foods

Article
Bioactive Phenolic Metabolites from Adriatic Brown Algae
Dictyota dichotoma and Padina pavonica (Dictyotaceae)
Ivana Generalić Mekinić 1, * , Vida Šimat 2, * , Viktorija Botić 1 , Anita Crnjac 1 , Marina Smoljo 1 , Barbara Soldo 3 ,
Ivica Ljubenkov 3 , Martina Čagalj 2 and Danijela Skroza 1

1 Department of Food Technology and Biotechnology, Faculty of Chemistry and Technology, University of Split,
R. Boškovića 35, HR-21000 Split, Croatia; viktorijabotic9@gmail.com (V.B.); anitaaklanac@gmail.com (A.C.);
marinasmoljo.1@gmail.com (M.S.); danci@ktf-split.hr (D.S.)
2 Department of Marine Studies, University of Split, R. Boškovića 37, HR-21000 Split, Croatia;
martina.cagalj@unist.hr
3 Department of Chemistry, Faculty of Science, University of Split, R. Boškovića 33, HR-21000 Split, Croatia;
barbara@pmfst.hr (B.S.); ivica.ljubenkov4@gmail.com (I.L.)
* Correspondence: gene@ktf-split.hr (I.G.M.); vida@unist.hr (V.Š.);
Tel.: +385-21329458 (I.G.M.); +385-21510192 (V.Š.)

Abstract: In this study, the influences of temperature (20, 40 and 60 ◦ C) and extraction solvents (water,
ethanol) on the ultrasound-assisted extraction of phenolics from the Adriatic macroalgae Dictyota



dichotoma and Padina pavonica were studied. The extracts were analysed for major phenolic sub-

 groups (total phenolics, flavonoids and tannins) using spectrometric methods, while the individual
Citation: Generalić Mekinić, I.; phenolics were detected by HPLC. The antioxidant activities were evaluated using three methods:
Šimat, V.; Botić, V.; Crnjac, A.; Smoljo, Ferric Reducing/Antioxidant Power (FRAP), scavenging of the stabile 2,2-diphenyl-1-picrylhydrazyl
M.; Soldo, B.; Ljubenkov, I.; Čagalj, (DPPH) radical and Oxygen Radical Antioxidant Capacity (ORAC). The aim of the study was also to
M.; Skroza, D. Bioactive Phenolic find the connection between the chemical composition of the extracts and their biological activity.
Metabolites from Adriatic Brown
Therefore, principal component analysis (PCA), which permits simple representation of different
Algae Dictyota dichotoma and Padina
sample data and better visualisation of their correlations, was used. Higher extraction yields of the
pavonica (Dictyotaceae). Foods 2021,
total phenolics, flavonoids and tannins were obtained using an alcoholic solvent, while a general
10, 1187. https://doi.org/10.3390/
conclusion about the applied temperature was not established. These extracts also showed good
foods10061187
antioxidant activity, especially D. dichotoma extracts, with high reducing capacity (690–792 mM
Academic Editors: TE) and ORAC values (38.7–40.8 mM TE in 400-fold diluted extracts). The PCA pointed out the
Oscar Martinez-Alvarez and Maria significant influence of flavonoids and tannins on the investigated properties. The results of this
Elvira López-Caballero investigation could be interesting for future studies dealing with the application of these two algae in
foods, cosmetics and pharmaceuticals.
Received: 27 April 2021
Accepted: 23 May 2021 Keywords: antioxidants; biological activity; brown seaweeds; extraction mode; phenolic compounds
Published: 25 May 2021

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in 1. Introduction
published maps and institutional affil-
Macroalgae (seaweeds), which are organisms that inhabit marine or freshwater habi-
iations.
tats, have attracted the interest of researchers as they are a proven source of various
biologically active metabolites. These components are generated as a protection against
free radicals or other oxidizing substances that affect macroalgae since they are exposed to
adverse environmental conditions [1–5].
Copyright: © 2021 by the authors.
Based on their photosynthetic pigment type, type of storage material and composition
Licensee MDPI, Basel, Switzerland.
of cell wall polysaccharides, macroalgae are usually divided into three groups: green, red
This article is an open access article
and brown algae [6,7]. Brown algae constitute the largest class (with up to 2000 species) [8]
distributed under the terms and
and are probably the most investigated, as these species contain a special group of bio-
conditions of the Creative Commons
logically active phenolics called phlorotannins [7,9]. The phenolic compounds present in
Attribution (CC BY) license (https://
brown algae species are derived from polymerised phloroglucinol units via the acetate
creativecommons.org/licenses/by/
4.0/).
malonate pathway [10]. Phlorotannins play a role in protecting algae against herbivores,

Foods 2021, 10, 1187. https://doi.org/10.3390/foods10061187 https://www.mdpi.com/journal/foods

Foods 2021, 10, 1187 2 of 10

bacteria and fouling organisms. They are also involved in protection against oxidative
damage, as their antioxidant activity is 10 to 100 times more powerful than that of other
polyphenols [11,12]. They have also been broadly investigated since they have a large spec-
trum of positive biological properties such as UV protective actions and anti-inflammatory,
anti-angiogenic, anti-allergic and antidiabetic effects [13,14]. Phlorotannins exist in soluble
or in cell-wall-bound forms. Based on the number and distribution of hydroxyl groups and
the nature of the structural linkages between phloroglucinol units, they can be divided into
phlorethols, fuhalols, fucols, fucophlorethols and eckols [5,12,15,16]. Seaweeds are also an
excellent source of other biologically active compounds such as polysaccharides (fucoidan,
laminarin and alginates), peptides, polyunsaturated fatty acids, pigments (carotenoids),
minerals, sterols, etc. [17,18].
In recent decades, great attention has been devoted to the search for new, bioactive-
compound-rich and inexpensive natural sources of valuable phytochemicals with beneficial
biological activities. Seaweeds from diverse habitats have great potential due to their
valuable nutritional profile, low caloric value and medicinal benefits. They are often
used as vegetables (fresh or dried) and/or ingredients in numerous dishes, and they are
also often used as valuable ingredients in the formulation of functional foods [17–19].
The brown macroalgae Dictyota dichotoma and Padina pavonica (Dictyotaceae) are species
widespread in the Adriatic Sea, but there are limited or scarce data on their chemical
composition [20,21], especially their phenolic content [22]. Due to the long duration and
application of high temperatures in conventional extraction protocols, negative effects on
their health-promoting components are often observed. Therefore, there is an increasing
trend toward the investigation of novel, environmentally friendly extraction technologies
for the preparation of highly valuable extracts. Among the different new technologies,
the use of ultrasound-assisted extraction is probably one of the easiest, cheapest and,
consequently, most widely used [5,15,18,23].
The aim of this study was to investigate the biological potential of D. dichotoma and
P. pavonica and the impact of the extraction mode (ultrasound, solvent and temperature
regime) on their phenolic profile. Furthermore, the connection between the phenolic profile
of the algae and related activity was investigated in order to find the extraction parameters
best suited for producing bioactive extracts.

2. Materials and Methods

2.1. General
All chemicals, reagents and solvents used were of adequate analytical grade and
were obtained from Kemika (Zagreb, Croatia) and Sigma-Aldrich (St. Louis, MO, USA).
Spectrophotometric measurements were performed on a SPECORD 200 Plus, Edition
2010 (Analytik Jena AG, Jena, Germany) and a Synergy HTX Multi-Mode Reader (BioTek
Instruments, Inc., Winooski, VT, USA). The high-performance liquid chromatography
(HPLC) system used was the Perkin Elmer Series 200 with a UV/VIS detector (Perkin-Elmer
Inc., Shelton, CT, USA), and the phenolic compounds were separated on an UltraAqueous
column (C18, 250 × 4.6 mm, 5 mm, Restek, Bellefonte, PA, USA).

2.2. Seaweed Material and Preparation of Extracts

Seaweed materials, Dictyota dichotoma (Hudson) J. V. Lamouroux and Padina pavonica
(L.) Gaill. were collected in August 2020 on the coast of Čiovo Island (Central Dalmatia,
Croatia, 43.523492◦ N, 16.285571◦ E). Zvjezdana Popović Perković, a marine botanist from
the University of Split’s Department of Marine Studies, confirmed the botanical identity of
the algal materials. The algal biomass was harvested by hand and then washed thoroughly
with fresh water to remove epiphytes. The materials (algae thalli) were air-dried for 15 days
in a shaded and aerated place at room temperature, and then, they were pulverised (1 min
in high-speed grinder, Model 980, Moulinex, France) and used for the preparation of
extracts.
Foods 2021, 10, 1187 3 of 10

The algal extracts were prepared by ultrasound-assisted extraction (Sonorex RK 103H,

Bandelin, Berlin, Germany) using different solvents: ethanol and water at three different
temperatures of 20 (room temperature, RT), 40 and 60 ◦ C. For algal extracts, 1 g of dried
and ground samples was weighed and 10 mL of solvent was added. The duration of
the extraction was 1 h, following which the suspensions were filtered and centrifuged
(10 min, 4000 rpm, Centric 322 A, Tehtnica, Slovenia). Extractions were carried out in three
repetitions for each plant material, and all sample extracts were combined in a total extract
that was used for further analysis.

2.3. Phenolic Composition

2.3.1. Spectrophotometric Analysis of Phenolic Subgroups
The total phenolic content in samples was determined by the Folin–Ciocalteu method [24]
and the results are expressed as mg of gallic acid equivalents per litre of extract (mg GAE/L).
Total flavonoids were determined using the colorimetric method reported by
Yang et al. [25]. The results are expressed as mg of quercetin equivalents per litre of extract
(mg QE/L).
Tannins were detected using vanillin-HCl according to the procedure described by
Julkunen-Titto [26]. In this assay, catechin was used as the standard and the results are
expressed as mg catechin equivalents per litre of extract (mg CE/L).

2.3.2. HPLC Analysis of Individual Phenolics

For the separation, quantification and identification of individual phenolics, the HPLC
method was used. The method was described in the study of Generalić Mekinić et al. [27].
The flow rate was 0.8 mL/min and the signal was monitored at 280 nm. The following
solvents were used: solvent A was water/phosphoric acid (99.8:0.2, v/v) and solvent B was
methanol/acetonitrile (50:50, v/v). The detected phenolic acids were identified by com-
paring their retention times and absorption spectra with those acquired for corresponding
standards and by sample spiking. The compounds were quantified using external standard
calibration curves. Phloroglucinol was detected using the same method but the signal was
monitored at 267 nm, where the maximal absorption spectrum of this compound has been
recorded. The results are expressed as mg of compound per litre of extract (mg/L).

2.4. Antioxidant Activity

2.4.1. Ferric Reducing/Antioxidant Power (FRAP)
The reducing activity of the samples was measured as FRAP value according to the
procedure reported by Benzie and Strain [28]. This method is used to measure the ability
of samples to reduce ferric-tripyridyltriazine (Fe3 + -TPTZ) to a ferrous-tripyridyltriazine
complex (Fe2 + -TPTZ) at a low pH value (3.6). Diluted extracts (10 µL) were added to a
freshly prepared FRAP reagent (300 mM acetate buffer:TPTZ in 40 mM HCl:20 mM FeCl3
× 6H2 O = 10:1:1) (300 µL) and the absorbances were measured at 593 nm. The results
obtained by this method are expressed in millimoles of Trolox equivalents per litre (mM
TE) [25].

2.4.2. Radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH)

The free radical scavenging activity against DPPH· was determined according to
the procedure described by Katalinić et al. [29]. The decrease in absorbance of the initial
DPPH solution (300 µL) after addition of the sample (10 µL) was monitored at 517 nm after
1 h. The results of the “quenching” reactions of antioxidants with DPPH are expressed in
micromoles of Trolox equivalents per litre (µM TE).

2.4.3. Oxygen Radical Antioxidant Capacity (ORAC)

An ORAC assay was performed according to the procedure described by Generalić
Mekinić et al. [27]. For the measurements, 25 µL of the diluted sample/phosphate buffer
(blank)/standard (Trolox) was added to a well of a black 96-well plate. After that, 150 µL
Foods 2021, 10, 1187 4 of 10

of the fluorescein was added, and after incubation for 30 min, 25 µL of the 2,20 -azobis(2-
methylpropionamidine) dihydrochloride solution was added to initiate the reaction. The
reaction was monitored every minute for 80 min, and the results are expressed as mM TE.

2.5. Statistical Analysis

For data analysis, STATISTICA (Data Analysis Software System, v. 13, StatSoft Inc.,
Tulsa, OK, USA) was used. Pearson’s correlation coefficient and principal component
analysis (PCA) were used for determining the relations between the variables. All data are
expressed as mean ± standard deviation (SD).

3. Results and Discussion

Phenolic compounds are plant secondary metabolites, and the isolation, characteri-
sation and investigation of their biological activities have been the aim of much research.
Generally, phenolics, but also the subgroup of phlorotannins, which are the dominant
brown algal phenolics, are proven to have positive pharmacological and nutraceutical
properties [2,12,14]. These health-promoting activities of phenolics are critical for neu-
tralising the effects of oxidizing agents (such as free radicals) that are generated during
metabolism due to exposure to extreme conditions (e.g., UV radiation, salinity, temper-
ature, high oxygen concentrations, etc.). The concentration of phenolics in brown algae
is influenced by different abiotic and biotic factors, while their analysis is influenced by
their chemical nature, the applied extraction procedure, storage conditions, the presence of
interfering substances, etc. [5,30]. In this study, different extraction modes were used to
obtain the most effective algae extracts with the highest share of phenolics. Recent trends
in algal research have also focused on the investigation and application of innovative
technologies in improving the extraction efficiencies, and ultrasound-assisted extraction is
a low-cost method that is often used [5,18,31,32].
As it is well known that the extraction rate may be improved by the modification of
process variables, the ultrasound-assisted extractions of algal samples were performed at
different temperatures (20, 40 and 60 ◦ C) and using different solvents (water and ethanol).
The compound chemical nature is a restricting factor in finding a suitable extraction solvent
system [5]. Although researchers use various organic solvents in order to obtain extracts
with a high share of phenolics [3,27–30,32], water and ethanol are preferred in the food,
pharmaceutical and cosmetic industries due to economic, toxicological and environmental
reasons [33]. Due to variations in the polarity of the phenolic compounds, it is expected
that hydroalcoholic mixtures could be the most suitable solvents. This is confirmed by the
results of this study, as a better extraction yield was obtained using alcoholic solvents in
contrast to water solvents. Figure 1 shows the total contents of phenolics, flavonoids and
tannins in extracts of D. dichotoma and P. pavonica.
The total phenolic content ranged from 127 mg GAE/L in the water extract of D.
dichotoma prepared at 40 ◦ C, to 423 mg GAE/L in the ethanolic extract of P. pavonica.
Although the use of high temperatures usually leads to a kinetic improvement, it is often
limited by the fact that most phenolics are not thermostable, so heat treatments could reduce
the total extracted amount [34]. Garcia-Vaquero et al. [31] also reported that ultrasound-
assisted extraction at 40 ◦ C (for 30 min) resulted in phenolic extracts with the highest yield
and maximal antioxidant capacity. As can be seen from the obtained results, the extracts
prepared at RT in all cases, except for ethanolic extracts of P. pavonica, yielded the highest
amount. The share of phenolics in the ethanolic extracts of D. dichotoma prepared at RT was
29% higher than that in the water extracts, and 56% and 46% higher in the ethanolic extracts
prepared at 40 and 60 ◦ C, respectively. Although there were no significant differences
between the phenolic potential of the D. dichotoma and P. pavonica water extracts, the
content of phenolics in the ethanolic extracts was significantly higher. Other authors also
investigated phenolics from D. dichotoma [2,23,34–36] and P. pavonica [3,22,23,33,34], but
comparison of the results is difficult due to the employment of different extraction protocols
(solvents used, time of extraction, temperature, additional actions such as stirring, use of
Foods 2021, 10, 1187 5 of 10

novel techniques, etc.) or due to results’ expression using different standard compounds.
However, the results of our study on the influence of drying and extraction methods on P.
pavonica phenolics showed better extraction yields in water extracts (more than twofold
higher results were obtained) [23]. Figure 1b also shows the distribution of flavonoids
among samples. It can be seen that the ethanolic extracts are richer in these valuable
compounds, especially extracts of D. dichotoma (concentration range from 871 mg QE/L
in extract prepared at 60 ◦ C to 975 mg QE/L in extract prepared at RT). Kosanic et al. [22]
also reported a higher content of flavonoids in extracts of D. dichotoma than in P. pavonica,
while Čagalj et al. [23] obtained a higher content of flavonoids in ethanolic extracts. All
other samples contained significantly lower amounts, from 23 to 160 mg QE/L. Similar
results were obtained for the total tannins (Figure 1c), where, again, the highest amounts
Foods 2021, 10, x FOR PEER REVIEW 5 of 10
of these compounds were detected in the ethanolic extracts of D. dichotoma (from 0.34 to
0.39 mg CE/L).

Figure 1. Total contents of (a) phenolics, phloroglucinol in Dictyota dichotoma (D) and
phenolics, (b) flavonoids, (c) tannins and (d) phloroglucinol
Padina pavonica (P) extracts.

individual
The total phenolic
phenolic content acids in the
ranged D. dichotoma
from 127 mg GAE/Land P. inpavonica extracts
the water detected
extract by
of D. di-
HPLC are presented in Table 1, while the phloroglucinol content is shown
chotoma prepared at 40 °C, to 423 mg GAE/L in the ethanolic extract of P. pavonica. Alt- in Figure 1d. Ac-
cordingthe
hough to the
usepresented results, the dominant
of high temperatures phenolic
usually leads to aacid in the
kinetic extracts of D.itdichotoma
improvement, is often
was trans-ferulic acid, with the highest concentrations in the ethanolic
limited by the fact that most phenolics are not thermostable, so heat treatments extract prepared at
could re-
duce P. pavonica,
RT. Inthe the dominant
total extracted amountphenolic was protocatechuic
[34]. Garcia-Vaquero acid.
et al. [31] reportedtrans-ferulic
Although
also that ultra-
acid was also found
sound-assisted in the
extraction at P.
40pavonica
°C (for 30 extracts, its concentration
min) resulted in phenolicwas onlywith
extracts significant in
the high-
the water extract prepared at RT (1.22 mg/L). While the levels of o-coumaric
est yield and maximal antioxidant capacity. As can be seen from the obtained results, the acid in the
P. pavonica
extracts extracts
prepared at were
RT inlow in all except
all cases, samples,for the concentrations
ethanolic extracts ofofP.its para- isomer
pavonica, yieldedwere
the
significantly higher in the ethanolic extracts than in the water extracts.
highest amount. The share of phenolics in the ethanolic extracts of D. dichotoma prepared Similar results
were
at RTobtained the D.than
was 29%inhigher dichotoma
that inextract, where
the◦water the highest
extracts, and 56%concentration was detected
and 46% higher in the
in the ethanolic extract prepared at 20 C (2.07 mg/L). Generally, the
ethanolic extracts prepared at 40 and 60 °C, respectively. Although there were no signifi- concentrations of
hydroxycinnamic
cant acid derivatives
differences between the phenolic potentialo-coumaric
(p-coumaric, and t-ferulic
of the D. dichotoma and acid) were higher
P. pavonica water
in the D. dichotoma extracts, while the P. pavonica extracts were richer in
extracts, the content of phenolics in the ethanolic extracts was significantly higher. Other hydroxybenzoic
(protocatechuic and p-hydroxybenzoic) acids. According to the results for phloroglucinol
authors also investigated phenolics from D. dichotoma [2,23,34–36] and P. pavonica
[3,22,23,33,34], but comparison of the results is difficult due to the employment of differ-
ent extraction protocols (solvents used, time of extraction, temperature, additional actions
such as stirring, use of novel techniques, etc.) or due to results’ expression using different
standard compounds. However, the results of our study on the influence of drying and
Foods 2021, 10, 1187 6 of 10

content (Figure 1d), it is apparent that the D. dichotoma extracts were richer in this substance,
with an almost threefold higher content in the water extracts than in the EtOH extracts. On
the other hand, the P. pavonica ethanolic extracts contained significantly lower amounts
of phloroglucinol, while its quantification in the water extracts was not possible (it was
present, but at an amount below the quantification limit).

Table 1. Phenolic acids in Dictyota dichotoma (D) and Padina pavonica (P) extracts detected by HPLC.

Compound Concentration (mg/L of Algal Extract)

Sample Protocatechuic p-hydroxybenzoic p-coumaric t-ferulic o-coumaric
∑
Acid Acid Acid Acid Acid
D H2 O 20 ◦ C 0.62 ± 0.05 0.22 ± 0.04 1.00 ± 0.02 1.41 ± 0.15 0.16 ± 0.01 3.41
D H2 O 40 ◦ C 0.05 ± 0.01 0.19 ± 0.02 0.72 ± 0.01 1.28 ± 0.10 0.10 ± 0.01 2.34
D H2 O 60 ◦ C 0.30 ± 0.00 0.13 ± 0.01 0.44 ± 0.02 0.68 ± 0.02 0.02 ± 0.00 1.57
D EtOH 20 ◦ C 0.05 ± 0.00 0.42 ± 0.05 2.07 ± 0.20 1.57 ± 0.07 0.17 ± 0.01 4.29
D EtOH 40 ◦ C 0.23 ± 0.04 0.30 ± 0.01 1.75 ± 0.15 1.37 ± 0.01 0.14 ± 0.00 3.78
D EtOH 60 ◦ C 0.14 ± 0.00 0.32 ± 0.06 1.77 ± 0.01 1.33 ± 0.03 0.12 ± 0.03 3.66
P H2 O 20 ◦ C 1.68 ± 0.03 0.51 ± 0.56 0.03 ± 0.00 1.22 ± 0.10 0.02 ± 0.00 3.46
P H2 O 40 ◦ C 1.70 ± 0.02 0.60 ± 0.02 0.02 ± 0.00 0.07 ± 0.00 0.03 ± 0.00 2.42
P H2 O 60 ◦ C 1.42 ± 0.10 0.60 ± 0.01 0.02 ± 0.00 0.07 ± 0.01 0.03 ± 0.00 1.55
P EtOH 20 ◦ C 1.34 ± 0.03 0.75 ± 0.02 0.69 ± 0.07 0.21 ± 0.01 0.01 ± 0.01 3.00
P EtOH 40 ◦ C 1.05 ± 0.09 0.76 ± 0.03 0.79 ± 0.04 0.24 ± 0.05 0.02 ± 0.00 2.86
P EtOH 60 ◦ C 1.15 ± 0.08 0.66 ± 0.07 0.88 ± 0.16 0.33 ± 0.05 0.03 ± 0.00 3.06

Due to the presence of phlorotannins, it has been reported that brown algae species
possess higher antioxidant activity than green and red algae do. It is well known that the
concentrations of these compounds vary according to numerous factors, such as species,
season, age, geographical location and environmental conditions [7,9]. In this study,
antioxidant activity was evaluated by means of a multiple-method approach, using three
assays: Ferric Reducing/Antioxidant Power (FRAP), scavenging of the stabile 2,2-diphenyl-
1-picrylhydrazyl (DPPH) radical and Oxygen Radical Antioxidant Capacity (ORAC). The
obtained results are presented in Table 2. According to the presented results for the reducing
activity of the samples, the highest activities were detected for the ethanolic extracts of
D. dichotoma (from 690 to 792 mM TE), while all other samples had more than threefold
lower activity. Although in most cases, the antioxidant activity of the samples shows a
high correlation with phenolic content, this study only confirmed the significant impact of
the flavonoid content on the reducing activity of the extracts (r = 0.9873, p < 0.0001).

Table 2. Antioxidant activity of Dictyota dichotoma (D) and Padina pavonica (P) extracts detected by
the FRAP, DPPH and ORAC methods.

FRAP DPPH ORAC

Sample
(mM TE) (µM TE) (mM TE)
D H2 O 20 ◦ C 139 ± 16 196 ± 47 26.7 ± 2.4
D H2 O 40 ◦ C 147 ± 3 144 ± 27 26.6 ± 1.5
D H2 O 60 ◦ C 232 ± 7 125 ± 36 20.6 ± 4.5
D EtOH 20 ◦ C 792 ± 9 113 ± 31 39.1 ± 0.8 *
D EtOH 40 ◦ C 717 ± 17 84 ± 6 40.8 ± 2.4 *
D EtOH 60 ◦ C 690 ± 21 109 ± 9 38.7 ± 2.3 *
P H2 O 20 ◦ C 187 ± 7 30 ± 10 25.7 ± 1.2
P H2 O 40 ◦ C 147 ± 8 84 ± 6 22.5 ± 1.9
P H2 O 60 ◦ C 106 ± 9 56 ± 11 26.2 ± 0.8
P EtOH 20 ◦ C 202 ± 8 501 ± 66 46.2 ± 1.4
P EtOH 40 ◦ C 231 ± 12 595 ± 13 55.8 ± 3.0
P EtOH 60 ◦ C 214 ± 24 645 ± 25 51.2 ± 0.7
* The result obtained for samples deleted 400-times.
Foods 2021, 10, 1187 7 of 10

Generally, very low DPPH activity of the samples was detected, with the ethanolic
extracts of P. pavonica being the most active (from 501 to 645 µM TE). Similar results were
obtained in the studies of Kosanić et al. [22] and Khaled et al. [35], where the free radical
scavenging activities of all three investigated algae species (D. dichotoma, P. pavonica and
Sargassum vulgare) were low (IC50 values significantly higher in comparison to ascorbic
acid, BHA and α-tocopherol).
Finally, for antioxidant activity measurements, an ORAC assay was also used. This
method reflects classical radical chain breaking activity and measures inhibition of peroxyl-
radical-induced oxidation [37]. Again, as can be seen in Table 2, ethanolic extracts were
superior in comparison with the water extracts of both algae species. While the activity of
the P. pavonica ethanolic extracts was about twofold higher than the activity of the water
extracts, the ethanolic extracts of D. dichotoma showed excellent activity. In order to obtain
results, these extracts were previously diluted to 1:400.
In order to describe and identify the similarities and differences among the samples, a
principal component analysis (PCA) was used. The correlation loadings of the first two
principal components (PCs) shown in Figure 2 suggest high correlations of the studied
parameters. The variables of flavonoids and tannins were strongly characterised by PC1
and showed a high mutual correlation (r = 0.9752, p < 0.001). Furthermore, those parameters
that showed similar characteristics with the FRAP value and p-coumaric content in the
extracts positively correlated (Figure 2a). The total phenolic content and the free radical
scavenging assays (DPPH and ORAC) were characterised by PC2. The first two PCs
described 86.81% of the initial data variability. The score plot (Figure 2b) shows the
position of the measured parameters in the multivariate space of the first two PCs. The
clear separation between samples indicates the differences between the solvent used and
not the applied temperature during the extraction. The water extracts of both algae are
grouped in the upper part of the plot, showing no significant difference between them,
and are characterised by a low content of phenolics. Based on the contents of flavonoids
Foods 2021, 10, x FOR PEER REVIEW 8 of 10
and tannins, the ethanolic extracts were separated into two opposite parts and, due to the
higher phenolics content, are positioned in lower part of the plot.

(a)
4
Figure 2. Cont.

D H20 60°C
2 D H20 40°C
P H20 60°C
Foods 2021, 10, 1187 8 of 10

(a)
4

D H20 60°C
2 D H20 40°C
P H20 60°C

Factor 2: 31.30% 1 D H20 20°C P H20 20°C

P H20 40°C
0 D EtOH 60°C
D EtOH 40°C
-1 D EtOH 20°C

-2
P EtOH 20°C

-3
P EtOH 60°C
P EtOH 40°C
-4

-5
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5

Factor 1: 55.51%
(b)
Figure 2.Figure 2. Correlation
Correlation of loading
of the (a) the (a) loading plot
plot and (b)and (b) plot
score scoreofplot
theofPCA.
the PCA.

4. Conclusions
The obtained results from the PCA are in accordance with the Pearson product moment
correlations between
Brownvariables
algae arethat showed
a valuable a high
source of correlation
biologicallybetween total phenolics
active compounds. and on the
Research
both FRAPphenolic
(r = 0.8005, p =of0.0018)
profile and ORAC
the Adriatic values
algae D. dichotoma and P. ppavonica
(r = 0.9483, < 0.001),
is FRAP
importantandfor the
phlorotannin content (r =of0.8951,
interpretation p = 0.001),potential
their biological flavonoids and
and, tannins for
therefore, (r =their
0.9744, p < 0.001),
potential use in the
flavonoidsfood,
and reducing
cosmetic and/or (r = 0.9739, p <industries.
activitypharmaceutical 0.001) and DPPH
The and
results of ORAC (r =also
this study 0.8033,
confirmed
p = 0.0017).

4. Conclusions
Brown algae are a valuable source of biologically active compounds. Research on
the phenolic profile of the Adriatic algae D. dichotoma and P. pavonica is important for the
interpretation of their biological potential and, therefore, for their potential use in the food,
cosmetic and/or pharmaceutical industries. The results of this study also confirmed that
the investigated algal species are a rich source of phenolics. The ethanolic extracts contained
higher concentrations of phenolics, while the extraction temperature did not influence the
extraction yield. D. dichtoma contained higher concentrations of hydroxycinnamic acid
derivatives, while P. pavonica were richer in hydroxybenzoic acids. The tested extracts also
showed good antioxidant potential using all three antioxidant assays, with flavonoids and
tannins probably being responsible for this activity. This is still a relatively new scientific
area, and further research should be directed toward the investigation of other extraction
parameters or novel technologies focused on the yield of extracted bioactive compounds,
especially phlorotannins. Furthermore, it would be interesting to investigate the other
biological properties of brown algae extracts.

Author Contributions: Conceptualisation, I.G.M., V.Š. and D.S.; methodology, I.G.M. and D.S.; for-
mal analysis, V.B., A.C., M.S., B.S., I.L. and M.Č.; data curation, I.G.M., D.S. and V.Š.; writing—original
draft preparation, I.G.M.; writing—review and editing, V.Š., B.S., I.L., M.Č. and D.S.; supervision,
I.G.M., D.S. and V.Š.; project funding acquisition, V.Š. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was supported by the PRIMA program under Project BioProMedFood
(Project ID 1467). The PRIMA program is supported by the European Union.
Foods 2021, 10, 1187 9 of 10

Data Availability Statement: Data are available on request.

Acknowledgments: This research was supported by the PRIMA program supported by the European
Union (Project BioProMedFood, ID 1467).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

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