Abstract (English):
Introduction. New natural antioxidants remain a relevant research task of food science. Natural antioxidants neutralize free radicals in food systems, as well as in human body. The antioxidant properties of seaweed have attracted scientific attention for many years. However, most experiments featured non-polar extracts while aqueous extracts still remain understudied. The present research objective was to evaluate the antioxidant properties of hydrothermal extracts of edible seaweed from the Northern Coast of the Sea of Japan. Study objects and methods. The study featured hot-water and autoclave (30 and 60 min) extracts of three edible seaweed species from Russia’s Far East. The research focused on dry matter yield, total phenol content, phenolic profile, antiradical properties, hydroxylion (OH•) scavenging activity, and superoxide radical (O2•−) scavenging activity. Results and discussion. The hot-water extracts appeared to have a higher yield than the autoclave extracts. The hot-water extract of red-purple seaweed Gracilaria verrucosa had the highest yield – 15.90%. The extract of brown seaweed Sargassum miyabei demonstrated the highest total phenol content. The phenolic profile of the extracts revealed 10 compounds, syringic acid and epicatechin being the major ones. The radical scavenging activity of the extracts varied from 48.2 to 88.9%, the highest value was observed in the hot-water extract of S. miyabei. The autoclave S. miyabei extracts also had a high radical scavenging activity, which exceeded other samples by 5.0–13.3%. The hot-water (30 min) extract of G. verrucosa had the lowest antiradical activity. Hot-water and autoclave extracts of S. miyabei showed the best OH• scavenging activity. Only the samples of G. verrucosa demonstrated signs of superoxide radical scavenging. Conclusion. The extracts of brown seaweed S. miyabei proved to be the most active. The hot-water and autoclave extracts had the highest total phenol content and the strongest DPPH and OH• inhibitory activity.

Keywords:
Marine plants, phenols, extract, antioxidant activity, radicals, Sea of Japan

INTRODUCTION
Modern science associates many diseases with
the destructive effect of oxidants, or free radicals.
Peroxidation is the most grievous consequence of
free radicals entering the cell. Lipid oxidation in
biological membranes is a highly destructive process
that triggers liver damage, carcinogenesis, and aging.
Lipid peroxidation is a major cause of the defective cell
proliferation [1, 2]. Oxidized lipid compounds react
with proteins, thus lowering enzymatic activity, causing
cleavage of polypeptide chains, initiating DNA damage,
accelerating aging, and aggravating such diseases as
cancer and atherosclerosis. The aldehyde groups of
these compounds form intermolecular crosslinks, which
violate the structure of macromolecules and disorganize
their functioning. By oxidizing lipids, free radicals
cause glaucoma, cataracts, cirrhosis, ischemia, etc. [3].

The negative effects of oxidative stress can be mitigated
by antioxidants, which neutralize potentially harmful
reactive free radicals in the body cells before they
cause lipid and protein oxidation. As a result, they can
reduce potential mutations and prevent cancer and heart
disease [4, 5].
Plants contain effective antioxidant substances.
Polyphenols or bioflavonoids of plant origin, when they
work in tandem, are extremely efficient against free
radicals. Therefore, any study of vegetables, fruits, and
spices as an alternative source of antioxidants will be
highly relevant, considering that synthetic antioxidants
are potentially toxic and carcinogenic. The past two
decades have seen a lot of studies in phytochemicals
from various types of terrestrial plant materials [6–8].
In this respect, seaweed attracts much less
scientific attention than higher plants. Seaweed makes
up a significant part of the marine flora. It contains
compounds of various structures and bioactivities,
which provide antioxidant, antibacterial, antiinflammatory,
and anticarcinogenic properties [9–11].
Although seaweed is rich in polysaccharides and
minerals, it is seldom used in food technology. A
science-based proof of its antioxidant activity could
increase its value as a food or supplement and expand
its market. When exposed to light and oxygen, seaweed
produces free radicals and other strong oxidants.
However, the absence of oxidative damage in its
structural components and its resistance to oxidation
during storage suggest that seaweed has a good
antioxidant system. Seaweed extracts are known to be
rich in such hydrophilic components as polyphenols
and soluble polysaccharides [9, 12–15]. In particular,
brown seaweed is rich in natural antioxidants,
e.g. such phenolic compounds as phlorotannins and such
carotenoids as fucoxanthin and isoprenoids [16].
Many seaweed extracts possess strong antioxidant
properties [17–21]. However, there have been no
publications on the antioxidant activity of seaweed
from the Northern Coast of the Sea of Japan. Moreover,
the antioxidant activity of seaweed has been studied
mainly in non-polar extracts and very seldom – in
aqueous extracts [22, 23]. Seaweed owes its antioxidant
properties due to its hydrophilic and hydrophobic
compounds. Therefore, its antioxidant activity in
hydrothermal extracts needs more research because
hydrothermal treatment is an integral part of processing.
This study featured three species of seaweeds that
are part of human and animal diet in the Primorye
Territory of Russia’s Far East.
Codium flagile from the family of Codiaceae is a
species of green seaweed found in the Indo-Pacific and
the Atlantic. In Russia, it grows on the Northern Coast of
the Primorye Territory. Its thallus is prostrate, branched,
pillow-shaped or upright. It can be 5–30 cm long. In
Oceania and Asia, and especially in Korea and Russia’s
Primorye, C. flagile is usually served raw or boiled.
C. flagile extracts are widely used in medical
cosmetology as it synthesizes water-retaining
substances.
Gracilaria verrucosa, Gracilariaceae family, is
a species of red-purple seaweed found in the lowboreal-
tropical Atlantic-Indo-Pacific. In Russia, this
seaweed grows in the Far East, in the Sea of Japan and
the Sea of Okhotsk. It has branched, cylindrical or flat,
gristly thallus, which is purple-red or fading to green.
G. verrucosa is 20–40 cm long, sometimes reaching
up to 100 cm. It grows in the littoral and sublittoral
sea zones at a depth of up to 2 m. The species prefers
estuarine and sheltered coastal areas and stony, siltysandy
and sandy bottom, interspersed with stones
and shell rock. Classified as a potentially commercial
species, G. verrucosa is a fast-growing seaweed with
a biomass of 4.5 kg/m2, which makes it convenient for
cultivation. In Southeast Asia, it is used for food and
as a tonic substance in folk medicine. G. verrucosa can
serve as a raw material for agar and various food and
feed additives. It is known to contain minerals, proteins
with antibiotic properties, B vitamins, phycocolloids,
etc. However, agar remains the only commercial product
related to this raw material.
Sargassum miyabei, Sargassaceae (Decne) Kutz.
family, is a species of brown seaweed that thrives
in cold waters in the low-boreal-subtropical areas
of Asian. Its thallus is branched, rough, and thick,
2.0–2.5 m in length, olive green. It grows in the littoral
and sublittoral at a depth of 11 m on rocky, stony, and
silty-sandy bottom, near semi-sheltered and open coastal
areas, in separate bushes or small clusters, clustering
with kelp seaweeds and sea grasses. It is a potentially
commercial perennial species, with the biomass up to
15 kg/m2, the mass of one thallus up to 7 kg, and
population density of 2–6 specimens per 2 m2. In the
Russian Far East, it grows in the Sea of Japan and the
Sea of Okhotsk. It is used in the food industry and
as a raw material for therapeutic and prophylactic
medications, food and feed additives. S. miyabei is
known to contain vitamins A, В1, В6, B12, minerals,
polysaccharides with immunostimulating and antitumor
activity, etc. [24].
The research objective was a comparative analysis
of hydrothermal extracts of the following seaweeds:
green C. flagile, brown S. miyabei, and red G. verrucosa,
obtained by hot-water or autoclave methods for 30 and
60 min. The extracts were compared according to the
following parameters: total phenols, phenolic profile, and
antioxidant activity, i.e. DHHP radicals, OH• scavenging
activity, and O2
•− scavenging activity.
STUDY OBJECTS AND METHODS
Materials. All samples of Codium flagile, Gracilaria
verrucosa, and Sargassum miyabei were harvested in
the coastal sea waters of the Sea of Japan in the northern
Primorye Territory in May-July 2018 (Table 1).
Chemicals and reagents. 1,1-diphenyl-2-
picrylhydrazyl (DHHP), ВНТ-2,6-di-tert-butyl-
4-methylphenol (ionol), and tannic acid were
purchased from Sigma-Aldrich (USA). The Folin-
Ciocalteu phenolic reagent was purchased from Fluka
(Switzerland). All the other reagents were of analytical
grade.

Table 1 Place and time of harvest
Seaweed Place Time
Codium
flagile
Olga Bay of the Sea of Japan,
Primorye Territory, Russia
May 2018
Gracilaria
verrucosa
Valentina Bay of the Sea of Japan,
Primorye Territory, Russia
June 2018
Sargassum
miyabei
Valentina Bay of the Sea of Japan,
Primorye Territory, Russia
July 2018
265
Tabakaev A.V. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 262–270
concentrations (0.1–1.0 mg/mL) of extracts in MeOH
(3.0 mL) were added to a 40 mM H2O2 solution (3.0 mL).
The optical density was measured at 230 nm after
10 min of incubation against an empty solution of
phosphate buffer without H2O2.
The O2
•− scavenging activity was determined by the
formula:
O2
•− scavenging activity (%) = [A0−А1/A0]×100 (4)
where A1 is optical density of the extract and A0 is optical
density of the control solution.
Calculation of ЕС50 (mg/mL) was the next step.
Statistical analysis. The data were obtained as mean
and standard deviation (SD) and analyzed by one-way
ANOVA using SPSS 11.5 for Windows. The difference
in mean values was significant at P < 0.05.
RESULT AND DISCUSSION
The hot-water and autoclave hydrothermal extracts
obtained from three edible seaweeds from the Northern
Coast of the Sea of Japan demonstrated different dry
matter yields. Figure 1 shows the effect of processing
time and method on the extraction yield.
A comparative analysis of the quantitative yield
resulted in the following descending order: Gracilaria
verrucosa > Codium flagile > Sargassum miyabei.
The hot-water extract of the red-purple seaweed
G. verrucosa had the highest yield – 15.90%, which
was 1.17–1.58 times higher than that in the autoclave
samples. The extraction time increased the yield by
55–57% for hot-water extracts and by 16.3–47.5% for
autoclave extracts. The hot-water G. verrucosa extract
and the autoclave C. flagile and S. miyabei extracts
experienced the greatest effect of extraction time.
However, a high yield did not equal high antioxidant
activity, which depends on strong antioxidants,
e.g. polyphenols.
Figure 2 illustrates the content of phenols in the
seaweed extracts.
A comparative analysis of the total phenols showed
that S. miyabei had the hot-water extract with the
highest phenolic level, about 3–4 times higher than
the other hot-water extracts. The autoclave extract of
S. miyabei had similar level of total phenols. The
extract of C. flagile had the lowest total phenols among
the hot-water extracts, while G. verrucosa had the
lowest total phenols among the autoclave samples. The
fact that brown seaweeds proved richer in fenols than
green and red seaweeds confirms the data obtained
by other scientists [31]. However, the methods were
slightly different. The total phenol content in the
autoclave C. flagile extract was twice as high as in the
hot-water extract, while in the autoclave extract of
S. miyabei it was lower than in the respective hot-water
extracts (Fig. 2).
Such hydrophilic polyphenols as phlorotanins,
which are bipolar and occur in brown seaweeds, can act
as antioxidant components and thus help the seaweed
overcome oxidative stress [32, 33].
Identification of phenolic compounds. Individual
phenolic compounds were tested using HPLC in extracts
obtained after 60 min of hydrothermal treatment because
a longer processing time provided a higher level of total
phenols. Table 2 shows the content of the main phenolic
compounds in the extracts.
Table 2 demonstrates 10 phenolic compounds in
the composition of edible seaweed extracts from the
Northern Coast of the Sea of Japan. Chlorogenic acid
was the first of the phenolic compounds to elute; it was
not detected in the hot-water C. flagile extract. Similarly,
2,5-dihydroxybenzoic acid was not detected in both
extracts of G. verrucosa and the autoclave extract of
C. flagile. Caffeic, coumaric, ferulic, salicylic, and
* Significant difference Codium flagile
vs. Sargassum miyabei; Р < 0.05; n = 3
** Significant difference Codium flagile
vs. Gracilaria verrucosa; Р < 0.05; n = 3
*** Significant difference Sargassum miyabei
vs. Gracilaria verrucosa; Р < 0.05; n = 3
Figure 1 Yield of seaweed extracts from the Northern Coast
of the Sea of Japan
* Significant difference Codium flagile vs. Sargassum miyabei; Р < 0.05; n = 3
** Significant difference Codium flagile vs. Gracilaria verrucosa;
Р < 0.05; n = 3
*** Significant difference Sargassum miyabei vs. Gracilaria verrucosa;
Р < 0.05; n = 3
Figure 2 Total phenols in the seaweed extracts from
the Northern Coast of the Sea of Japan
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Hot-water extract, 30 min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Hot-water extract, 60 min
*
**
*
***
*
C. flagile G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
C. flagile
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Autoclave extract, min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Autoclave extract, 60 min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин 30 мин АК ***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
0
5000
10000
15000
20000
25000
Total phenol content mg tannic
acid/ 100g dry extract
ЭК 30 мин ***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
0
5
10
15
20
25
30
35
ЕС50, mg/mL
ЭК 30 Hot-water extract, 30 min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Total phenol content mg tannic
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Hot-water extract, 60 min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
0
5000
10000
15000
20000
25000
C. flagile Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea ЭК 30 мин ЭК 60 мин АК 30 ***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the ЭК 30 мин ЭК 60 мин АК 30 Autoclave extract, min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
5000
10000
15000
20000
25000
C. flagile Total phenol content mg tannic
acid/ 100g dry extract
Seaweeds ЭК 30 мин ЭК 60 мин ***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
Seaweeds ЭК 30 мин ЭК 60 мин Autoclave extract, 60 min
266
Tabakaev A.V. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 262–270
syringic acids, epigallocatechin gallate, epicatechin
gallate, as well as epicatechin were registered in all the
samples. Syringic acid and epicatechin appeared to be
the major phenolic compounds for all the extracts.
All the autoclave extracts showed higher levels of
chlorogenic and syringic acids compared to the hotwater
samples, which had a higher content of ferulic acid
and epigallocatechin gallate.
A comparative analysis of individual phenolic
compounds showed that S. miyabei extracts had the
highest content of epicatechin, which was maximal
in the hot-water sample, and syringic acid, which was
maximal in the autoclave sample. The C. flagile extracts
demonstrated the highest content of ferulic acid and
epigallocatechin gallate, which were the most abundant
in the hot-water sample. The G. verrucosa extracts had
the highest content of chlorogenic acid, especially in
the autoclave sample, coumaric acid (hot-water sample),
salicylic acid (autoclave sample), and epicatechin
gallate (autoclave sample). Thus, the phenolic profile
of the extracts depended on the type of seaweed and
the method of hydrothermal treatment. Phenolic acids,
which are the main class of phenolic compounds, are
found in seaweed in significant quantities. Typical
phenols are known to possess antioxidant properties.
Other publications prove that seaweed is rich in phenolic
compounds, e.g. catechin, caffeic acid, epicatechin,
epicatechin gallate, etc. [34, 35].
Antioxidant activity. Radical scavenging
properties. DPPH possesses a nitrogen free radical
Table 2 The main phenolic compounds in the seaweed extracts from the Northern Coast of the Sea of Japan
Compound Retention
time,
min
Content, mg/g
Sargassum miyabei Codium flagile Gracilaria verrucosa
Hot-water
extract
Autoclave
extract
Hot-water
extract
Autoclave
extract
Hot-water
extract
Autoclave
extract
324 nm
Chlorogenic Acid 8.12 2.48 ± 0.01 4.12 ± 0.02 n.d. 0.15 ± 0.00 6.78 ± 0.23 10.82 ± 0.33
Caffeic Acid 10.49 1.69 ± 0.05 2.80 ± 0.12 2.69 ± 0.01 5.09 ± 0.02 4.51 ± 0.14 3.09 ± 0.09
2,5-dihydroxybenzoic Acid 17.43 0.12 ± 0.01 0.45 ± 0.02 0.85 ± 0.00 n.d. n.d. n.d.
Coumaric Acid 20.56 6.02 ± 0.21 8.71 ± 0.29 10.28 ± 0.32 9.04 ± 0.03 11.64 ± 0.48 8.47 ± 0.29
Ferulic Acid 24.19 8.83 ± 0.43 7.12 ± 0.30 15.21 ± 0.56 10.27 ± 0.40 3.15 ± 0.09 2.70 ± 0.08
Salicylic Acid 44.92 11.05 ± 0.42 10.58 ± 0.37 6.87 ± 0.02 7.18 ± 0.03 8.48 ± 0.30 12.48 ± 0.55
277 nm
Epigallocatechin Gallate 8.13 15.19 ± 0.59 10.48 ± 0.43 35.89 ± 0.97 20.17 ± 0.79 18.29 ± 0.75 14.50 ± 0.59
Epicatechin 10.11 33.52 ± 0.98 31.84 ± 0.83 20.15 ± 0.86 24.76 ± 0.95 30.48 ± 1.07 26.53 ± 1.08
Epicatechin Gallate 13.00 4.07 ± 0.15 2.11 ± 0.07 8.97 ± 0.25 9.15 ± 0.38 12.07 ± 0.43 14.20 ± 0.56
Syringic Acid 14.78 57.40 ± 2.14 63.18 ± 2.46 17.67 ± 0.54 28.94 ± 1.05 25.76 ± 0.90 30.87 ± 1.14
n.d. – not detected
Table 3 Antiradical properties of the seaweed extracts from the Northern Coast of the Sea of Japan
Seaweed Antiradical properties
Radical scavenging
activity,%
ЕС50, μg/mL , min Antiradical efficacy, μg/L·c
30 min 60 min 30 min 60 min 30 min 60 min 30 min 60 min
Hot-water extract
Codium flagile 58.9 ± 2.8 72.8 ± 3.5 25.69 ± 1.20 20.04 ± 0.67* 22.01 ± 1.00 17.89 ± 0.80 0.0018 ± 0.0000 0.0028 ± 0.0001
Gracilaria
verrucosa
48.2 ± 2.3 76.1 ± 3.7** 28.06 ± 1.30 21.29 ± 0.96 36.21 ± 1.70 27.12 ± 1.30 0.0010 ± 0.0000 0.0017 ± 0.0001
Sargassum
miyabei
72.6 ± 3.5 88.9 ± 4.4 14.53 ± 0.60 10.39 ± 0.09 10.59 ± 0.50 8.09 ± 0.40 0.0065 ± 0.0003 0.0120 ± 0.0005***
Hot-water extract
Codium flagile 65.3 ± 3.2 80.6 ± 4.0 14.59 ± 0.21 11.23 ± 0.36 17.23 ± 0.36 15.52 ± 0.60 0.0040 ± 0.0002 0.0057 ± 0.0003
Gracilaria
verrucosa
50.1 ± 2.5 78.4 ± 3.8** 30.83 ± 0.58 22.36 ± 1.47 32.36 ± 1.47 20.04 ± 1.00 0.0010 ± 0.0000 0.0022 ± 0.0006
Sargassum
miyabei
75.2 ± 3.6*** 85.6 ± 4.1 12.12 ± 0.15* 11.15 ± 0.08 12.30 ± 0.08 9.08 ± 0.40 0.0067 ± 0.0003 0.0099 ± 0.0005
Ionol 94.0 ± 4.5 8.75 ± 0.3 8.0 ± 0.4 0.0160 ± 0.0700
* Significant difference Codium flagile vs. Sargassum miyabei; Р < 0.05; n = 3
** Significant difference Codium flagile vs. Gracilaria verrucosa; Р < 0.05; n = 3
*** Significant difference Sargassum miyabei vs. Gracilaria verrucosa; Р < 0.05; n = 3
267
Tabakaev A.V. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 262–270
and is easily destroyed by a free radical scavenger. The
DPPH radical assay was used to test the ability of the
antioxidant compounds found in the extracts to act as
proton radical scavengers or hydrogen donors. Table 3
demonstrates the antiradical properties of the extracts.
The autoclave extracts appeared to be more active
than the hot-water extracts. The radical scavenging
activity varied within wide limits of 39.6–88.9%.
The maximal value was observed in the hot-water
S. miyabei extract, and its radical scavenging
activity was 12.8–28.4% higher than that of the
other extracts and only 10.4% lower than that of
ionol. The autoclave extracts of S. miyabei also
had a high radical scavenging activity, which
exceeded that of the other extracts by 5.0–13.3%.
A comprehensive assessment of the antiradical
activity showed that the hot-water (30 min) extract of
G. verrucosa had the lowest values. The hot-water
extract of S. miyabei had the most pronounced
antiradical properties. Its EС50 concentration was
1.89–2.41 times lower than the other extracts, its
time was 2.21–9.79 times shorter, and its antiradical
efficiency was 4.29–24.00 times higher.
The extract of S. miyabei, which had the highest
total phenol content, also had a much lower effective
concentration of ЕС50. The autoclave extracts demonstrated
a strong correlation between the level of total
phenols (Fig. 2) and the effective concentration of EС50.
When the total phenol level was high, the effective EС50
concentration was low. Therefore, the polyphenolic
components in the seaweed extracts were capable of
acting as free radical scavengers. For hot-water extracts,
however, this ratio was weak. Since the DPPH radical
analysis was not specific for any particular antioxidants,
the antiradical activity of these extracts result not only
from phenols, but also from various other water-soluble
antioxidants, e.g. polysaccharides, folic acid, thiamine,
and ascorbic acid [36, 37].
The OH• scavenging activity and absorption
of superoxide radicals. Antioxidant activity was
determined according to the ability of the antioxidant
components to inhibit the oxidation of deoxyribose
by the reactive hydroxyl-ion radical (OH•), formed as a
result of the Fenton-type reaction. The reaction involves
two antioxidant defense mechanisms: suppression of the
generation of OH• from H2O2 by binding with metal ions
and the direct transfer of one electron to the generated
radical. Figure 3 illustrates the OH• scavenging activity
of the seaweed extracts.
Both autoclave and hot-water S. miyabei extracts
demonstrated the highest OH• scavenging activity,
while that of the other extracts was significantly lower.
A comparative analysis showed similar EС50 values and
ranking. A longer extraction time increased the OH•
scavenging activity, but the increase was insignificant.
Brown seaweeds are known to contain floratanins,
which chelate heavy metals [32, 38]. Probably, the
high content of floratanins predetermined the high OH•
scavenging activity of S. miyabei extracts. However,
seaweeds have a complex composition, and its phenols
are not the only compounds that exhibit antioxidant
activity, which is also typical of carotenoids and
polysaccharides [39]. Hydrothermal extracts of
some green and brown seaweeds contain sulfated
heteropolysaccharides, which strongly inhibit OH•
activity in vitro [40–42].
Figure 4 demonstrates the O2
•− scavenging activity of
the extracts.
Although O2
•− is a relatively weak oxidizing agent,
it decomposes into stronger oxidative forms, such
as singlet oxygen and OH•. The autoclave extract of
G. verrucosa was the only sample to demonstrate
O2
•− scavenging activity. All hot-water extracts showed
O2
•− inhibitory activity. Figure 4 shows that the
maximal inhibitory activity belonged to the C. flagile
extract. Surprisingly, S. miyabei revealed no signs of
O2
•− scavenging. The extraction time only slightly
increased the O2
•− absorption.
* Significant difference Codium flagile vs. Sargassum miyabei; Р < 0.05; n = 3
** Significant difference Codium flagile vs. Gracilaria verrucosa;
Р < 0.05; n = 3
*** Significant difference Sargassum miyabei vs. Gracilaria verrucosa;
Р < 0.05; n = 3
Figure 3 OH• scavenging activity of the seaweed extracts from
the Northern Coast of the Sea of Japan
* Significant difference Codium flagile vs. Gracilaria verrucosa;
Р < 0.05; n = 3
Figure 4 O2
•− scavenging activity of the seaweed extracts
from the Northern Coast of the Sea of Japan
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
S.miyabei
Japan
мин АК 60 мин
***
*
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
S.miyabei
of Japan
мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Hot-water extract, 30 min
*
**
***
*
verrucosa S.miyabei
of the Sea of Japan
АК 30 мин АК 60 мин
***
*
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
**
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Hot-water extract, 60 min
***
*
C. flagile S.miyabei
G.verrucosa
Seaweeds of the Sea of Japan
мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
C. flagile
G.verrucosa
Seaweeds of the Sea of Japan
мин ЭК 60 мин АК 30 мин АК 60 мин
Autoclave extract, 30 min
***
*
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
Autoclave extract, 60 min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
Hot-water extract, 30 min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
Hot-water extract, 60 min
*
**
*
***
*
C. flagile G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
Autoclave extract, 30 min
*
**
*
***
*
0
4
8
12
16
C. flagile G.verrucosa S.miyabei
Yield, %
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
*
**
0
5000
10000
15000
20000
25000
C. flagile S.miyabei
Total phenol content mg tannic
acid/ 100g dry extract
G.verrucosa
Seaweeds of the Sea of Japan
ЭК 30 мин ЭК 60 мин АК 30 мин АК 60 мин
***
* **
0
50
100
150
200
C. flagile
ЕС50, mg/mL
G.verrucosa S.miyabei
Seaweeds of the Sea of Japan
*
0
0
5
10
15
20
25
30
35
C. flagile
ЕС50, mg/mL
G.verrucosa
Seaweeds of the Sea of Japan
Autoclave extract, 60 min
268
Tabakaev A.V. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 262–270
The established difference in the O2
•− absorption
between the hot-water and autoclave extracts proved
that the two methods extracted different types of
antioxidants. The lack of correlation between the
total content of phenols and О2
•− inhibition means that
such activity demonstrated by all three species could
not be explained solely by the presence of phenolic
antioxidants. Sulfated polysaccharides obtained from
the hot-water extracts of green and brown seaweeds
inhibited the activity of О2
•− in vitro [40–42].
CONCLUSION
The hot-water extraction proved more effective
than the autoclave extraction in terms of OH• a nd О 2
•−
scavenging activity. However, the autoclave extracts had
better antiradical properties. The extracts of the brown
seaweed Sargassum miyabei showed significantly higher
antiradical and antioxidant activity than the extracts of
the red seaweed Gracilaria verrucosa and the green
seaweed Codium flagile. The relationship between OH•
scavenging and antioxidant activities in these samples
indicated that it came from the hydrophilic polyphenolic
antioxidants. The hydrothermal extracts of the seaweeds
proved to be a promising source of antioxidants for
human consumption.
CONTRIBUTION
The authors contributed equally to the manuscript
and are equally responsible for any potential
plagiarism.
CONFLICT OF INTEREST
The authors declare that there is no conflict

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