Journal of Applied Phycology (2020) 32:1441–1453

https://doi.org/10.1007/s10811-019-02013-2

Ultrasound-assisted extraction of phlorotannins and polysaccharides

from Silvetia compressa (Phaeophyceae)
Benjamín Vázquez-Rodríguez 1 & Janet A. Gutiérrez-Uribe 2 & Marilena Antunes-Ricardo 2 & Liliana Santos-Zea 2 &
Lucia Elizabeth Cruz-Suárez 1

Received: 2 July 2019 / Revised and accepted: 3 December 2019 / Published online: 20 January 2020
# Springer Nature B.V. 2020

Abstract
Silvetia compressa is a brown seaweed native to the coast of Baja California, Mexico. It is a rich source of phlorotannins and
polysaccharides, two compound families with important nutraceutical applications. Optimal conditions for obtaining highly
concentrated phlorotannin and polysaccharide extracts from S. compressa were determined using Box-Behnken experimental
design combined with response surface methodology (RSM). The effect of extraction temperature (X1: 50–65 °C), ultrasound
power density (X2: 1.2–3.8 W cL−1), solvent/seaweed ratio (X3: 10–30 mL g−1 seaweed meal), and ethanol concentration (X4:
25–100% ethanol in water) on phlorotannin and polysaccharide yield was explored. Experimental results were fitted to a second-
degree polynomial model, while model fitness was assessed using analysis of variance (ANOVA). From this analysis, optimal
phlorotannin and polysaccharide extraction conditions were determined for the evaluated parameters (X1 = 50 °C, X2 = 3.8 W
cL−1, X3 = 30 mL g−1 seaweed meal, and X4 = 32.33%). Under these conditions, experimental phlorotannin and polysaccharide
yields were 0.73% and 23% (w/w), respectively. Phlorotannin extraction was significantly enhanced by ultrasound power density,
while polysaccharide extraction improved when using low ethanol concentration in the solvent, therefore both families of
compounds were obtained combining those parameters. Experimental data agreed with predictions from the RSM model,
indicating suitability of the obtained model and the success of RSM in optimizing the extraction conditions. In addition, the
identification of the main compounds in the phlorotannin extract was carried out by HPLC-MS-TOF; 8 of the 12 identified
phenolic compounds belonged to the phlorotannin family, 4 from the fuhalol group, 3 being eckol derivatives, and 1 being a
phloroglucinol subunit.

Keywords Brown seaweed . Phaeophyceae . Phenolic compounds . Phytochemical recovery . Response surface methodology

Introduction
* Janet A. Gutiérrez-Uribe
jagu@tec.mx
In the last 20 years, seaweeds have become a common source
of phytochemicals (Plaza and Iba 2008; Samarakoon and Jeon
* Lucia Elizabeth Cruz-Suárez
lucia.cruzsr@uanl.edu.mx
2012; Rebours et al. 2014). Several marine brown algae ex-
tracts from different species are used as food and additives in
Benjamín Vázquez-Rodríguez cosmetic and food industries (Hifney et al. 2016). Advances in
bnjmnvzqzrdrgz@gmail.com
phytochemistry have identified various marine brown algae
Marilena Antunes-Ricardo genera (e.g., Eisenia, Cystoseira, Macrocystis, Sargassum,
marilena.antunes@tec.mx
Laminaria, and Ascophyllum) as potential sources of chemical
Liliana Santos-Zea compounds with nutraceutical, pharmaceutical, prebiotic, and
lilianasantos@tec.mx cosmetic applications (Balboa et al. 2013). The nutraceutical
1
effect of brown algae is primarily due to two families of chem-
Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo
León, Ave. Universidad SN, Cd. Universitaria, 66455 San Nicolás de
ical compounds: phlorotannins (PT) and polysaccharides (PS).
los Garza, NL, Mexico Extracted fractions containing phlorotannin and PS, separated
2
Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Centro
or in mixture, have shown hypoglycemic, hypolipidemic, an-
de Biotecnología-FEMSA, Av. Eugenio Garza Sada 2501 Sur, ticancer, antiviral, antibacterial, bacteriostatic (Bogolitsyn et al.
64849 Monterrey, NL, Mexico 2019), antioxidant (Wijesekara et al. 2011; Gil-Chavez et al.
1442
2013), or prebiotic bioactivity (Charoensiddhi et al. 2016;
Kong et al. 2016).
The phenolic compounds known as phlorotannins are com-
posed of phloroglucinol units bonded in different ways to
form diverse structures. Phlorotannins are highly hydrophilic
molecules accumulated in brown seaweeds and whose prima-
ry function is to protect them from environmental stress and
from consumption by herbivores (Wijesekara et al. 2011).
Phlorotannin content varies greatly between seaweed species,
with brown seaweeds having the highest amount (0.5–15%
dry weight) (Pavia et al. 2003; Rajauria 2015). Traditional
solvent extraction has been used for obtaining phenolic and
phlorotannin extracts, mainly using ethanol, methanol, and
acetone (Kadam et al. 2015a).
Brown seaweeds have a polysaccharide content of up to
76% of algal dry weight (Rioux and Turgeon 2015). Seaweed
cell walls differ from those of terrestrial plants; they contain
uncommon polyuronides and polysaccharides that may be
methylated, acetylated, pyruvylated, or sulfated (Wells et al.
2017). The main polysaccharides present in brown seaweed
cell walls are the intercellular matrix alginate (18–48% dry
weight) and fucose containing polysaccharides such as lami-
narin and fucoidan (2–10% dry weight) (Rioux et al. 2007;
Rioux and Turgeon 2015). Alginate is the main commercial
polysaccharide in brown algae, while fucoidan and laminarin
represent a by-product from alginate extraction (Wijesinghe
and You-Jin 2012; Rioux and Turgeon 2015). For brown sea-
weed polysaccharide extraction, several methods have been
reported, but traditional hot water and ethanolic extraction
are the most frequent solvents used (Garcia-Vaquero et al.
2017).
Ultrasound-assisted extraction (UAE) is an emergent tech-
nology that shortens extraction time from several hours to
minutes (Carrera et al. 2012; Živković et al. 2018) and can
reduce the amount of solvent used (Kadam et al. 2015c;
Chemat et al. 2017). Application of ultrasound waves to the
extraction medium generates cavitation in the liquid and the
implosion of these cavitation bubbles results in erosion and
particle breakdown (Grosso et al. 2015; Chemat et al. 2017).
Recently, UAE has been used to enhance the extraction of
several bioactive compounds from brown seaweeds, such as
phlorotannins (Lee et al. 2013; Kadam et al. 2015b, 2015c;
Rodrigues et al. 2015; Dang et al. 2017) and polysaccharides
(Rodrigues et al. 2015; Garcia-Vaquero et al. 2017; Moreira
et al. 2017).
Silvetia compressa is an edible brown algae native to the
northern coast of Baja California, Mexico, and has potential
industrial applications as fertilizer, forage, and as raw material
for the extraction of alginate and other colloids employed as
thickeners in the food industry (Aguilar Rosas et al. 2002).
Seaweeds of the Silvetia genus are also used as food prepara-
tion ingredients in Asian countries (Pereira 2016). Due to its
natural abundance, several lines of research have studied
J Appl Phycol (2020) 32:1441–1453
reproduction in Silvetia species, as well as its industrial, nu-
traceutical (antiviral, antioxidant and hypoglycemic), and bio-
remediation applications (Girardi et al. 2014; Morán-
Santibañez et al. 2016; Múzquiz et al. 2019). Previous reports
showed that the hydroethanolic extraction of Mexican S.
compressa at different conditions produced yields between
18 and 37% (Múzquiz et al. 2019; Tapia-Salazar et al.
2019). Additionally, polyphenols with high antiviral and anti-
oxidant activity (Morán-Santibañez et al. 2016) as well as α-
glucosidase and α- amylase inhibitory activities have been
obtained (Múzquiz et al. 2019).
In this study, a Box-Behnken experimental design (BBD)
using response surface methodology (RSM) was applied to
determine the impact of extraction temperature, power densi-
ty, solvent/seaweed ratio, and ethanol concentration on
phlorotannin and polysaccharide extraction yields in S.
compressa. This study sought to optimize phlorotannin and
polysaccharide extraction conditions in order to maximize
yield and high level of both families of compounds.
Furthermore, these methods allow the development of statis-
tical models that can assess the relevance, as well as the sig-
nificance, of each factor’s effects along with the interaction
effects between the factors.
Materials and methods
Seaweed material and reagents
Silvetia compressa (J. Agardh) E.Serrão, T.O.Cho, S.M.Boo,
and Brawley (Phaeophyceae, Fucales) samples were collected
at La Escalera, Baja California, Mexico (31° 30′ 59.1″ N 116°
38′ 51.1″ W) from December 2014 to January 2015. The thalli
were collected, washed several times with seawater to remove
sand and epiphytes, drained on clean stone or clotheslines, and
sun-dried inside a greenhouse shadow for several days until
reaching a 10.9% moisture content. Subsequently, samples
were ground (Pulvex model 200, Mexico), passed through a
500 μm sieve, and vacuum-packed for future use.
Experimental design and statistical analysis
The BBD in combination with RSM was used to evaluate the
effect of extraction parameters on phlorotannin and polysac-
charide extraction yield and to optimize conditions for
obtaining higher phlorotannin and polysaccharide yields.
The experimental design consisted of four factors on three
levels with a central point (Table 1) and consisted of 27 ran-
domized runs with 2 replicates (Table 2). The independent
variables employed in this design were extraction temperature
(X1, 55–65 °C), ultrasound power density (X2, 1.2–3.8 W
cL−1), solvent/seaweed ratio (X3, 10–30 mL g−1 dry seaweed
meal), and ethanol concentration (X4, 25–100%). The range
J Appl Phycol (2020) 32:1441–1453
Table 1 Independent variables and their levels used for Box-Behnken
design
Independent variable −1 0 1
Extraction temperature—X1 (°C) 50 57.5
Power density—X2 (W cL−1) 1.2 2.4
Solvent/seaweed ratio—X3 (mL g−1 dw) 10.0 20.0
Ethanol concentration—X4 (%) 25.0 62.5
of temperatures and concentrations of ethanol used in this
study were selected based on previous literature research (Xi
et al. 2009; Múzquiz et al. 2019). The temperature range pro-
posed is in vein with those reported for the extraction of ma-
rine plants, which typically are below 60 °C (Tanniou et al.
2014; Puspita et al. 2017). Response variables were fitted to a
second given-order polynomial model (Eq. 1), where Y is the
predicted response (solids, polysaccharide, and phlorotannin
extraction yields); β0 is the model constant coefficient; β1, β2,
β3, and β4 are the linear coefficients; β11, β22, β33, and β44
are the quadratic coefficients; β12, β13, β14, β23, β24, and β34
are the cross-product coefficients; and X1, X2, X3, and X4
represent the independent variables extraction temperature,
power density, solvent/seaweed ratio, and ethanol concentra-
tion, respectively.
Y ¼ β 0 þ β1 X 1 þ β2 X 2 þ β 3 X 3 þ β4 X 4 þ β12 X 1 X 2
þ β13 X 1 X 3 þ β14 X 1 X 4 þ β23 X 2 X 3 þ β 24 X 2 X 4
þ β34 X 3 X 4 þ β11 X 21 þ β 22 X 22 þ β33 X 23 þ β 44 X 24
The BBD design and multiple linear regression analysis
were conducted using JMP v.14 software. An analysis of var-
iance (ANOVA) at a significance level of 0.05 was run for the
experimental data, and optimal extraction conditions for the
three response variables were obtained using the desirability
function of the RSM.
Extract preparation
To extract phlorotannins and polysaccharides from dried
ground seaweed, experimental samples were suspended in
the extraction solvent with their corresponding solvent/
seaweed ratio. Extracts were placed in an ice bath and soni-
cated (SFX150 Cell Disruptor, Branson, USA) for 30 min
with their corresponding ultrasonic intensities, using a 1/16″
micro tip with an immersion depth of 10 cm. All extraction
treatments were mixed at 250 rpm for 90 min after sonication
in an agitated hot plate at their corresponding extraction tem-
perature. Extracts were then centrifuged at 685×g for 15 min
at 4 °C (SL16R Thermo Fisher Scientific, USA); the superna-
tant was transferred to an evaporator (Rocket Synergy
1443
evaporator, Genevac, UK) to remove ethanol and water.
Then, crude extracts were stored at − 80 °C until further anal-
ysis (Xi et al. 2009; Múzquiz et al. 2019).
65
Solids extraction yield
3.8
30.0
Solids extraction yield (Y1) was determined as the total
100.0
amount of solids recovered from seaweed samples and
expressed as gram of hydroethanolic extract per gram of dry
seaweed meal (g EXT g−1 dw).
Total polysaccharide content
Total polysaccharide content was determined using a gravi-
metric method. The concentrated extract was dissolved in
5 mL of pure methanol and centrifuged for 10 min at
2739×g. The methanol supernatant was collected for
phlorotannin quantification and identification, while the pre-
cipitate was dried under a nitrogen flow and weighed. The
total polysaccharide extraction yield (Y2) was determined by
the amount of solids recovered after the methanol precipita-
tion and calculated as gram of polysaccharide per gram of dry
seaweed meal (g PS g−1 dw).
Identification and quantification of phlorotannins
in seaweed extracts
Phlorotannin identification and quantification of the selected
ð1Þ seaweed extract was carried out following the methodology
described by Muzquiz et al. (2019) using high-performance
liquid chromatography coupled with a diode array detector
(HPLC-DAD, Agilent Technologies 1200 series, USA).
HPLC was performed through a Luna C18 reverse column
(250 mm, 4.6 mm, 5 μm particle size; Phenomenex, USA)
with a flow rate of 1 mL min−1 at 30 °C and an injection
volume of 10 μL. The mobile phase consisted of (A) acidified
water (1% formic acid) and (B) 100% methanol. The elution
was performed as follows: 0–5 min 10% B, 5–30 min 20–60%
B, 30–35 min 60% B, 35–40 min 60–20% B, and 40–50 min
10% B (post time). Spectral data from all peaks were accumu-
lated in the 230–550 nm range and chromatograms were re-
corded at 270 nm. Chromatographic data were collected using
HP-Agilent Software for LC. Phloroglucinol (Sigma-Aldrich,
USA) was used as a quantification standard. Phlorotannin
extraction yield (Y3) was expressed as mg of phloroglucinol
equivalents (PGE) per g dry seaweed (mg PGE g−1 dw) using
Eq. 2 as a calibration curve (R2 = 0.999), where y is the peak
area and x is the concentration in mg PGE g−1 dw.
y ¼ 1:7075x þ 0:9081 ð2Þ
HPLC-MS-TOF model G1969A Agilent 1100 adjusted to
the same chromatographic conditions described above was
1444 J Appl Phycol (2020) 32:1441–1453

Table 2 Box-Behnken design with coded independent variables, and experimentally observed responses in the extracts

Run X1 (°C) X2 (W cL−1) X3 (mL g−1 dw) X4 (%) Y1 (g EXT g−1 dw) Y2 (g PS g−1 dw) Y3 (mg PGE g−1 dw)

1 57.5 2.4 20 62.5 0.35 0.1866 9.57

2 50 1.2 20 62.5 0.30 0.1667 3.31
3 57.5 2.4 30 25 0.48 0.1987 6.66
4 65 1.2 20 62.5 0.31 0.1740 7.05
5 57.5 2.4 30 100 0.11 0.0184 3.12
6 57.5 2.4 30 100 0.13 0.0166 2.97
7 57.5 2.4 10 25 0.36 0.2064 3.63
8 57.5 2.4 20 62.5 0.33 0.1874 9.04
9 50 3.8 20 62.5 0.33 0.1762 6.69
10 57.5 2.4 10 100 0.11 0.0472 1.74
11 50 3.8 20 62.5 0.34 0.1912 6.29
12 50 1.2 20 62.5 0.28 0.1666 4.27
13 57.5 2.4 10 25 0.35 0.2075 3.61
14 65 3.8 20 62.5 0.36 0.1648 6.97
15 65 1.2 20 62.5 0.31 0.1823 8.67
16 57.5 2.4 10 100 0.12 0.0326 1.70
17 57.5 2.4 30 25 0.45 0.2076 4.73
18 65 3.8 20 62.5 0.34 0.1745 9.83
19 50 2.4 20 25 0.49 0.2135 7.73
20 57.5 3.8 10 62.5 0.29 0.1685 10.93
21 57.5 1.2 10 62.5 0.27 0.1539 9.85
22 65 2.4 20 100 0.16 0.0477 7.50
23 65 2.4 20 25 0.50 0.2158 8.91
24 57.5 1.2 30 62.5 0.35 0.1798 5.77
25 50 2.4 20 100 0.15 0.0464 3.92
26 50 2.4 20 100 0.16 0.0452 3.37
27 65 2.4 20 100 0.14 0.0466 6.59
28 57.5 1.2 10 62.5 0.27 0.1464 7.89
29 57.5 3.8 10 62.5 0.28 0.1555 10.82
30 57.5 2.4 20 62.5 0.33 0.1770 8.34
31 50 2.4 20 25 0.52 0.2178 7.04
32 57.5 3.8 30 62.5 0.34 0.1915 7.72
33 57.5 1.2 30 62.5 0.32 0.1765 4.47
34 65 2.4 20 25 0.41 0.2014 8.31
35 57.5 3.8 30 62.5 0.37 0.1831 8.26
36 57.5 2.4 20 62.5 0.31 0.1678 7.52
37 65 2.4 10 62.5 0.31 0.1192 4.86
38 50 2.4 30 62.5 0.60 0.1918 5.24
39 57.5 3.8 20 100 0.21 0.0531 2.37
40 50 2.4 10 62.5 0.29 0.1800 6.51
41 65 2.4 30 62.5 0.35 0.1874 3.63
42 57.5 1.2 20 25 0.47 0.2444 3.93
43 57.5 2.4 20 62.5 0.28 0.1558 6.84
44 57.5 1.2 20 100 0.17 0.0653 2.50
45 65 2.4 10 62.5 0.35 0.1191 5.02
46 57.5 3.8 20 25 0.43 0.2513 5.47
47 65 2.4 30 62.5 0.37 0.1985 5.01
48 57.5 1.2 20 100 0.19 0.1098 2.76
49 57.5 2.4 20 62.5 0.33 0.2258 6.11
50 57.5 3.8 20 100 0.17 0.0499 2.62
51 50 2.4 30 62.5 0.53 0.2037 6.25
52 50 2.4 10 62.5 0.30 0.2005 5.77
53 57.5 3.8 20 25 0.47 0.2216 5.92
54 57.5 1.2 20 25 0.50 0.2266 4.02

dw dry seaweed meal, EXT hydroethanolic extract, PS polysaccharides, PGE phloroglucinol equivalent

used to identify bioactive compounds. Mass spectra were col-
lected using electrospray source in positive mode (ESI+) un-
der the following conditions: m/z range 150 to 1500, nitrogen
gas at 300 °C, drying gas flow rate 8 L min−1, nebulizer pres-
sure 20 psi, capillary voltage 4000 V, and fragment voltage
70 V. Extracted ion chromatograms were obtained using
Analyst QS 1.1 software (Applied Biosystems, USA) and
considering accurate mass from each bioactive compound or
its adducts with Na or K with an error range of 0.01 units.
Mass spectra were used to identify the different compounds
based on their fragmentation patterns. These were subsequent-
ly compared and verified on natural compound mass spectra
J Appl Phycol (2020) 32:1441–1453
databases and previous reports from phlorotannins identified
in brown seaweeds.
Results
Effect of extraction parameters on solids extraction
yield
The effect of solvent/seaweed ratio on extraction yield is shown
on Fig. 1b, d, f. Extraction yield ranged from 0.11 to 0.60 g EXT
g−1 dw1. The highest solids extraction yield was obtained with
extraction parameters X1: 50 °C, X2: 2.4 W cL−1, X3: 30 mL g−1
dw, and X4: 62.5% EtOH. In contrast, the lowest extraction yield
was obtained when extraction parameters were X1: 57.5 °C, X2:
2.4 W cL−1, X3: 30 mL g−1 dw, and X4: 100% EtOH. Extraction
temperature had a significant quadratic effect on solids yield,
decreasing in the range of 50–57.5 °C and increasing in the range
of 57.5–65 °C, with a slight, but significant, linear tendency to
decrease yield and a significant negative interaction with the
solvent/seaweed ratio (Table 3).
Power density (X2) was the only non-significant parameter on
solids extraction yield (Table 3). Extraction temperature (X1),
solvent/seaweed ratio (X3), and ethanol concentration (X4) were
significant parameters (p < 0.05). Only the interaction between
the X1X3 and X3X4 showed a significant impact on the response
variables. Equation (3) shows the second-order polynomial mod-
el describing the solids extraction yield response to the extraction
parameters that had a significant effect:
Y 1 ¼ 0:32−0:01X 1 þ 0:04X 3 −0:15X 4 −0:06X 1 X 3 −0:02X 3 X 4 ð3Þ
þ 0:03X 21 −0:02X 24
Effect of extraction parameters on polysaccharide
extraction yield
Polysaccharide extraction yield ranged from 0.0166 to
0.2513 g PS g−1 dw (Table 2). The highest polysaccharide
concentration was obtained with extraction parameters X1:
57.5 °C, X2: 3.8 W cL−1, X3: 20 mL g−1 dw1, and X4: 25%
EtOH. The lowest polysaccharide concentration was obtained
when extraction parameters were X1: 57.5 °C, X2: 2.4 W cL−1,
X3: 30 mL g−1 dw, and X4: 100% EtOH. Equation (4) shows
the second-order polynomial model that describes the poly-
saccharide extraction yield from S. compressa based on the
extraction parameters that showed significant effects:
Y 2 ¼ 0:1834 þ 0:0139X 3 −0:0847X 4 þ 0:0312X 1 X 3 −0:0469X 24 ð4Þ
Solvent/seaweed (X3) had a linear positive effect on the ex-
traction of polysaccharide from S. compressa meal (p ≤ 0.05).
The interaction among extraction parameters X1 (temperature)
and X3 was positively significant for polysaccharide extraction,
1445
contrasting with the negative interaction between X1 and X3 ob-
served for the total solids yield (Table 3). Ethanol concentration
(X4) had significant negative effects on the polysaccharide ex-
traction yield both at linear and quadratic levels. As ethanol con-
centration increased, polysaccharide extraction yield in the ex-
tract decreased, peaking at high ethanol concentrations (Fig. 2c,
e, f), which was more pronounced than for solids extraction yield
(Fig. 1c, e, f).
Effect of extraction parameters on phlorotannin
extraction yield
The highest phlorotannin concentration was obtained with
extraction parameters X1: 57.5 °C, X2: 3.8 W cL−1, X3:
10 mL g−1 dw, and X4: 62.5% EtOH. The lowest phlorotannin
concentration was obtained when extraction parameters were
X1: 57.5 °C, X2: 2.4 W cL−1, X3: 10 mL g−1 dw, and X4: 100%
EtOH. Phlorotannin extraction yield ranged from 1.70 to
10.93 mg PGE g dw−1 (Table 2). Temperature increase fa-
vored phlorotannin leaching to the extraction solvent, as
shown in the RS from Fig. 3a–c.
Three of the four selected extraction parameters had a sig-
nificant effect on the phlorotannin extraction yield from S.
compressa meal; temperature (p = 0.0395), power density
(p = 0.013), and ethanol concentration (p = 0.004) (Table 3).
Only two quadratic levels were significant for phlorotannin
extraction yield: solvent/seaweed ratio (X3) and the ethanol
concentration (X4). No interactions among the extraction pa-
rameters were significant for phlorotannin extraction.
However, in the power density range tested in this study, there
was no significant quadratic effect and only a significant linear
positive effect, which is clearly shown on Fig. 3a (especially at
low temperature) and d.
Equation (5) shows the second-order polynomial model
that describes the phlorotannin extraction yield from S.
compressa based on the extraction parameters that presented
significant effects:
Y 3 ¼ 7:90 þ 0:66X 1 þ 0:80X 2 −1:20X 4 −1:11X 23 −2:68X 24 ð5Þ
Optimization of extraction parameters and model
validation
Table 2 shows the experimental conditions and the EXT,
phlorotannin, and polysaccharide yields obtained for each exper-
iment. Using the obtained statistical models, the optimum values
for the extraction were X1: 50 °C, X2: 3.8 W cL−1, X3: 30 mL g−1
dw, and X4: 32.33 %EtOH. The predicted values for the response
variables at these extraction conditions were Y1: 0.5596 g EXT
g−1 dw, Y2: 0.2288 g PS g−1 dw, and Y3: 7.4836 mg PGE g−1 dw.
The experimental EXT, polysaccharide, and Phlorotannin extrac-
tion yields were Y1: 0.5572 ± 0.0220 g EXT g−1 dw, Y2: 0.2207
1446 J Appl Phycol (2020) 32:1441–1453

Fig. 1 3D response surface of extraction yield (g EXT g−1 dw) of S. compressa affected showing the interactive effect of temperature (X1,°C), power
density (X2, W cL−1), solvent/seaweed ratio (X3, mL g−1 dw), and ethanol concentration (X4, %) on the response variable

Table 3 Regression coefficients

and R2 of predicted second-order Term Y1 (g EXT g−1 dw) Y2 (g PS g−1 dw) Y3 (mg PT g−1 dw)
polynomial models for the inves-
tigated responses from S. Regression P value Regression P value Regression P value
compressa extracts coefficient coefficient coefficient

Intercept
0.32** < 0.0001 0.1834** < 0.0001 7.90** <0.0001
Linear
X1 − 0.01* 0.0256 − 0.0035ns 0.4558 0.66* 0.0395
X2 0.00ns 0.2399 − 0.0004ns 0.9228 0.80* 0.0130
X3 0.04** < 0.0001 0.0139** 0.0056 − 0.35ns 0.2602
X4 − 0.15** < 0.0001 − 0.0847** < 0.0001 − 1.20** 0.0004
Quadratic
X11 0.03** 0.0035 − 0.0018ns 0.7987 − 0.33ns 0.4820
X22 − 0.00ns 0.7868 0.0019ns 0.7880 − 0.34ns 0.4601
X33 − 0.00ns 0.9834 − 0.0106ns 0.1440 − 1.11* 0.0220
X44 − 0.02** 0.0060 − 0.0469** < 0.0001 − 2.68** < 0.0001
Cross Product
X1X2 − 0.00ns 0.9139 − 0.0063ns 0.4413 − 0.54ns 0.3202
X1X3 − 0.06** < 0.0001 0.0312** 0.0005 − 0.05ns 0.9171
X2X3 0.00ns 0.9139 − 0.0006ns 0.9349 0.21ns 0.6892
X1X4 0.01ns 0.3335 0.0021ns 0.7994 0.53ns 0.3274
X2X4 0.01ns 0.3335 − 0.0095ns 0.2669 − 0.46ns 0.3930
X3X4 − 0.02* 0.0280 − 0.0046ns 0.5744 − 0.18ns 0.7287
Coefficient of 0.9474 0.9167 0.7214
determination (R2)

* Significant at p < 0.05; ** highly significant at p < 0.01; ns not significant

J Appl Phycol (2020) 32:1441–1453
Fig. 2 3D response surface of polysaccharide extraction (g PS g−1 dw) of S. compressa showing the interactive effect of temperature (X1,°C), power
density (X2, W cL−1), solvent/seaweed ratio (X3, mL g−1 dw), and ethanol concentration (X4,%) on the response variable
± 0.030 g PS g−1 dw, and Y3: 7.73 ± 0.2319 PGE mg g−1 dw
respectively, suggesting that the obtained model adequately de-
scribes the extraction process using UAE.
Identification of phlorotannin present in the extracts
Chromatograms showed 12 peaks (Fig. 4). Of these 12 com-
pounds, 10 belonged to the phlorotannin family, 1 belonged to
the phenolic acids family (quinic acid), and 1 belonged to the
flavonoid family (acacetin derivative) (Table 4). Among the iden-
tified phlorotannin compounds, four belonged to the fuhalol fam-
ily (peaks 1, 9, 11, and 12), five were eckol derivatives (peaks 3,
5, 6, 7, and 10), and one was a phloroglucinol subunit (peak 8).
Discussion
The negative linear tendency and pronounced negative effect
in the low range went against what was expected since incre-
ments in the extraction temperature should have produced an
increase in solubility and diffusivity (Goula 2012). The in-
creased solids extraction yield at higher temperatures was at-
tributed to the softened seaweed tissues, with weakened cell
wall integrity that favored the release of extractable matter
(Wan Aida et al. 2011).
The solvent/seaweed ratio (X3) positively affected the total
amount of extractable matter that can be solubilized in the extract
prior to reaching its saturation state. Ethanol concentration (X4)
1447
negatively affected compounds diffusion to the extraction sol-
vent, increasing this negative effect at high ethanol concentra-
tions (highly significant linear and quadratic tendencies). This
was due to the solvent’s polarity change. An increase in the
solvent/seaweed ratio typically increases the amount of solutes
present in the extract due to a higher leaching rate (Zhang et al.
2007). This may be attributed to more extractable matter being
able to permeate from the cellular matrix to the extraction solvent
(Bucić-Kojić et al. 2007; Prasad et al. 2009).
The use of low ethanol concentrations resulted in the
highest extraction yields, which reflects the hydrophilic nature
of most components within S. compressa. Ethanol concentra-
tions below 50% allow the extraction of polysaccharides and
protein fractions present in the algae matrix, while ethanol
concentrations between 60 and 70% allow the extraction of
phlorotannins (He et al. 2013).
Similar solvent/seaweed ratio and ethanol concentration
effects on the amount of extractable matter and the bioactive
chemical profile have been reported for several plant matrices
such as Chelidonium majus (Grosso et al. 2014), Matrica
riarecutita flowers (Zeković et al. 2014), acorns (Onem
et al. 2014), and brown seaweeds, such as Ascophyllum
nodosum, Pelvetia caniculata (Tierney et al. 2013), and
Macrocystis pyrifera (Leyton et al. 2016). Broadly, higher
solvent/solute ratios and lower ethanol concentrations in the
extraction solvent resulted in higher extraction yields.
The hydrophilic nature of polysaccharides present in sea-
weeds is the driving force of the extraction, resulting in
1448 J Appl Phycol (2020) 32:1441–1453

Fig. 3 3D response surface of phlorotannin extraction (mg PGE g−1 dw) of S. compressa affected showing the interactive effect of temperature (X1,°C),
power density (X2, W cL−1), solvent/seaweed ratio (X3, mL g−1 dw1), and ethanol concentration (X4,%) on the response variable

polysaccharide-rich extracts obtained when using hot water or a
polar solvent at low concentration. The effect of ethanol con-
centration in polysaccharide extraction has been well-studied in
several biological matrices, such as Ganoderma lucidum (Ma
et al. 2013), as well as in microalgae such as Chlorella
pyrenoidosa (Shi et al. 2007) and brown seaweeds such as
Fucus vesiculosus (Kadam et al. 2013), Sargassum sp. (Hahn
et al. 2012), Ecklonia radiata (Charoensiddhi et al. 2017),
Eisenia bicyclis (Ermakova et al. 2013), Laminaria saccharina
(Garcia-Vaquero et al. 2017), Ascophyllum nodosum (Foley
et al. 2011), and Sargassum glaucescens (Huang et al. 2016).
The decrease in polysaccharide yield with the increase in etha-
nol concentration occurs due to precipitation and low solubility
of polysaccharide in ethanol. When ethanol is present, the di-
electric constant of the extraction solvent decreases and the
sulfate esters and positive ions in the media form ionic bonds,
resulting in a precipitate rich in PS (Hahn et al. 2012; Ma et al.
2013). The use of alkaline enzymatic extraction reported a great
enhancement for the carbohydrates and phlorotannins, present
in Macrocystis pyrifera maximizing up to 22.1 and 0.5% their

Fig. 4 Brown seaweed S. compressa extract HPLC chromatogram (6) dieckol, (7) eckol derivative, (8) 3 phloroglucinol units, (9)
recorded at 270 nm. Peak identification: (1) dihydroxytetrafuhalol, (2) dihydroxypentafuhalol, (10) phlorofucofuroeckol A, (11) pentafuhalol,
quinic acid, (3) 7-phloroeckol, (4) acacetin derivative, (5) eckstolonol, and (12) trifuhalol
J Appl Phycol (2020) 32:1441–1453
Table 4 Features of the identified and compounds in S. compressa extracts using HPLC-DAD and HPLC-MS-TOF
Peak ID Proposed compound Accurate mass [M+] (m/z)
1 Dihydroxytetrafuhalol – 546
2 Quinic acid 192.0633 192
3 7-Phloroeckol – 496
4 Acacetin derivative – 357
5 Eckstolonol – 370
6 Dieckol 742.0806 742
7 Eckol derivative – 545
8 3 Phloroglucinol units – 374
9 Dihydroxypentafuhalol – 671
10 Phlorofucofuroeckol A – 602
11 Pentafuhalol – 638
12 Trifuhalol 390.0587 390
extraction compared to more traditional methods (Leyton et al.
2017); a cost analysis regarding the alkaline media treatment
and the use of enzymes should be assed against UAE.
The use of high temperatures causes a decrease in the di-
electric constant of water, resulting in a more efficient solvent
for the extraction of polyphenolics (Prasad et al. 2009). In
addition, higher temperatures increase the molecular move-
ment of the extraction solvent, thus increasing permeation
and extraction of the target compounds (He et al. 2013).
Increases in X1 (temperature) break the phenolic-matrix, en-
hancing polyphenol extraction (Prasad et al. 2009). Other au-
thors have reported similar behaviors in phlorotannin extrac-
tion from Saccharina japonica, Ecklonia cava, and M.
pyrifera (He et al. 2013; Leyton et al. 2016; Yoon et al. 2017).
The effect of the power density has been shown to display a
parabolic behavior in phlorotannin extraction yield (Chemat
et al. 2017). Extractions performed at high ultrasonic intensities
can decrease phlorotannin concentrations, which may be related
to free radicals in the extraction solvent formed by the high
amplitude and prolonged ultrasound waves (Luque de Castro
and Priego-Capote 2007; Carrera et al. 2012). Degradation of
phenolic compounds related to free radical formation due to
high ultrasonic intensities and prolonged ultrasonication pe-
riods has been reported in matrices such as pomegranate
(Živković et al. 2018), grape peel (Carrera et al. 2012), hemp
(Teh and Birch 2014), and other brown seaweeds (Han et al.
2011; Dang et al. 2017). Polyphenols and phlorotannins act as
reducing agents and become oxidized by these free radicals
(Tiwari et al. 2009; Carrera et al. 2012). Several authors have
reported an increase in phlorotannin extraction in brown sea-
weed using ultrasonic assisted extraction. Ultrasonic waves can
reduce extraction time, facilitate the liberation of high molecu-
lar weight phlorotannins, and enhance phlorotannin extraction
up to 30.5% compared to control experiments where no ultra-
sound was used (Kadam et al. 2013, 2015b, 2015c; Lee et al.
1449
UV max (λmax) Ref
227 (Montero et al. 2016; Agregán et al. 2017)
312, 378 (Agregán et al. 2017)
275 (Cho et al. 2019)
266 (Agregán et al. 2017)
215, 267 (Iwai 2008)
235 (Iwai 2008; Agregán et al. 2017)
297 (Agregán et al. 2017)
352 (Tierney et al. 2014; Vissers et al. 2017)
407 (Montero et al. 2016)
405 (Trifan et al. 2019)
405 (Montero et al. 2016)
367, 470 (Montero et al. 2016)
2013; Obluchinsksya et al. 2015; Moreira et al. 2017). The
enhancement of phlorotannin extraction might be attributed to
their release from damaged cell walls and even cells due to a
ultrasonic mechanical effect, which triggers an instantaneous
release of the plant extract components into the surrounding
medium (Shirsath et al. 2012).
Ultrasonic treatment in extraction operations enhances the
release of phytochemicals from the vegetal matrix by the
means of (1) cellular tissue disruption by cavitation, (2) and
an increase in the solid to liquid mass transfer by acoustic
streaming (Panda and Manickam 2019). Higher ultrasound
intensities or power densities increases bubbles size formed
in the solvent. Bubbles size affect both the cellular matrix and
the extraction solvent by promoting more violent implosions
capable of particle size reductions and promoting a more tur-
bulent flow within the extraction solvent (micro-scale gener-
ation of more eddy diffusion and internal diffusion). The in-
crease of ultrasound intensity or power density also affects
extraction solvent temperature; depending on the properties
of the target molecule, it can enhance the extraction or degrade
the target compound (Shirsath et al. 2012). Increments in pow-
er density and ultrasound intensity have enhanced the extrac-
tion of phenolics compounds due acoustic streaming in oreg-
ano (Santos-Zea et al. 2018) and cavitation effects in the
brown seaweed Ascophyllum nodosum (Kadam et al. 2015b).
As members of the polyphenol family, phlorotannins have a
polar physicochemical nature and are highly soluble in polar
solvents such as methanol, ethanol, and acetone (Tierney et al.
2013). The mixture’s ethanol/water ratio may enhance
phlorotannin extraction by two means: (1) due to the mixture’s
polarity, a higher aqueous extraction solvent can be obtained
due to the breaking of hydrogen bonds releasing more phenolic
compounds to the media and, (2) by augmenting the contact
area between the seaweed and the solvent by means of particle
swelling (Živković et al. 2018). These results agree with
1450
preceding studies where increments of polar solvent in the
extraction solvent improved phlorotannin and polyphenol ex-
tractions (He et al. 2013; Tierney et al. 2013; Sharmila et al.
2016). Pure ethanol does not enhance the extraction of PT, and
it has been reported that elevated solvent concentrations (eth-
anol, methanol, and acetone) higher than 60% can extract less
polar components dissolved with greater ease, leading to a
reduction in the extract’s phlorotannin concentration (He
et al. 2013).
EXT yield values were 38.79% and 35.56% higher than
those previously reported by Muzquiz et al. (2019) and
Tapia-Salazar et al. (2019) for the hydroethanolic extraction
of S. compressa. The phlorotannin extraction yield compared
to previous reports was lower. The total phenolic compounds
(TPC) in S. compressa determined by the Folin-Ciocalteu (FC)
method (Múzquiz et al. 2019; Tapia-Salazar et al. 2019) had
concentrations 11.6- and 16.9-fold higher than the total
phlorotannin concentration obtained in this study. However,
the phlorotannin yield obtained in the present study was in
accordance with the phlorotannin content in brown seaweeds
by others (Kadam et al. 2015a). Differences in the obtained
results can be attributed to several factors, mainly to the quan-
tification method and the standard used. The TPC determina-
tion by FC method is not a selective method and the discrep-
ancies between the FC determination and the HPLC method
can be attributed to (1) an overestimated TPC value due to the
presence of several compounds, such as ascorbic acid, sugars,
aromatic amines, sulfur dioxide, organic acids, and Fe(II)
(Lester et al. 2012; Sánchez-Rangel et al. 2013), and (2) the
detection of several phenolic compounds different from the PT
by the FC, so their contribution to the TPC is not involved in
the HPLC method (Porgali and Büyüktuncel 2012).
The fuhalol family comprised around the 40% of PT present
in the extract. These compounds are phloroglucinol units
linked by ether bonds and have been reported in several brown
seaweed genera, such as Bifurcaria, Sargassum, Carpopyllum,
Laminaria, Ascophyllum, and Pelvetia (Pal Singh and Bharate
2006; Tierney et al. 2014; Li et al. 2017; Gonçalves-Fernández
et al. 2019). Eckol derivatives represented 50% of the
phlorotannins present in the extract. This is a family of
phlorotannins composed of phloroglucinol subunits linked by
dibenzo[1,4]dioxin bonds, mainly identified in Ecklonia/
Eisenia brown seaweed genera (Pal Singh and Bharate
2006). These findings agree with previous studies that report
on the main natural sources of phlorotannins, mainly in brown
and some red seaweeds (Pal Singh and Bharate 2006;
Gonçalves-Fernández et al. 2019).
Conclusion
In this study, UAE proved to be effective for obtaining an
extract rich in phlorotannins and polysaccharides from S.
J Appl Phycol (2020) 32:1441–1453
compressa. Extraction yields obtained for extract (total
solids), phlorotannins, and polysaccharides were 0.5572 g
EXT g−1 dw, 7.73 mg PGE g−1 dw, and 0.2207 g PS g−1 dw
respectively at the optimized extraction parameters [i.e., tem-
perature (50 °C), power density (3.8 W cL−1), solvent/
seaweed ratio (30 mL dw−1), and ethanol concentration
(32.33%)]. There was no significant difference between the
obtained statistical models and the experimental data. This
study demonstrates how extraction yield is highly correlated
with polysaccharide extraction, the latter being one of the
more abundant fractions present in S. compressa seaweed.
The increase in phlorotannin extraction assisted by ultra-
sound allows to fully exploit the advantages of this technolo-
gy. Nowadays, extraction of polyphenols can be performed at
the pilot and industrial scales, increasing the extraction yields
of natural products of high commercial value for their use in
pharmaceutical, food, and cosmetic industries (Martinez and
Ossa 2016). Results from this study effectively demonstrate
that UAE can enhance and positively affect phlorotannin ex-
traction from S. compressa. The enhanced phlorotannin ex-
traction by means of UAE validates its use. The optimization
of this process is the first step toward the (1) extract stabiliza-
tion and subsequent (2) process scale-up for the (3) develop-
ment of bioactive products containing high levels of polysac-
charides and phlorotannins. These are important consider-
ations given the growing market for antioxidants and
prebiotics.
Acknowledgments This work was supported by the Mexican National
Council of Science and Technology (CONACyT, Research project
238458, Ciencia Basica 2014). The authors acknowledge financial and
infrastructure support from Universidad Autonoma de Nuevo León
(Programa Maricultura), and Tecnológico de Monterrey (NutriOmics,
Research Group).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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