed the higher values for the different
in vitro
activities studied. Here, we provide
valuable insights on the effects of consuming F. vesiculosus algae depending on the way of cooking
and on the origin of the algae. This study can be an opening door for future development of an active
ingredient, seaweed, derived for pharmaceutical and food industries.

Foods 2020,9, 955 17 of 20
5. Conclusions
When required to collect algae to prepare food with high bioactivity it is better to select the
ocean than the river estuary. To prepare the algae, drying in the oven should not be used; it is
advisable to dry through lyophilization and prepare the soup. The alga can be washed without
losing many of its bioactive compounds. This can lead to a meal containing phlorotannins and small
peptides with the capacity to inhibit AChE and reduce diet cholesterol intestinal permeation as well as
hypercholesterolemia through the inhibition of HMGR.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2304-8158/9/7/955/s1,
Figure S1: Chromatogram LC-HRMS/MS in negative mode of F. vesiculosus from ocean (blue) and from Tagus (black).
Author Contributions:
Formal analysis and investigation, R.A, and L.G.; observations, L.A. and SEM;
conceptualization and supervision, M.L.S.; supervision of the mass spectrometry, P.D.V.; algae harvesting
and identification, R.M.; methodology, M.L.S.; resources, M.L.S.; writing—original draft preparation, R.A., L.A.,
R.P., M.L.S. and R.P., writing—review and editing, L.A. and M.L.S., supervision, M.L.S.; project administration
and funding acquisition, M.L.S. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by FCT (Portugal) through financing of Research Centers with the contract
program: BioISI: UIDB/04046/2020 and UIDP/04046/2020; CESAM (UIDP/50017/2020+UIDB/50017/2020) and
MARE (UIDP/04292/2020+UIDB/04292/2020); research project PTDC/BIA-BQM/28355/2017; R.A. is recipient of a
fellowship from BioSys PhD programme PD65-2012 (PD/BD/142861/2018) from FCT (Portugal). Supported also by
Bioisi MassSpec Facility.
Acknowledgments:
The authors wouldlike to thank Rita Guedes forher technicalsupportwith theLC-HRMSassistance.
Conflicts of Interest: The authors declare no conflict of interest.
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