Food Chemistry 341 (2021) 128262

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Main bioactive phenolic compounds in marine algae and their mechanisms T

of action supporting potential health benefits
C. Jimenez-Lopeza,b, A.G. Pereiraa,b, C. Lourenço-Lopesa, P. Garcia-Oliveiraa,b, L. Cassanic,
M. Fraga-Corrala,b, M.A. Prietoa, , J. Simal-Gandaraa,
⁎ ⁎

a
Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, University of Vigo, Ourense Campus, E-32004
Ourense, Spain
b
Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolonia, 5300-253 Bragança, Portugal
c
Research Group of Food Engineering, Faculty of Engineering, National University of Mar del Plata, RA7600 Mar del Plata, Argentina

ARTICLE INFO ABSTRACT

Keywords: Given the growing tendency of consumers to choose products with natural ingredients, food industries have
Phenolic compounds directed scientific research in this direction. In this regard, algae are an attractive option for the research, since
Algae they can synthesize a group of secondary metabolites, called phenolic compounds, associated with really pro­
Extraction techniques mising properties and bioactivities. The objective of this work was to classify the major phenolic compounds,
Bioactivities
compare the effectiveness of the different extractive techniques used for their extraction, from traditional sys­
Industrial applications
tems (like heat assisted extraction) to the most advance ones (such as ultrasound, microwave or supercritical
fluid extraction); the available methods for identification and quantification; the stability of the enriched extract
Chemical compounds studied in this article:
Phloroglucinol (PubChem CID359) in phenolic compounds and the main bioactivities described for these secondary metabolites, to offer an over­
Gallic acid (PubChem CID370) view of the situation to consider if it is possible and/or convenient an orientation of phenolic compounds from
Ferulic acid (PubChem CID445858) algae towards an industrial application.
Sinapic acid (PubChem CID637775)
Catechin (PubChem CID9064)
Catechin gallate (PubChem CID6419835)
Epigallocatechin (PubChem CID72277)
Eckol (PubChem CID145937)
Triphloroethol A (PubChem CID21626545)
Eckstolonol (PubChem CID10429214)

1. Introduction many obstacles (i.e. adverse weather, herbivory), and allow them to
interact and adapt to their surroundings; thus, the same plant species
Algae, fruit, vegetables, and other edible plant are natural sources of growing in different locations may have different concentrations of
phytochemical compounds (Acosta-Estrada, Gutiérrez-Uribe, & Serna- compounds, or even different compounds in their constitution (Azmir
Saldívar, 2014). Once introduced in usual diet, phytochemicals exert et al., 2013; Lobo & Lourenço, 2007; Santos, Abreu, & Saraiva, 2016).
physiological effects in humans, and for this reason they are also called Focusing on phenolic compounds, they constitute a large and di­
bioactive compounds. Some examples of beneficial phytochemical versified group of secondary metabolites found mainly in plants, al­
compounds are vitamins, carotenoids (lycopene, β-carotene, and xan­ though other organisms also can synthesize them, such as algae.
thophylls) and phenolic compounds (Komes et al., 2011). Understanding the mechanisms of action of these compounds and their
These functional phytochemicals can be divided into two major interaction with the human body become relevant due to their multiple
groups: primary metabolites and secondary metabolites. Primary me­ and beneficial health effects (Barbosa-Pereira et al., 2014). From a
tabolites, namely carbohydrates, amino acids, lipids, and nucleic acids, chemical point of view, phenolic compounds are formed by a structural
are responsible for the development and growth of organisms. core based on a hydroxyl group bonded directly to a phenol, which
Secondary metabolites, on the other hand, are a group of compounds gives them the ability to capture free radicals, reactive oxygen species
that, while not essential, give plants the ability to survive and overcome and chelated metal ions (Silva, 2013). Those phenolic compounds that

⁎
Corresponding authors.
E-mail addresses: mprieto@uvigo.es (M.A. Prieto), jsimal@uvigo.es (J. Simal-Gandara).

https://doi.org/10.1016/j.foodchem.2020.128262
Received 4 February 2020; Received in revised form 11 September 2020; Accepted 27 September 2020
Available online 01 October 2020
0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

present more than one phenolic ring in their structure are called ingredients in the food industry, or to incorporate them in cosmetic
polyphenols, and they are synthesized in nature as response to en­ formulations and pharmaceutical preparations with therapeutic pur­
vironmental harmful stimuli such as UV radiation, pathogen attacks poses (Abad, 2013). Those are practical, useful and interesting chal­
and insects or wounds (Dias, Sousa, Alves, & Ferreira, 2016; Lopes lenges that are discussed on the present manuscript, which collects and
et al., 2018). The different classes and subclasses of polyphenols are compare published literature from documents regarding algae-con­
usually based on their chemical structures, what refers to the number of tained phenolic compounds’ extraction, classification, stability, identi­
phenolic rings they possess and the structural elements bonded to these fication and bioactivities. Databases consulted with this purpose were
rings. In this context, the main groups of phenolic compounds are: Scopus, Algaebase and ScienceDirect.
phenolic acids, coumarins, flavonoids, stilbenes, tannins, lignans and
lignin (Costa et al., 2013; Silva, 2013). They are associated with very 2. Classification of phenolic compounds from algae
diverse biological activities beneficial to health, and are present in
numerous and abundant species of seaweed, especially, in the group of Phenolic compounds are present in an immense variety of terrestrial
brown algae (Fernando, Nah, & Jeon, 2016). and marine plants, due to their importance and contribution to or­
Currently, the discovery, development and market launch of new ganisms’ growth and survival, also helping in the defense against pa­
natural products that can be used as functional ingredients is becoming thogens and predators. These compounds can be synthetized following
very important, since those natural metabolites could replace the syn­ pentose phosphate, shikimate or phenylpropanoid pathways.
thetic ones, associated with several health problems or disorders. Regarding their chemical structure, all phenolic compounds com­
Undoubtedly, algae represent a natural source of products of interest, prise at least one aromatic phenolic ring with one or more hydroxyl
such as phenolic compounds (Al-Saif, 2014). These compounds are substituents that can be highly polymerized, which allows their classi­
present in most classes of algae (Liu, Hansen, & Lin, 2011), and parti­ fication, with more than 8000 different structures being currently
cipate in various survival processes, such as defense and protection known. To carry out its classification, different criteria can be followed.
against different factors, whether they are abiotic, such as ultra-violet One of them is based on the number of carbons in the molecule (Fig. 1).
(UV) radiation, or biotic, such as the attack of pathogenic micro­ Other types of classifications are based on their level of distribution
organisms or other living beings. Most of these compounds show also (shortly distributed, widely distributed and polymers), or according to
biological functions such as antioxidant or antimicrobial properties. their characteristics or properties (soluble and insoluble). From a nu­
Several studies have reported that there is a correlation between the tritional point of view, this last classification is very useful since in­
number of phenolic compounds present and the antioxidant potential soluble phenolic compounds will not cross the intestinal barrier to
that an alga shows (Klejdus, Plaza, Snóblová, & Lojková, 2017). reach the blood. This variety of classifications occurs since phenolic
The huge diversity of algae refers not only to phytoplankton, which compounds comprise a large number of heterogeneous structures that
is considered a microalga, but also to macroalgae or seaweed, so, in range from a very simple to a highly polymerized structural level (de
total, more than 11,000 different species are known. As a general Giada, 2016; Vermerris & Nicholson, 2006). Some of them show
classification, phytoplankton is made up of diatoms (Bacillariophyta), bioactivities represented in Table 1.
dinoflagellates (Dinophyta), green and yellow–brown flagellates
(Prasinophyta, Prymnesiophyta, Cryptophyta, Chrysophyta, and 2.1. Simple phenolic
Rhaphidiophyta) and blue-green microalgae (Cyanophyta). On the
other hand, macroalgae are classified into three large groups: green This group is formed by phenols that present hydroxyl substituents
algae (Chlorophyta), brown algae (Phaeophyta) and red algae in different positions: ortho, meta, and para (1,2-, 1,3- and 1,4-, re­
(Rhodophyta), depending on the types of pigments they contain, which spectively). Moreover, simple phenolics sometimes show three func­
allows them to inhabit at different depths (Sithranga Boopathy & tional groups. In this case, it can be meta-tri substitution (1,3,5-) or vic-
Kathiresan, 2013). tri substitution (1,2,6-) (Vermerris & Nicholson, 2006). Examples of
Although secondary metabolites normally do not fulfill primary compounds that belong to this group are catechol, hydroquinone, and
functions such as organism growth, there are always exceptions. Great phloroglucinol, the last one being found exclusively in macroalgae
examples are a group of phenolic compounds known as phlorotannins, (Tsimogiannis & Oreopoulou, 2019). A study showed that catechol was
which are found exclusively in algae, especially in brown algae, and detected in 27 Japanese seaweed, which correspond to green or red
participate in the development and growth of the cell walls (Liu et al., algae (Yoshie-Stark & Hsieh, 2003). It is quite common to find phenols
2011). Phlorotannins can be defined as polymers of phloroglucinol with bromine substituents which are called bromophenols. Fig. 2 con­
units (1,3,5-tryhydroxybenzene), and are synthesized in algae through tains the chemical structures of some relevant phenolic compounds that
the acetate – malonate pathway (also known as polyketide pathway), can be found in algae.
originating compounds of wide ranges of molecular size (126–650 kDa)
(Agregán et al., 2017; Eom, Kim, & Kim, 2012). They are hydrophilic 2.2. C6-CN phenolic compounds
compounds that contain both phenolic and phenoxy groups in their
structure and can be divided into four large subgroups: phlorethols and This group is formed by compounds whose basic skeleton is C6-CN
fuhalols (they have both bonds), fucols (they contain phenyl bonds), being N 1 ≤ N ≤ 3. Within this group, three subdivisions can be made
fucophloroethols (they have ether and phenyl bonds), and eckols (with in C6-C1, C6-C2, and C6-C3. (Fig. 1). C6-C1 correspond to phenolic acids
dibenzodioxin bonds). Several phlorotannins have been associated with and aldehydes, which are characterized by the presence of a carboxyl
the exertion of biological properties, beneficial for the human health, group as a substitute on a phenol (Vermerris & Nicholson, 2006). One of
such as antioxidant or antimicrobial activities (Li, Wijesekara, Kim, & the most abundant simple phenolic acids belonging to this group is
Li, 2011). gallic acid, commonly used as standard in total phenolic compounds
Among the properties associated with phenolic compounds present quantification. It can be found in some algae in high concentrations, as
in seaweed, it is worth highlighting their antioxidant activity because is the case of the brown alga Halopteris scoparia (Linnaeus) Sauvageau,
their capacity of exerting a scavenging action is related to further which also contains gentisic acid. Other simple acids have been also
bioactivities, such as anti-inflammatory, antitumoral, hypocholester­ found in algae, like 4-hydroxybenzoic acid in brown alga Undaria pin­
olemic, anticoagulant, antiviral and antimicrobial activities. For this natifida (Harvey) Suringar (Mekinić et al., 2019). Compounds belonging
reason, there is a growing interest in the study and development of to the C6-C2 group are known as phenylethanoids and include hydro­
innovative strategies to optimize the extraction of these bioactive xylated and/or methoxylated derivatives of phenylacetic acid, acet­
compounds from seaweeds; aiming to use them as functional ophenone, and phenethyl alcohol. They are not very common in nature.

2
C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

Fig. 1. Classification of phenolic compounds according their basic structures (Adapted from (de Giada, 2016; Tsimogiannis & Oreopoulou, 2019; Vuolo et al., 2019)).

Among the C6-C3 category are phenylpropanoids, among which we can acid and sinapic acid. Coumarins have the same skeleton as cinnamic
highlight hydroxycinnamic acids, cinnamic aldehydes, monolignols, acids, but they have an oxygen heterocycle as part of the C3-unit. They
phenyl propenes, coumarins, isocoumarins, and chromones. They can have been found in green algae such as Dasycladus vermicularis (Scopoli)
be found in the red alga Tichocarpus crinitus (S.G.Gmelin) Ruprecht Krasser, and they can be also be found as sulfated metabolites, as in the
(Ishii, Okino, Suzuki, & Machiguchi, 2004; Tsimogiannis & Oreopoulou, case of 7-hydroxycoumarin-3,6-disulfate (Hartmann, Ganzera, Karsten,
2019). Among the cinnamic-like acids, it is worth highlighting p-cou­ Skhirtladze, & Stuppner, 2018). Regarding isocoumarins, they differ
maric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid, chlorogenic from coumarins in that the position of the carbonyl and oxygen groups

Table 1
Main bioactive compounds in marine algae. ().
Adapted from Freile-Pelegrín & Robledo, 2013
COMPOUNDS ALGAL CLASS ALGAE SPECIE

Bromophenols Rhodophyceae Pterocladia capillacea, Odonthalia corymbifera, Rhodomela confervoides, Jania rubense
Phaeophyceae Padina arborescens, Sargassum siliquastrum, Lobophora variegata, Meroditerpenoids Sargassum fallax
Chlorophyceae Codium fragile, Avrainvillea longicaulis, Avrainvillea nigricans, Avrainvillea rawsonii
Terpenoids Rhodophyceae Laurencia sp., Callophycus serratus
Mycosporine-like amino acids Rhodophyceae Porphyra sp.
Chlorophyceae Prasiola spp.
Tichocarpol Rhodophyceae Tichocarpus crinitus
Phlorotannins Phaeophyceae Eisenia bicyclis, Ecklonia cava, Ecklonia kurome, Ecklonia stolonifera, Ishige okamurae, Eisenia arborean
Colpol Phaeophyceae Colpomenia sinuosaq
Coumarins Phaeophyceae Dasycladus vermicularis
Vanillic acid Phaeophyceae Cladophora socialis

3
C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

Fig. 2. Representative chemical structures of phenolic compounds found in marine algae.

are reversed (Freile-Pelegrín & Robledo, 2013; Vermerris & Nicholson, (xanthonoids), C6-C2-C6 category (stilbenoids, anthraquinones, and
2006). anthrones), C6-C3-C6 category (flavonoids) and C6-C7-C6 (diarylhepta­
noids). C6-C3-C6 category can be divided into three groups depending
on the arrangement of the C3 group that links the two benzene rings.
2.3. C6-CN-C6 phenolic compounds They are chalcones (linear C3-chain), aurones (cyclization of chal­
cones), and flavonoids (six-member heterocycle) (Vermerris &
Within this group, four subdivisions can be made: C6-C1-C6 category

4
C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

Nicholson, 2006). In a deeper structural classification, flavonoids can case of marine algae, these compounds are generally found free in the
be further divided into 13 classes, being the most important ones fla­ intracellular space and concentrated around organelles sensitive to UV
vanols, flavones, isoflavones, anthocyanins, and flavanones (de Giada, rays. As for its molecular structure, they are formed by cyclohexenone
2016). A study concerning 27 species of Japanese red algae showed that or cycloheximide chromophore conjugated to an amino acid residue or
hesperidin was found in all of them, being Caulerpa serrulata (Forsskål) its imino alcohol (Carreto & Carignan, 2011; Freile-Pelegrín & Robledo,
J.Agardh the one that showed the major concentration. Said study 2013; Rosic, Braun, & Kvaskoff, 2015).
concluded that in general, red algae have a higher content in flavonoids
than green and brown algae (Yoshie-Stark & Hsieh, 2003). Other re­ 3. Extraction technologies for phenolic compounds
cognized, well-known and abundant flavonoids found in algae, be­
longing to flavan-3-ols subgroup, are catechin and epicatechin, catechin Although there are several extraction methodologies for obtaining
gallate and epigallocatechin. They are widely present in brown algae phenolic compounds, two general types of extraction techniques are
species, such as Eisenia bicyclis (Kjellman) Setchell, Sargassum fusiforme found: conventional extraction techniques and non-conventional ex­
(Harvey) Setchell and Saccharina japonica (Areschoug) C.E.Lane traction techniques. The traditional techniques refer to simple solid-
(Mekinić et al., 2019). solvent extractions, and the non-conventional ones include pressurized
liquid extraction, microwave-assisted extraction, ultrasound-assisted
2.4. Lignans extraction and subcritical CO2 extraction, among others.

This type of phenolic compounds is a dimer or oligomer that is 3.1. Traditional techniques
formed due to the union of monolignols – p-coumaryl alcohol, coniferyl
alcohol, and sinapyl alcohol (Vermerris & Nicholson, 2006). Although it Traditional extractions usually include maceration, also known as
was thought it would only be present in terrestrial plants, it has also heat assisted extraction, percolation, and Soxhlet extraction (Aires,
been discovered in calcified intertidal red seaweed Calliarthron chei­ 2017). The use of this kind of extractions is a current practice, widely
losporioides Manza (Freile-Pelegrín & Robledo, 2013). employed around the world. The most common extractive solvents
applied are methanol, ethanol, acetone, water, and ethyl ethanoate in
2.5. Lignins different combinations, and they are selected based on the polarity of
the molecules to extract. Phenolic compounds mostly tend to hydro­
Among polymeric phenols, it is worth mentioning tannins and lig­ philicity, so solvents such as hydroalcoholic mixtures are very effective
nins. These last ones are the most abundant organic polymers in nature, for this process. Some authors have demonstrated that using a combi­
in which their structure is composed of complicated monolignol (cou­ nation of solvent with acids, such as citric acid, tartaric acid, or hy­
maryl, coniferyl, and sinapyl alcohol) and lignan (dimers of mono­ drochloric acid could improve the extraction efficiency of those com­
lignols) units, nonlinearly and randomly linked forming a polymer with pounds (Santos-Buelga, Gonzalez-Manzano, Dueñas, & Gonzalez-
a three-dimensional network. However, they haven’t been extensively Paramas, 2012; Vieira et al., 2018).
studied in marine organisms, such as algae (Tsimogiannis & Within the traditional extractions techniques, it is worth mentioning
Oreopoulou, 2019). that the Soxhlet extraction gets better results in terms of yield, although
this technique also presents some disadvantages such as the degrada­
2.6. Tannins tion of thermolabile compounds (as anthocyanins, hydrolysable tannins
or some phenolic acids) or the requirement of relatively large amounts
Tannins are usually divided into three chemically groups based on of solvents and the long times of processing. Normally, the Soxhlet
their structures: hydrolysable tannins (or pyrogallol-type tannins), fla­ technique is used for the extraction of lipophilic compounds (Santos-
vonoid-based condensed tannins (or polyflavonoid tannins, catechol- Buelga et al., 2012).
type tannins, pyrocatechollic type tannins, nonhydrolyzable tannins, or Regarding heat-assisted extraction, it can be divided into two steps.
flavolans) and phlorotannins. Hydrolysable tannins are derived from The first one is the faster step and consists of a compounds transference
simple phenolic acids and their carbohydrates' hydroxyl groups are from the matrix surface to the solvent. The second one is slower, thus
partially or completely esterified with phenolic groups. Flavonoid- consists of diffusion from the inside part of the matrix to the solvent.
based condensed tannins are formed through biosynthesis of flavins and Critical and determining parameters in this process are the type of
catechins, being scarce the information on the content of flavonoids in sample and solvent selected, and the temperature and time of the ex­
algae. Phlorotannins are oligomers of phloroglucinol that are ex­ traction, so many kinds of approaches can be done using this technique,
clusively found in marine algae, especially in brown algae. according to the large amplitude of the variables involved. The main
Phlorotannins represent the most studied group of phenolic compounds disadvantage is that it is needed a filtration, clarification or decantation
obtained from algae, since it can come to represent up to 25% of the to separate the solid parts when the extraction is finished. Besides, it
alga dry weight (Freile-Pelegrín & Robledo, 2013). Some remarkable usually requires large number of solvents and longs times, a char­
examples of these particular tannins contained just in algae are triph­ acteristic also shared by percolation, a method consisting in a container
loroethol-A, eckol, dieckol, and eckstolonol (Mekinić et al., 2019). where the powder sample is placed, and through which the extractive
solvent is discharged drop by drop from one extreme to another by
2.7. Phenolic terpenoids gravity (Aires, 2017).

This type of compounds have been found in brown (mer­ 3.2. Innovative techniques
oditerpenoids: plastoquinones, chromanols, chromenes) and red mac­
roalgae (diterpenes and sesquiterpenes), more common in Sargassaceae 3.2.1. Pressurized liquid extraction (PLE)
and Rhodomelaceae (Freile-Pelegrín & Robledo, 2013). This type of extraction, also known as extraction with pressurized
solvent (PSE), is characterized by using high pressures (10 to 15 MPa),
2.8. Mycosporine-like amino acids (MAAs) short processing times and temperature ranges that can comprise be­
tween 50 and 200 °C, all using low volumes of non-toxic solvents in the
MAAs have been discovered in many different marine and fresh­ extraction, and thus being considered a green technology (Table 2 part
water species including cyanobacteria, fungi, algae (cyanobacteria, A). The application of four different solvents (hexane, ethyl acetate,
Rhodophyta, and some microalgae, among others) and animals. In the pure ethanol and 50% ethanol) for the extraction of phenolic

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C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

Table 2
Different experimental conditions used for the extraction of bioactive compounds from marine algae.
ALGAE SPECIE EXTRACTION CONDITIONS & YIELDS REFERENCES

A) Pressurized Liquid Extraction (PLE)

L. ochroleuca 1 g algae dw; 1450 bar; 80–160 °C; 37–52% yield. (Otero et al., 2019)
S. muticum 2 g of algae dw; 1500 bar; 120 °C; ethanol:water (75:25, v/v); 20 min; 40% (Sánchez-Camargo et al., 2016)
yield.
Phaeophyta species from Brittany coasts 10 g algae dw; 1500 bar; 75 °C; dichloromethane:methanol (1:1, v/v). (Zubia, Fabre, & Shannon, 2009)
A. nodosum, P. canaliculata, F. spiralis & U. intestinalis 2.5 g algae dw; 1500 bar; 120 °C; acetone:water (80:20, v/v); 60 min; ≈ (Tierney et al., 2013)
20% yield.
C. vulgaris, S. vulgare, Porphyra spp., C. abies-marina, S. 1 g algae dw; 1500 bar; 100/200 °C; 5 min; 12–68% yield (Plaza, Amigo-Benavent, del Castillo,
muticum, U. pinnatifida & H. incurvus Ibáñez, & Herrero, 2010)

B) Microwave Assisted Extraction (MAE)

S. vestitum 0.5 g algae dw; 960 W; 1.25 min; ethanol 70%; 6% yield (Dang et al., 2018)
A. nodosum, L.japonica, L. trabeculate & L. nigrecens 30 g algae dw; 110 °C; 2450 MHz; 15 min; methanol 70%; 5–20% yield. (Yuan et al., 2018)
C. racemosa 4 g algae dw; 50 °C; 200 W; 40 min; ethanol:water 60%; 7% yield. (Li et al., 2012)
E. prolifera 5 g algae dw; 500 W; 25 min; ethanol:water 30%; 10% yield. (HongYu, Bin, ChunGuang, & YinFeng,
2010)

C) Ultrasound Assisted Extraction (UAE)

H. banksii 1 g algae dw; 50 KHz; 150 W; 50 mL; ethanol 70%; 30 °C; 60 min. (Dang et al., 2017)
L. obtuse 1 g algae dw; 40 KHz; 250 W; 30 mL; ethanol 95%; 50 °C; 45 min. (Topuz, Gokoglu, Yerlikaya, Ucak, &
Gumus, 2015)
A. nodosum & L. hyperborea 4–10 g algae dw; 20 KHz; 750 W; 40–200 mL; distilled water (0.03 M HCl); (Kadam et al., 2015a; Kadam, Tiwari,
10–15 min. Smyth, & O’Donnell, 2015)
E. cava 1 g algae dw; 40 KHz; 200 W; 100 mL; different solvents (water 100%, (Lee & Kim, 2015)
methanol 50%, methanol 100% ; 30 °C; 6–15 min.
S. muticum, O. pinnatifida & C. tomentosum 2 g algae dw; 60 KHz; 400 W; 50 mL; deionized water; 50 °C; 60 min. (Rodrigues et al., 2015)
L. japonica 1 g algae dw; 20 KHz; 200 W; 15 mL; ionic liquid [BMIM][BF4]; 60 min. (Han, Zhu, & Row, 2011)

D) Subcritical Water Extraction (SWE)

S. muticum 2 g algae dw; 1500 psi; 50–200 °C; 20 min. (del Sánchez-Camargo, 2017)
S. platensis 2.5 g algae dw; 1500 psi; 115–170; 9–15 min. (Herrero, Martín-Álvarez, Señoráns,
Cifuentes, & Ibáñez, 2005)
F. serratus, L. digitata, G. gracilis & C. fragile 2.5 g algae dw; 1500 psi; 120 °C; 25 min. (Heffernan et al., 2014)
C. abies-marina, Porphyra spp., S. vulgare, S. muticum, 1 g algae dw; 1500 psi; 120–200 °C; 20 min. (Plaza et al., 2010)
U. pinnatifida & H. incurvus

E) Supercritical CO2 Extraction (SC-CO2)

U. pinnatifida 10 g algae dw; 250 bar; 60 °C; 0.12 L/h; 0.83 h; yield of 780 mg/g. (Roh, Uddin, & Chun, 2008)
P. valderianum 10 g algae dw; 500 bar; 50 °C; 120 L/h; 1.5 h; yield of 3.97 mg/g. (Chatterjee & Bhattacharjee, 2014)
C. glomerata, U. flexuosa & C. fragilis 10 g algae dw; 300 bar; 40 °C; 300 L/h; 2 h; yield of 30.20 mg/g. (Fabrowska, Ibañez, Łęska, & Herrero,
2016)

compounds from the brown alga Laminaria ochroleuca Bachelot Pylaie compounds from brown algae showed good results (Yuan et al., 2018).
at 100 bar was tested, observing that the highest extraction yield (37% A study regarding the optimization of the extraction of phenolic com­
for 80 °C and 52% for 160 °C) was obtained using ethanol diluted in pounds from brown alga Sargassum vestitum (R.Brown ex Turner)
water (Otero, López-Martínez, & García-Risco, 2019). C.Agardh was carried out to maximize the extraction yields. The con­
Several studies showed the advantages of this extractive method clusion was that the most important variable affecting this methodology
when carried out in the absence of light and oxygen, because it favors is solvent nature and concentration, followed by radiation time and
the conservation of compounds of interest, such as phenolic compounds power (Dang, Bowyer, Van Altena, & Scarlett, 2018).
(Tierney et al., 2013).
3.2.3. Ultrasound-assisted extraction (UAE)
3.2.2. Microwave-assisted extraction (MAE) This method uses ultrasound waves with a frequency between
The foundation of this method consists of the use of microwave 20 kHz and 100 kHz which originate bubbles due to the pressure dif­
potency that causes changes in cell structures due to electromagnetic ference that is created. Then these bubbles collapse and cavitation
waves. This electromagnetic energy is transformed into calorific energy occur, causing near liquid–solid interfaces breakdown of particles with
by two mechanisms: ionic conduction and dipole rotation (Kalil, the consequent release of bioactive compounds to the matrix. The main
Moraes, Sala, & Burkert, 2017). This technique can be performed in advantages of the application of UAE on the phenolic compounds ex­
open (operate at atmospheric pressure) or closed vessels (pressure traction from algae include low temperatures, short times and low
higher than atmospheric), and it is mainly used for the extraction of amount of solvent (Ciko et al., 2018). However, it must be noticed that
polyphenols and polysaccharides (Ciko, Jokić, Šubarić, & Jerković, ultrasonication time can increase temperature, what can compromise
2018). phenolic compounds’ stability. The parameters to optimize in this kind
Fundamental parameters that must be taken into account for the of extractions are frequency, power, temperature, time, and solid:sol­
optimization of this type of extraction are power and frequency of vent ratio (Heleno et al., 2016) (Table 2 part C).
microwaves, solid-to-solvent ratio, temperature, pressure, and time
(Table 2 part B) (Pinela et al., 2016). It is important to notice that high 3.2.4. Subcritical water extraction (SWE)
microwave power and elevated temperatures may destroy phenolic This method consists of applying water at higher temperatures than
compounds. Regarding extraction solvents, these compounds are easily its boiling point (100–374 °C) under high pressure (10–60 bar) to
dissolved when using hydroalcoholic mixtures with intermediate maintain its liquid state for a short time (5–10 min). One of the most
ethanol concentrations (Ciko et al., 2018). important factors to take into account in this type of extraction is the
The application of this extraction technique to obtain phenolic variability of the dielectric constant with temperature (Herrero,

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C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

Cifuentes, & Ibañez, 2006). Main parameters that should be taken into 5. Robust identification of phenolic compounds
account and that can be optimized when using this methodology are
pressure, time, and temperature, as well as selecting an appropriate Phenols have been detected in samples using very varied techni­
solvent (Zakaria & Kamal, 2016) (Table 2 part D). ques, such as spectrometric, biological, or analytical ones.
Spectrometric assays are usually employed as a screening tool which
3.2.5. Supercritical CO2 extraction (SC-CO2) allows estimating the antioxidant capacity of the samples which is
This method allows multiple combinations of temperature and mostly related to the total content of phenolic compounds. These in
pressure. Besides, CO2, which is a non-toxic gas, is used as a super­ vitro methods can be classified into two groups, based on the transfer
critical fluid so fluid behaves like liquid and gas simultaneously which reaction of a single electron or a hydrogen atom (Vuolo, Lima, &
makes extraction easier. The polarity of CO2 can be modified by the use Maróstica Junior, 2019). The first group includes methods such as
of co-solvents such as ethanol, and in this way also extract polar com­ Trolox equivalent antioxidant capacity (ABTS or TEAC) assay, the ferric
ponents. As low temperatures and pressure are used, the thermal de­ reducing ability of plasma (FRAP) assay, the 2,2-diphenyl-1-picrylhy­
gradation of phytochemicals is prevented (Aires, 2017) (Table 2 part E). drazyl (DPPH) radical scavenging capacity assay or the β-carotene test
(Rojas & Buitrago, 2019; Vuolo et al., 2019). During the redox reaction,
4. Stability of phenolic compounds colorimetric changes are observed, correlated with the concentration of
antioxidant species present in the sample (Vuolo et al., 2019). On the
Phenolic compounds possess high stability when they are in the other group, some examples are the oxygen radical absorbance capacity
original fresh matrix and are well preserved at low temperatures, but, (ORAC) assay, peroxyl scavenging capacity (PSC), or the total peroxyl
when extracted, it is important to take certain precautions to avoid radical-trapping antioxidant parameter (TRAP) assay. Substrates and
degradation. In the case of using algae as raw material to obtain phe­ antioxidant molecules compete to join peroxyl radical and this reaction
nolic compounds, it is advisable to carry out the extraction as quickly as is monitored and evaluated (Vuolo et al., 2019). Another important
possible once the raw material has been acquired. If this is not possible, assay to analyze the effect of phenols is that based on the use of in vivo
the conservation of the matrix is more effective after the application of models, mainly performed on mice and rats, but also guinea-pigs,
any of the following methodologies: freezing, freeze-drying or drying in rabbits and fish (Martins, Barros, & Ferreira, 2016; Rojas & Buitrago,
a steam room. The most common technique used to this purpose is 2019; Vuolo et al., 2019). The antioxidant activity of phenolic com­
drying in the air at room temperature, since drying at high tempera­ pounds is evaluated by the effect displayed on parameters associated
tures normally leads to loss of volatile compounds and unnecessary with the redox homeostasis, such as catalase, superoxide dismutase,
degradation of phenolic compounds (Wong & Chikeung Cheung, 2001). glutathione peroxidase, malondialdehyde, peroxidase, ferric reducing
Phenolic compounds present in algae are especially sensitive to heat antioxidant power, heme oxygenase-1, and many other factors (Martins
and light, particularly UV radiation (López, Caleja, Prieto, Sokovic, et al., 2016; Vuolo et al., 2019). These two categories of assays, espe­
Calhelha, Barros, & Ferreira, 2019). These factors may lead to reduc­ cially the spectrometric ones, are widely applied in the field of phenols
tions in the biological properties of the compounds, due to their de­ but they only permit to estimate and evaluate the number of phenols in
composition, as was shown in a study in which two algae extracts from a sample. To identify and quantify the chemical profile of phenolic
Sargassum muticum (Yendo) Fensholt and Bifurcaria bifurcata R.Ross compounds analytical methods must be used. This method allows the
were submitted to different processes to compare their effects: drying in study of many different molecules in just one experiment; however, this
oven at ~55 °C for 4 h, and greenhouse drying exposed to light, at throughput is subject to the chromatographic separation of the com­
~23 °C for 72 h, resulting in both methods producing significant re­ ponent of the samples. High-performance liquid chromatography
ductions (Agregán et al., 2017; Le Lann, Jégou, & Stiger-Pouvreau, (HPLC) is the most common technique applied for the field of phenols
2008; Lim & Murtijaya, 2007). In general, all drying processes that although other techniques such as gas chromatography (GC) have been
involve temperatures above 40 °C are associated with losses of phenolic used. Regarding identification, many detectors have been coupled to
compounds (Lim & Murtijaya, 2007; Wong & Chikeung Cheung, 2001), HPLC such as the ultraviolet (UV) or photodioarray detectors (PDA).
although there are always exceptions, such as some polyphenols, which Nevertheless, the most relevant instruments to identify or determine
did not suffer apparent degradation during a drying process carried out molecular structures are the HPLC or GC coupled to mass spectrometry
at 60 °C (Larrauri, Rupérez, & Saura-Calixto, 1997). Some authors state (MS) or nuclear magnetic resonance (NMR). Therefore, since analytical
that there are three possible mechanisms responsible for the degrada­ methods are the only identification tool, different examples employed
tion of these compounds at high temperatures: to identify phenolic compounds from algae are described below.
The phenolic content of five microalgae (Chaetoceros calcitrans
(i) the breaking of bonds between phenolic compounds and cell walls; (Paulsen) H.Takano, Isochrysis galbana Parke, Skeletonema costatum
(ii) partial degradation of cell walls polymers due to oxidative changes (Greville) Cleve, Odontella sinensis (Greville) Grunow, and
in the structures of aromatic compounds and; Phaeodactylum tricornutum Bohlin) and one brown macroalga, S. japo­
(iii) thermal degradation of the polyphenols themselves caused by nica, C.Mayes, Druehl & G.W.Saunders, were evaluated using HPLC
oxidative enzymes (e.g. peroxidases), whose deactivation does not coupled to a UV detector. Gallic acid was the most abundant phenolic
occur immediately (Maillard & Berset, 1995). compound, among all 10 analyzed. I. galbana showed the maximum
content of gallic acid with a value of 13.6 mg/g of dry weight (DW).
Another possible cause of degradation of some phenolic compounds Other phenols, including rosmarinic, syringic, or chlorogenic acids,
is a direct exposure to sunlight, hence greenhouse drying is also not a were found in lower concentrations with variable ranges of con­
good idea. In fact, in the extractions carried out in the dark, greater centration 0.1–7.2, 0.9–2.2, and 0.1–2.0 mg/g of DW, respectively (Foo
amounts of total phenolic compounds with potent antioxidant activity et al., 2017).
values were obtained (Le Lann et al., 2008). The use of the drying Similar phenolic compounds were observed in the study of (Klejdus
technique was able to avoid the loss of heat-sensitive products, how­ et al., 2017), where rapid resolution liquid chromatography-tandem MS
ever, there were changes in the native conformation of certain mole­ was used to determine the phenolic compounds present in three brown
cules that could lead to a reduction in bioactive capacities (Franks, algae, Cystoseira abies-marina (S.G.Gmelin) C.Agardh, U. pinnatifida,
1998). Therefore, the best option is the extraction of phenolic com­ and S. muticum, and in the red algae Chondrus crispus Stackhouse. Re­
pounds directly from fresh algae, at low temperatures, and in the ab­ sults showed that p-hydroxybenzoic acid, gallic acid, p-hydro­
sence of light, although, if this is not possible, extraction from frozen xybenzaldehyde, vanillic acid, 3,4-dihydroxybenzaldehyde, and proto­
and/or freeze-dried matrices is recommended (Le Lann et al., 2008). catechuic acid were the phenolic compounds found in higher

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C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

concentration in these algae, while ferulic, p-coumaric, caffeic, syringic, mechanism is mainly through scavenging of free radicals, inhibition of
and chlorogenic acid were found in a lower concentration. (Onofrejová lipid peroxidation, and also activating the endogenous antioxidant
et al., 2010) studied the phenolic compounds present in the brown alga system (Ferreira et al., 2017; Kumar & Goel, 2019; Vuolo et al., 2019).
U. pinnatifida and the red alga Porphyra tenera Kjellman using HPLC- This antioxidant potential of phenolic compounds is strongly related to
electrospray (ESI)-MS. P. tenera showed a higher content in phenolic its structure and many factors are known to affect it, such as the hy­
compounds than U. pinnatifida. For the first alga, p-hydroxybenzoic and droxyl groups, the glycosylation pattern, or the substituents. Firstly, the
salicylic acids were compounds most abundant, with values of 690 and hydroxyl group (–OH) are the donors of hydrogens and electrons, so its
530 ng/g, respectively. For the second one, salicylic p-hydroxybenzoic number and position influence directly the antioxidant ability of phe­
acid was also the most relevant phenols, found in concentrations of 226 nolic compounds. The presence of substituents on the aromatic ring
and 211 ng/g, respectively. affects the stabilization of the molecule and consequently the radical-
Among phenolic compounds, phlorotannins, present in brown algae, scavenge activity (Kumar & Goel, 2019; Vuolo et al., 2019). The gly­
have been quite studied. For example, phenolic compounds of four cosylation of the compounds has been reported to affect the antioxidant
brown algae (Ascophyllum nodosum (Linnaeus) Le Jolis, S. japonica, activity, as it interferes with structure, methylation, and the electron
Lessonia trabeculata Villouta & Santelices, and Lessonia nigrescens Bory) displacement of the molecule (Kumar & Goel, 2019; Vuolo et al., 2019).
were evaluated by LC-DAD-ESI-MS/MS (Yuan et al., 2018). Several Finally, methylation has been shown to reduce antioxidant ability
peaks were tentatively identified as phenolic acid derivatives, galloca­ (Vuolo et al., 2019).
techin derivatives and phlorotannins. Phlorotannins present in Fucus Numerous studies have demonstrated the antioxidant properties of
serratus Linnaeus were studied using an HPLC coupled to a quadrupole algae extracts rich in phenolic compounds. For example, a previously
time-of-flight (qTOF)-MS. Molecules with different polymerization de­ cited study evaluated the antioxidant properties of five microalgae and
grees, between and 6 and 23 phloroglucinol units (PGUs), were found one macroalgae species. Two of the microalgae, C. calcitrans and I.
to be the major one containing 8 to 13 PGUs (Heffernan, Smyth, galbana were demonstrated to be the most relevant ones since their
Fitzgerald, Soler-Vila, & Brunton, 2014). In another study, Ecklonia antioxidant activity was the strongest. Results also showed a clear
stolonifera Okamura extracts were analyzed with a validated HPLC-ESI- correlation between the phenolic content, especially gallic acid, and the
MS method. The major phlorotannins found were dieckol, eckol, and antioxidant capacity of the microalgae and macroalgae extracts tested
phlorofucofuroeckol-A, being the first one the most abundant (Goo, although carotenoids were also involved in this response (Foo et al.,
Choi, & Na, 2010). 2017). Similar results were observed for U. pinnafitida and P. tenera
Several studies have employed NMR to identify phenolic com­ extracts that demonstrated antioxidant activity determined by TEAC
pounds extracted from algae, especially phlorotannins. Phlorotannins assays and attributed to the different phenolic compounds identified in
present in the brown algae F. vesiculosus Linnaeus were evaluated by the samples, such as p-Hydroxybenzoic, salicylic, cinnamic and caffeic
HPLC-UV-PDA based on the presence of the representative monomer acid (Onofrejová et al., 2010). These results were. Antioxidant prop­
unit of this family of compounds, the phloroglucinol. Further analyses erties of brown macroalgae A. nodosum, S. japonica, L. trabeculata, and
performed by NMR confirm that this alga contains phlorotannin mo­ L. nigrescens were studied and significant statistical correlation between
lecules (Koivikko, Loponen, Pihlaja, & Jormalainen, 2007). NMR was the results of antioxidant and the phenolic content of this algae was
also used to study the phlorotannins profile of the brown algae S. mu­ demonstrated (Yuan et al., 2018).
ticum. Extracts purified using solid-phase extraction (SPE) were ana­ Green and red algae have been described to contain lower con­
lyzed. NMR-data showed a high content of a phlorotannin type known centrations of phenolic compounds than brown algae, therefore this last
as phlorethol that comprises different units of phloroglucinol linked group has been further studied which showed the group of the phlor­
through aryl–ether bonds. Although, phlorethol was the main compo­ otannins as the most relevant in these algae. Numerous studies have
nent differences in the composition and quantification of phlorethol demonstrated the antioxidant effects of phlorotannins in diverse cell
were found depending on the geographical distribution of the algae lines. For example, the antioxidant properties of extracts obtained from
(Tanniou et al., 2014). Similarly, the technique NMR allowed identi­ S. muticum were evaluated on Vero cells and HaCaT cells previously
fying and quantifying the variations of phloroglucinol content in the submitted to different pro-oxidant compounds: AAPH (2,2′-azobis-2-
brown algae Cystoseira tamariscifolia (Hudson) Papenfuss throughout methyl-propanimidamide) and H2O2 for the first cell line and UV-B for
the year (Jégou, Kervarec, Cérantola, Bihannic, & Stiger-Pouvreau, the second one. After the treatment with the extract rich in phenols, the
2015). production of ROS decreased in both cell lines showing that S. muticum
extracts revert the oxidation effects (Yu et al., 2019). Similarly, E. cava
6. Mechanisms of action for the bioactivity of phenolic extract demonstrated to be rich in diverse phlorotannins, such as eckol
compounds and dieckol) showed antioxidant effects on macrophage cells but also
on zebrafish embryos, reducing the production of reactive oxygen
Phenolic compounds have been reported to have different proper­ species (ROS) (Kim et al., 2014). The antioxidant effect of extracts from
ties of interest for diverse applications. These molecules exert a broad the same algae species, E. cava, determined to contain high amounts of
range of promising health benefits, being their antioxidant, anti­ dieckol, was investigated as the defense system in obese mice. Dietary
microbial, anti-inflammatory and cytotoxic effects some of the bioac­ supplementation with these extracts showed an increase of catalase and
tivities more studied (Ganzera & Sturm, 2018; Martins et al., 2016; glutathione peroxidases in the liver, reducing the damage caused by a
Vuolo et al., 2019). Besides, several other bioactivities have been as­ high-fat diet.
sociated with them, such as neuroprotective, immunomodulatory, car­
dioprotective, or diuretic (Ferreira, Martins, & Barros, 2017; Vuolo 6.2. Cytotoxic properties
et al., 2019). Thus, phenolic compounds are used in cosmetics, drugs,
and also in the food industry, to enhance food quality and nutritional In the last decades, phenolic compounds present in different marine
benefits and preserve foods (Kumar & Goel, 2019; Martins et al., 2016). algae species have been identified to possess antitumor and cytotoxic
capabilities (Guedes, da Silva, Aguiar, de Barros, & Pinotti, 2013). The
6.1. Antioxidant properties potent antioxidant activity of polyphenols has pointed them as very
promising potential anticancer agents. Phlorotannins are the main
Phenolic compounds have been known to be potent antioxidants, phenolic molecules present and isolated from algae, specifically from
preventing oxidative damage of nucleic acids, proteins, and other bio­ brown algae. The molecular structure of phlorotannins contains many
molecules (Ferreira et al., 2017; Vuolo et al., 2019). Their action hydroxyl groups that have been described as responsible for their

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C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

Table 3
Antitumor, anti-inflammatory and antimicrobial activities of phenolic compounds isolated from marine algae.
ALGAE SPECIE TESTED EXTRACTS ACTIVITIES REFERENCES

A) Antitumor activities
E. cava Ethyl-acetate extract (30% polyphenols) Inhibited migration and invasion of A549 cells (Lee, Kang, Jung, Kim, &
Kim, 2011)
A. esculenta Crude polyphenols extract Reduced viability of Caco-2 cells (Nwosu et al., 2011)
A. nodosum Crude extract (0.5% polyphenols) Showed antiproliferative effects (O’Sullivan et al., 2011)
L. nana Bromophenols Reduced tumour growth (Shi, Li, Guo, Su, & Fan,
2009)
E. cava Crude extract (60% polyphenols) Inhibited MMP-2 and MMP-9 activity (Kim et al., 2006)
U. lactuca Polyphenols extract Reduced viability of Caco-2 cells (Nwosu et al., 2011)
O. colensoi Bromophenols Showed moderate cytotoxic activity against leukaemia (Popplewell &
cells Northcote, 2009)
P. palmata Polyphenols extract Reduced viability of Caco-2 cells (Nwosu et al., 2011)

B) Anti-inflammatory activities
E. bicyclis Phloroglucinol, eckol, phlorofucofuroeckol A & Inhibition of LPS-induced nitric oxide (NO) production in (Jung, Jin, Ahn, Lee, &
dioxinodehydroeckol RAW 264.7 cells Choi, 2013)
E. arborea Phlorofucofuroeckol B Inhibitory effects on histamine release (Sugiura et al., 2006)
P. dentata Catechol & rutin Inhibition of LPS induced NO production in RAW 264.7 (Kazłowska et al., 2010)
cells
V. obtusaloba Vidalols A and B Inhibition of mouse ear inflammation (Wiemer, Idler, &
Fenical, 1991)
E. cava Dieckol Inhibition of LPS induced NO production in murine BV2 (Jung et al., 2009)
microglia
I. okamurae Diphlorethohydroxycarmalol Down-regulation of iNOS and COX-2 expression and NF-B (Heo et al., 2010)
activation in human umbilical vein endothelial cells and
RAW 264.7 cells
E. stolonifera Phlorofucofuroeckol A & B Inhibition of NO production by downregulating iNOS and (Lee et al., 2012)
prostaglandin E2 production in LPS stimulated RAW
264.7 cells
Ishige foliacea Octaphlorethol A Inhibition of pro-inflammatory cytokines, mitogen- (Manzoor et al., 2013)
activated protein kinase and NF-B pathways in CpG-
stimulated macrophage and dendritic cells

C) Antimicrobial activities
U. reticulata, C. occidentalis, C. Rutein, quercetin & kaempherol Inhibiting of Gram-positive and negative bacteria (E. coli, (Al-Saif, 2014)
socialis, D. ciliolata & G. P. aeruginosa, S.aureus, E. faecalis)
dendroides
J. rubens, C. mediterranea, P. Phenol, tannin & flavonoids Controlling the growth of V. fluvialis (Mohy El-Din & El-
capillacea Ahwany, 2016)
E. kurome 8′-bieckol, eckol, dieckol, phloroglucinol & Bactericidal activity (Methicillin-resistant Staphylococcus (Ahn et al., 2004)
phlorofucofuroeckol-A aureus (MRSA), B. cereus, C. jejuni, E. coli, S. enteritidis, S.
typhimurium, V. parahaemolyticus)
E. bicyclis Eckol, dieckol, dioxinodehydroeckol, Effective in inhibiting growth (S. aureus & MRSA) (Eom et al., 2012)
fucofuroeckol-A & phlorofucofuroeckol-A
E. cava Dieckol Fungicidal activity (T. rubrum) (Choi et al., 2010).
E. cava Eckol Potent antimicrobial activity (MRSA) (Choi et al., 2010).
C. rubrum, S. vulgare, S. fusiforme & Phenols Antimicrobial activity (S. aureus & K. pneumoniae) (El Shafay et al., 2016)
P. pavonia

antioxidant capacity. Thus, this group of phenols has been studied to results underline the importance of studying the effect of the molecules
prevent carcinogenic processes (Jiang & Shi, 2018). Some of the results in different in vitro cell lines to identify the best target molecule for each
obtained in these studies can be observed in Table 3 (Part A). carcinogenic model. That is the case of a study in which up to three
The cytotoxic activity of extract obtained from the red macroalga carcinogenic cell lines were used to evaluate the specificity of the cy­
Polysiphonia lanosa (Linnaeus) Tandy was evaluated using two colon totoxic and anti-proliferative effects of 27 marine algae species. Extracts
cell lines, known as DLD-1 and HCT-116. Extracts of this alga were from 14 species of Rhodophyta, 8 of Chlorophyta, and 5 of Phaeophyta
previously characterized using GC–MS showing the presence of lanosol were obtained using a solution of dichloromethane:ethanol (7:3) and
and some derivatives such as ethyl ether, an aldehyde, methyl ether, evaluated against three cancer cell lines: human laryngeal carcinoma
and an n-propyl ether. From these compounds, they were synthesized (Hep-2) cells, human cervical adenocarcinoma (HeLa) cells, and human
some additional ones. The structural determination of all these mole­ nasopharyngeal carcinoma (KB) cells. Udotea flabellum (J.Ellis & So­
cules allowed relating some chemical structures with their cytotoxicity. lander) M.Howec, Udotea conglutinata (J.Ellis & Solander) J.V.La­
The factors that triggered the cytotoxic effects were the number and mouroux (belonging to the genus Chlorophyta) and Bryothamnion tri­
position of the bromine substituent, the number of phenolic groups, and quetrum (S.G.Gmelin) M.Howe (Rhodophyta) showed low IC50 values,
the presence of n-propyl ether derivatives in the side chain. The mo­ thus the application of their extracts in low concentrations represent
lecules containing all these chemical structures were those showing the high cytotoxic effects against of the Hep-2 cell line (IC50 of 22.5, 22.2
strongest mortality against DLD-1 cells. In the case of this study, it was a and 8.2 μg/mL respectively). Similar IC50 values were found for the
synthetic compound named 3c, which increased with longer exposition brown algae extracts obtained from Lobophora variegata (J.V.La­
times up to 24 h with an IC50 of 2.7 µM. When all the molecules were mouroux) Womersley ex E.C.Oliveira and Dictyota caribaea Hörnig &
tested against HCT-116 cells the same 3c compound was the most active Schnetter when tested against KB cells (IC50 of 26.2 and 27.9 μg/mL
one however the n-propyl ether of lanosol showed a cytotoxicity 10-fold respectively) (Moo-Puc, Robledo, & Freile-Pelegrín, 2009). In other
higher than that observed with DLD-1 cells (Shoeib et al., 2004). These studies different species of brown (Scytosiphon lomentaria (Lyngbye)

9
C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

Link, Padina pavonica (Linnaeus) Thivy, and Cystoseira mediterranea agar assay. The flavonoid content for these algae was established in a
Sauvageau) and red algae (Hypnea musciformis (Wulfen) J.V.Lamouroux range from 0.12 to 0.46 mg/g and associated with their antimicrobial
and Spyridia filamentosa (Wulfen) Harvey and brown algae species have activity. The methanolic extracts were the ones that achieved greater
been assayed against different tumor cell lines, such as the human inhibition of the growth of V. fluviales, followed by the ethanolic ex­
breast adenocarcinoma (MCF-7) and the human prostate carcinoma tracts and, finally, the acetone extracts. Likewise, the extracts obtained
epithelium like (DU-145, LNCaP, and PC3). Extracts obtained with di­ from P. capillacea achieved greater inhibition halos than those obtained
chloromethane, chloroform, and ethanol of H. musciformis achieved from the other algae, proving that they contained mostly beneficial
cytotoxic activity Extracts from S. filamentosa, C. mediterranea and P. fatty acids, as well as 1,2-benzenedicarboxylic acid (Mohy El-Din & El-
pavonica showed reasonable cytotoxic activities against all the carci­ Ahwany, 2016).
nogenic cell lines. The strongest effect was observed when treating cells Other relevant molecules, also belonging to phenolic compounds
with S. filamentosa extract at a concentration of 100 μg/mL for 24 h. In and present in seaweed, are phlorotannins. Numerous extracts rich in
these conditions, it was demonstrated to reduce the cell viability of the these substances have been studied. As an example, phlorotannins ex­
four cell cultures at levels lower than 20% using, except for LNCap that tracted from Ecklonia kurome Okamura (8,8′-bieckol, eckol, dieckol,
reduce it up to 40%. In the case of C. mediterranea and P. pavonica, the phlorofucofuroeckol-A and phloroglucinol) were found to have bac­
viability of the cells was reduced to this same level (40%) when treating tericidal effects against a wide range of pathogenic microorganisms of
them for 48 h with extract at concentrations of 200 μg/mL (Taskin, current relevance, such as methicillin-resistant S.aureus (MRSA),
Caki, Ozturk, & Taskin, 2010). In other work, the same the human Bacillus cereus, Campylobacter jejuni, E. coli, Salmonella enteritidis,
breast adenocarcinoma (MCF-7) together with other models such as the Salmonella typhimurium, and Vibrio parahaemolyticus. After that, in vivo
promyelocytic leukemia (HL-60) or myelogenous leukemia (K562) cells assays were performed to discard toxic effects. The daily oral admin­
were used to test the cytotoxicity of two species collected from the coast istration of doses between 170 and 1500 mg of phlorotannins per kg of
of Hong Kong, Hydroclathrus clathratus (C.Agardh) M.Howe and Padina body weight for 14 days did not produce significant toxic adverse ef­
arborescens Holmes. Aqueous extracts could inhibit the growth of both fects in mice. Regarding the mechanism of action, phlorotannins have a
carcinogenic culture cell models, MCF-7 and HL-60, without producing harmful effect on bacterial proteins, preventing their growth
high toxicity in non-tumor cells. Although, the most promising result (Nagayama, Iwamura, Shibata, Hirayama, & Nakamura, 2002). Phlor­
was that obtained with a dichloromethane extract obtained from H. otannins were also found in E. bicyclis, specifically eckol, dieckol, di­
musciformis, which showed an IC50 of 3.8 μg/mL against K562 cells, oxinodehydroeckol, fucofuroeckol-A, 7-phloroeckol and phlor­
improving even the IC50 values achieved by the control substance, the ofucofuroeckol-A. These compounds showed IC50 32–64 µg/mL against
etoposide (Wang, Liang, Astronomo, Hsu, Hsieh, Burton, & Wong, S. aureus and MRSA (Eom et al., 2012b).
2008). Phenolic compounds found in algae have also been demonstrated to
Some authors point out that phenolic compounds can inhibit the possess antifungal properties, as reported by some studies performed
telomerase activity of tumor cells by suppressing their expression, thus with dieckol and eckol extracted and purified from E. cava. Both com­
achieving an anticancer effect (Guedes et al., 2013). pounds achieved a potent antifungal activity against Trichophyton ru­
brum Malmsten induced by two different mechanisms of action: the
6.3. Antimicrobial properties inhibition of cell metabolism and the disordered cell membrane.
Besides, the minimum inhibitory concentration (MIC) against MRSA
Antimicrobial activities are another well-known bioactivity related was between 125 and 250 µg/mL (Choi et al., 2010; Lee, Lee, Oh, Lee, &
to phenolic compounds, as the scientific literature shows and we discuss Chee, 2010).
below. Extracts obtained from different algae species belonging to Table 3 compiles additional examples in which extracts obtained
Chlorophyta (Ulva reticulata Forsskål, Caulerpa occidentalis (J. Ag.) from a wide variety of algae have been evaluated as antimicrobial
Boergs and Cladophora social Kützing), Phaeophyta (Dictyota ciliolata agents and show how phenolic compounds with different chemical
Sonder ex Kützing), and a Rhodophyta (Gracilaria dendroides Gargiulo, natures achieve potent antibacterial activities against a wide range of
De Masi & Tripodi), were tested against pathogenic bacteria, both microorganisms of current clinical importance (Table 3 part B). Cur­
Gram-positive and Gram-negative: Escherichia coli, Pseudomonas aeru­ rently, antibiotic resistance represents a worldwide threat to human
ginosa, Staphylococcus aureus, and Enterococcus faecalis. Different sol­ and animal health. Therefore, phenolic compounds, or synergistic
vents (ethanol, chloroform, petroleum ether, and water) were used to combinations thereof with other substances, such as fatty acids, halo­
perform the extractions. The highest and most significant antibacterial genated compounds or terpenes, offer possible renewed solutions to
activity was achieved using chloroform extracts of all the five species, combat the microbial resistance to antibiotics that arise (El Shafay, Ali,
followed by ethanolic ones. All the five chloroform-based extracts & El-Sheekh, 2016; Lee et al., 2014)
showed a stronger effective inhibition of the growth of E. coli and P.
aeruginosa than for S. aureus and E. faecalis. Growth was inhibited by all 6.4. Anti-inflammatory properties
these extracts, except that obtained from C. occidentalis. An interesting
result is that ethanol and chloroform extracts of the five species re­ Marine algae are responsible for many anti-inflammatory products
corded stronger inhibitory activities against E. coli than that for ampi­ on the market over the last few years (Abad, 2013). Phlorotannins,
cillin. In general terms, regarding the most effective species, the results present in brown algae, are particularly important for this bioactivity
pointed to the red algae G. dendroides, followed by U. reticulata, and because they are pro-inflammatory cytokines inhibitors capable of
thirdly, the brown algae D. ciliolata. The phenolic compounds rutein, acting in lipopolysaccharides (LPS) stimulated microglial cells as in­
quercetin, and kaempherol, each one belonging to the flavonoid group, ducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor
were present in all the extracts obtained and were the compounds re­ necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and inter­
sponsible for this antimicrobial capacity (Al-Saif, 2014). Other studies leukin-6 (IL-6) (Fernando et al., 2016; Ryu & Kim, 2012).
are in line with these results, showing that other phenolic compounds Extracts of Porphyria dentata C.Agardh, a red edible seaweed are
possess the capacity of inhibiting the growth of different pathogenic known worldwide in folk medicine for the treatment of various in­
microorganisms. The antimicrobial capability of three different algae flammatory diseases, has been also studied. Some of the phenolic
species, Jania rubens (Linnaeus) J.V.Lamouroux, C. mediterranea, and compounds that were identified were hesperidin, rutin, and catechol.
Pterocladiella capillacea (S.G.Gmelin) Santelices & Hommersand, was The extract containing these compounds inhibited the production of NO
analyzed testing different solvents (methanol, ethanol, acetone, and in LPS-stimulated RAW 264.7 cells. They also tested the isolated com­
chloroform) against Vibrio fluvialis using the halo inhibition method on pounds by themselves and catechol proved to be a potent suppressor of

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C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

the up-regulation of iNOS promoter and NF-κB enhancer. It had a lot 7. Conclusions and further prospects
better results than rutin and hesperidin alone was unable to inhibit
either activity (Kazłowska, Hsu, Hou, Yang, and Tsai (2010). A wide variety of compounds extracted from seaweed are currently
Vidalia obtusiloba (Mertens ex C.Agardh) J.Agardh is also a red alga used in various commercial product groups as functional food in­
that contains phenolic compounds with anti-inflammatory proprieties. gredients, as natural nutraceuticals obtained from marine environ­
Bromophenolic metabolites were isolated from this alga, namely vida­ ments, as biomedical materials, in cosmetic preparations, as well as in
lols A 313 and B 314 that act as anti-inflammatory compounds by in­ other products that promote human health (Sun et al., 2018).
hibiting the phospholipase A2 enzyme. Table 3 (part C) summarizes the The application of phenolic compounds extracted from algae as
phenolic compounds that have been isolated from algae and their anti- functional ingredients could also offer new opportunities to continue
inflammatory activities (El Gamal (2010). developing products for therapeutic, palliative or prophylactic pur­
poses, which have beneficial effects on human health. These facts, to­
gether with the growing consumers demand of products of natural
6.5. Other bioactivities origin, have aroused great interest in the industries, particularly in the
food, pharmaceutical and cosmetic industries, make algae an ideal
Dieckol is also associated with antidiabetic proprieties. When natural matrix from which to extract phenolic compounds for purposes
testing dieckol extracted from brown seaweed, (Kang et al., 2013) of industrial application, so that they behave as natural ingredients that
discovered that it also had antidiabetic potential because the levels of replace synthetic ones, associated with certain disorders or allergies, or
blood glucose, serum insulin, and body weight diminished when com­ as active and functional ingredients that provide beneficial health ef­
pared to a diet containing this molecule with a control (without fects. Besides, algae can be easily obtained, since their cultivation
dieckol). through aquaculture is currently booming and increasingly being op­
Another study tested A. nodosum extracts and their capability to timized, so that it is easy, fast, economic and productive, compared to
inhibit α-glucosidase and α-amylase. These proteins are responsible for the cultivation of land plants. Although “algaquaculture”, as some au­
starch digestion and blood glucose regulation and they are key enzymes thors define the cultivation of algae, is mainly focused on the produc­
that, thus act at very low levels. The extracts from this edible seaweed tion of algae with alimentary purposes, it could be also advantageous to
contained up to 4.5 mg/g DW and showed very effective inhibition of produce algae to extract bioactive compounds from them, such as
the α-amylase activity with an IC50 of 0.1 μg/ml GAE while it displayed phenolic compounds. Another option to obtain those beneficial mole­
a higher IC50, 20 μg/ml GAE, for the inhibition of the α-glucosidase The cules could be the use of invasive algae species that migrate to other
low concentrations that these extracts require to inhibit enzymatic ac­ areas causing environmental and economic damages, so they must be
tivities are easily achievable in the gut. Therefore, they represent an removed and eliminated.
affordable alternative to other anti-diabetic drugs (Nwosu et al. (2011). Furthermore, algae contain certain phenolic compounds that are
Algae extracts from E. stolonifera containing phlorotannin, dieckol, exclusively found in them in nature, phlorotannins. Those are re­
and eckol were studied, resulting in possessing antihypolipidemic ac­ sponsible of the exertion of bioactivities whose use could improve the
tivities. These compounds were tested in hyperlipidemic rats and were human health, raising algae value and uniqueness as raw natural ma­
able to significantly reduce the levels of LDL cholesterol, total choles­ terial. Although algae constitute a highly widespread renewable re­
terol, and triglyceride, and significantly increase the level of HDL source, most seaweed are underexploited and processed into fertilizers
cholesterol (Yoon, Kim, Chung, and Choi (2008). and animal feeds. Pre-processing operations and extraction processes
Marine organisms like algae are very exposed to extreme solar ra­ conditions play important roles in improving or reducing the phenolic
diation. Their defense mechanism is the production of a large variety of compounds content, thus altering their health benefits. This review
photo-protective and anti-photoaging compounds. These compounds collects the most recent applied techniques to extract phenolic com­
are capable of absorbing UV-A and UV-B rays and some of them can pounds and the suitable conditions of the different variables involved to
even scavenge the ROS produced and inhibit the formation of free ra­ avoid losses and guarantee their stability; as well as a classification of
dicals. Several extracts from different marine algae shown photo-pro­ the phenolics found in algae and a description of the biological prop­
tective functions and these extracts were rich in phenolic compounds erties associated with them. Regarding phenolic compounds’ identifi­
like shinorine, porphyra-334, palythene, eckstolonol, eckol, sarga­ cation and quantification, most studies report their results as “total
chromenol, tetraprenyltoluquinol chromane meroterpenoid, scyto­ phenolic compounds”, instead of providing a list of the specific mole­
nemin, and sargaquinoic acid, all compounds with photo-protective cules, since their identification is still difficult due to the lack of in­
capacity (H. D. Wang, Li, Lee, & Chang, 2017). formation concerning this area.
Eckstolonol isolated from E. cava was also tested, proving that this Therefore, algae seem to have a promising future within the phar­
phenolic compound was able to protect HaCat cells from photo-oxida­ maceutical and cosmetic industries (Barlow, Sims, & Quinn, 2016).
tive stress. Eckstolonol (200 µM) was shown to repair the damage However, numerous in vivo analysis beyond rats are necessary, that is,
produced by the UV-B rays due to the activation of catalase and su­ to move into the clinical phases of research so that bioactivities are
peroxide dismutase enzymes by removing the increased ROS (Jang tested and toxicity tests are conducted on human subjects in search of
et al. (2012). possible and promising applications of these compounds in different
Phenolic compounds extracted from marine algae also demon­ areas concerning the human being’s health. Likewise, it would be in­
strated antiviral activity, specifically anti-HIV compounds. (Ahn et al., teresting to elucidate more about phenolic compounds’ identification
2004) isolated two phlorotannins, 8,8′-bieckol and 8,4′’’-dieckol, and and the specific mechanisms of action associated with the structures of
these have shown an inhibitory effect on HIV-1 reverse transcriptase each compound, so that structure–activity relationships that allow
and protease in vitro. 6,6′-Bieckol, a phloroglucinol derivate, extracted molecular modifications can be established to achieve more appropriate
from E. cava also shown to be a potent inhibitor against HIV-1 and characteristics or properties for certain applications.
induced syncytia formation, lytic effects, and viral p24 antigen pro­
duction in vitro and in cellular experiments. Additionally, it was able to CRediT authorship contribution statement
inhibit the activity of HIV-1 reverse transcriptase enzyme with an IC50
of 1.07 µM, presenting no cytotoxicity at the tested concentrations C. Jimenez-Lopez: Writing - original draft. A.G. Pereira: Writing -
where it has inhibited HIV-1 replication almost completely (Artan et al., original draft. C. Lourenço-Lopes: Writing - original draft. P. Garcia-
2008). Oliveira: Writing - original draft. L. Cassani: Writing - original draft.
M. Fraga-Corral: Writing - original draft. M.A. Prieto: Writing -

11
C. Jimenez-Lopez, et al. Food Chemistry 341 (2021) 128262

original draft, Conceptualization, Methodology, Writing - review & Dang, T. T., Bowyer, M. C., Van Altena, I. A., & Scarlett, C. J. (2018). Optimum conditions
editing. J. Simal-Gandara: Writing - original draft, Conceptualization, of microwave-assisted extraction for phenolic compounds and antioxidant capacity of
the brown alga Sargassum vestitum. Separation Science and Technology (Philadelphia),
Methodology, Writing - review & editing. 53(11), 1711–1723.
Dang, T. T., Van Vuong, Q., Schreider, M. J., Bowyer, M. C., Van Altena, I. A., & Scarlett,
Declaration of Competing Interest C. J. (2017). Optimisation of ultrasound-assisted extraction conditions for phenolic
content and antioxidant activities of the alga Hormosira banksii using response surface
methodology. Journal of Applied Phycology, 29(6), 3161–3173.
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culture to improve the production of phenolic compounds: A review. Industrial Crops
interests or personal relationships that could have appeared to influ­ and Products, 82, 9–22.
ence the work reported in this paper. El Gamal, A. A. (2010). Biological importance of marine algae. Saudi Pharmaceutical
Journal, 18(1), 1–25.
El Shafay, S. M., Ali, S. S., & El-Sheekh, M. M. (2016). Antimicrobial activity of some
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