Journal of Food Measurement and Characterization

https://doi.org/10.1007/s11694-020-00758-w

ORIGINAL PAPER

Evaluation of bioactive chemical composition, phenolic,

and antioxidant profiling of different crude extracts of Sargassum
coriifolium and Hypnea pannosa seaweeds
Mohammad Khairul Alam Sobuj1 · Md. Ariful Islam2 · Md. Amdadul Haque3 · Md. Mohidul Islam1 ·
Md. Jahangir Alam2 · S. M. Rafiquzzaman2

Received: 29 July 2020 / Accepted: 19 November 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Seaweed as a source of functionally bioactive components has been well recognized due to its beneficial effects for human
health. However, beneficial health effects of seaweeds vary with the different aspects especially geographical location of
the seaweed. In the present study, two seaweeds (Sargassum coriifolium, Hypnea pannosa) were collected from Bangladesh
and crude extracts were prepared using methanol, ethanol and water as extraction solvent. Here, we determined preliminary
phytochemical composition, total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity of crude
extracts using different qualitative and quantitative in vitro assays. Overall, screening of phytochemicals revealed that both
the seaweeds contain diverse type bioactive compounds including terpenoid, saponin, phlobatannin, cardiac glycosides, phe-
nolic and flavonoid depending on the seaweed species and extraction solvents used. FT-IR spectroscopy also confirmed the
presence of phenols, carboxylic acid, ketones, ethers, aromatics, amides and sulfonates at varying degree. Methanol extract
of S. coriifolium and H. pannosa showed highest activity followed by ethanol and water extracts. Here, methanolic extract
of S. coriifolium showed highest amount of TPC (128.56 mg of GA/g), TFC (58.29 mg of quercetin/g), DPPH (75.01%,
­IC50 = 1.03 mg/ml), ABTS (72.24%, I­ C50 = 1.55 mg/ml), hydrogen peroxide (71.64%, I­ C50 = 1.98 mg/ml), phosphomolyb-
denum (28.08 mg of ASE/g) and reducing power assays (938.5208 mg of ASE/g). A positive correlation was found among
TPC and different antioxidant assays. These results confirmed the presence of diverse type of chemical composition with its
antioxidant activity which will be useful for pharmacological as well as in functional food application.

Keywords Sargassum coriifolium · Hypnea pannosa · Chemical composition · Antioxidant activity · Crude extracts

Introduction superoxide anion (­ O2−), hydroxyl radicals (OH⋅), hydrogen

The reactive oxygen species (ROS) are generated in living
organisms because of endogenous metabolic activity or from
exogenous sources [1, 2]. Generally, the oxygen molecule
reacts within the body and produces by-product element
called free radicals or reactive oxygen species (ROS) (e.g.,
* S. M. Rafiquzzaman
rafiquzzaman@bsmrau.edu.bd
1
Marine Fisheries and Technology Station, Bangladesh
Fisheries Research Institute, Cox’s Bazar 4700, Bangladesh
2
Department of Fisheries Biology and Aquatic Environment,
BSMRAU​, Gazipur, Bangladesh
3
Department of Agro‑Processing, BSMRAU​, Gazipur,
Bangladesh
peroxide ­(H2O2), and singlet oxygen (1O2) due to oxidation
process. ROS causes cellular and subcellular destruction by
different ways like peroxidation of tissue membrane lipids,
denaturing cellular proteins, DNA strands break down, and
cellular function disruption [1, 3]. Excessive amounts of
ROS creates oxidative stress which is thought to be involved
in the development of different non-curable diseases like
alzheimer’s, cancer, parkinson’s, heart failure, autism [4],
infection and chronic fatigue syndrome [5, 6]. Therefore, the
elimination of ROS from human body is one of the utmost
concerning approaches to defend against these diseases.
Elimination of oxidative stress can be done by con-
sumption of natural product which is rich in antioxidant
compounds to make balance between ROS production and
endogenous protection when the body is in under oxida-
tive stress. The common naturally occurring antioxidants

13
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include retinoids, ascorbic acid, flavonoids, polyphe-
nols and tocopherols plays a vital role in developing the
immunity against many diseases. Report showed that use
of synthetic antioxidant (e.g., butylated hydroxyl toluene
(BHT), butylated hydroxyanisole (BHA) and propyl gal-
late compounds in food preservative may cause blood clot-
ting, tumor and also effects of lung [7]. In recent years,
researchers are focusing to search the natural sources anti-
oxidant rich food materials rather than the synthetic one.
Previous research revealed that seaweeds to be an amusing
sources of bioactive natural compounds which is potential
as antioxidant compounds [8–11].
Seaweeds are enormous collection of macro algae refers
to several species of macroscopic, multicellular, marine
algae. Marine algae or seaweed lives naturally in shal-
low water which is exposed to ultraviolet sunlight and
various abnormal environmental conditions. There is a
long tradition in China, Japan, Korea, and Southeast Asia
of consuming seaweeds as part of the regular diet. The
most remarkable utilization of seaweed is found in phy-
cocolloid or hydrocolloid industry and cosmetic industry,
biofuel industry, pharmaceutical industry for the develop-
ment of drugs for Alzheimer’s disease, cancer and gastric
ulcer, waste water treatment industry, bioplastic indus-
try [12, 13]. Traditionally seaweeds are rich in bioactive
compounds possess strong anti-inflammatory, anti-pain
activities, antibacterial, antifungal and high antioxidant
properties [14, 15]. In addition, seaweed contains different
phytochemical composition such as tannins, saponins, phe-
nols and flavonoids in varying concentration [11, 16]. In
Bangladesh, diversity of seaweed is rich and reported
that there are 193 seaweed species belonging to 88 are
red (Rhodophyta), 51 green (Chlorophyta) and 54 brown
(Phaeophyta) group occurring on Bangladesh coast [17].
However, researches on the exploring of seaweed
resources in Bangladesh is poorly elicited although it has
immense potentialities. Most studies have been focused
on biochemical and proximate composition analysis of
the seaweed [18]. Very few researches on the analysis of
phytochemical composition with its antioxidant activity
of seaweeds collected from Bangladesh coast have been
found in the literature [11]. The composition and yield
vary according to season, age, species and geographic
location [19]. Therefore, in the present study, we screened
out functionally active chemical composition using dif-
ferent qualitative test and confirmed the presence of func-
tional group of compounds by FT-IR spectroscopic method
using different crude extracts of S. coriifolium and H. pan-
nosa. In addition, we also determined total phenolic con-
tent and antioxidant activities of crude extracts by different
in vitro spectroscopic assays with its correlation among
total phenolic content and different antioxidant activities.
13
M. K. A. Sobuj et al.
Materials and methods
Chemicals and reagents
Aluminum chloride, hydrochloric acid, ammonium molyb-
date, chloroform, sulfuric acid, ethanol, ethyl acetate, ferric
chloride, ferrous sulfate, Gallic acid, Folin-Ciocalteu’s phe-
nol reagent, glacial acetic acid, hydrogen peroxide, metha-
nol, potassium acetate, potassium persulfate, sodium nitrite,
sodium acetate, sodium carbonate, sodium phosphate,
2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were
purchased from Sigma (St. Louis, MO). Wholly chemicals
and reagents support in the study were of analytical grade.
Sample collection and preparation
Mature S. coriifolium and H. pannosa sample were col-
lected from the Saint Martin’s Island (92°32ʹ43.04ʺE and
20°61ʹ65.74ʺN) of Bay of Bengal of Bangladesh in Novem-
ber 2019. The seaweed was collected from exposed rock
exteriors and washed frequently with clean sea water to
eliminate rubbish and other foreign ingredients. The speci-
mens were transported in an icebox (4 °C) and carried out
it in the laboratory. The samples were rinsed with distilled
water to remove salt, sand, debris and any other contami-
nation. Then the cleaned seaweed was left to freeze dried
(VaCo 2, Zirbus, UK) at − 83 °C for two days to remove the
moisture, powdered, sealed in airtight bags and preserved in
a refrigerator at 4 °C for further analysis in the laboratory.
Preparation of seaweed extract
Seaweed extract was prepared according to the method of
Rafiquzzaman et al. [11]. Briefly, four gram of seaweed fine
powder was soaked in 100 ml of solvent (water, methanol
and ethanol) by maceration for the preparation of an extract
by solvent extraction. The sample was kept in the dark for
24 h with intermittent shaking for better extraction. After
incubation, the solution was filtered with Whatman filter
paper No 4 (20–25 µm) retaining hygienic conditions. The
methanol and ethanol extracts were then concentrated using
a Rotary Vacuum Evaporator (LRE-702A, Labocon, UK)
and Nitrogen Evaporator (AT-EV-50, Athena Technology,
India) at 36 °C and the water solvent was dried by the Freeze
Dryer (VaCo 2, Zirbus, UK) at − 83 °C.
Qualitative analysis of phytochemical substances
Newly prepared all crude extracts of seaweed were subjected
to qualitative assessments for the identification of various
Evaluation of bioactive chemical composition, phenolic, and antioxidant profiling of different…
classes of active phytochemical constituents such as terpe-
noid [20], phlobatannin [11], saponin [21] and cardiac gly-
cosides [22] following standard methods. General reactions
in these analyses exposed the presence or absence of these
compounds in the crude extracts tested.
Test for terpenoids (Salkowski test): Five ml of each
extract was added with 2 ml of chloroform and 3 ml of con-
centrated ­H2S04. Formation of a reddish brown color at the
interface indicates the presents of terpenoids of the sample
extracts [20].
Test for phlobatannins: Three ml of extract was mixed
with few drops of 1% hydrochloric acid and observed for the
precipitation. A red precipitate showed the positive results
for the presence of phlobatannin in the crude extract [11].
Test for saponin (frothing test): About 3 ml of each
extracts was treated with 3 ml of distilled water and sample
was shaken vigorously for a stable persistent of froth about
1 min. The frothing was added with 3 drops of olive oil and
shaken vigorously, then observed for the formation of an
emulsion, which indicates the presence of saponins in the
extracts [21].
Test for cardiac glycosides (Keller-Killani test): Five ml
of each extracts was mixed with 2 ml of glacial acetic acid
containing one drop of ferric chloride solution. Further 1 ml
of concentrated ­H2S04 was added into the solution. A brown
ring at the interface indicates the presence of a deoxysugar
characteristic of cardenolides. A violet ring may appear
below the brown ring (at the H ­ 2SO4 layer), while in the ace-
tic acid layer, a greenish ring may form just above the brown
ring which will gradually spread throughout this layer [22].
Fourier‑Transformed infrared (FT‑IR) spectroscopy
FT-IR analysis was performed to detect the characteristic
peaks in ranging from 500 to 4000 cm−1 and their func-
tional groups [23–26]. Different crude extracts of S. corii-
folium and H. pannosa were used to determine the presence
of characteristic peaks and their functional groups using
FT-IR spectroscopy (Perkin Elmer Spectrum 2). Analysis
was repeated twice and confirmed the spectrum in case of
all extracts.
Quantitative analysis of phytochemicals
Total phenolic content (TPC)
The concentration of total phenols in the crude extracts was
determined using Folin-Ciocalteu Phenol reagents and exter-
nal calibration with Gallic acid following by Karydas et al.
[27] with slight modification. Briefly, 0.5 ml extract solution
was added with 0.1 ml of FC reagent solution. After 15 min,
2.5 ml of saturated ­Na2CO3 (75 g/l) was added in the solu-
tion and allowed to stand for 30 min at RT and absorbance
was measured at 760 nm using the spectrophotometer
(T80 + UV/VIS Spectrophotometer, UK). The concentra-
tion of total phenolics was calculated as mg of Gallic acid
equivalent per gram. The calibration equation for Gallic acid
was Y = 0.0116X + 0.0162 ­(R2 = 0.9987).
Total flavonoid content (TFC)
Total flavonoid contents of crude extracts were deter-
mined using the aluminum chloride colorimetric method,
as described by Padhan, Nayak and Panda [28] with slight
modifications. Briefly, 1 ml extract solution was mixed with
3 ml methanol, 0.2 ml 10% aluminum chloride and 0.2 ml
1 M potassium acetate. The solution was then incubated at
RT for 30 min and absorbance was measured at 420 nm. The
concentration of total flavonoids was calculated as mg of
quercetin equivalent per gram. The calibration equation for
Quercetin was Y = 0.0102X − 0.0637 ­(R2 = 0.9693).
Evaluation of total antioxidant capacity
DPPH (2, 2‑diphenyl‑1‑picrylhydrazyl) assay
The radical scavenging activity of different extracts was
determined by using DPPH assay according to Doh, Dunno
and Whiteside [29] with minor modification. Extracts solu-
tion was mixed with 0.15 mM DPPH solution (prepared in
ethanol) with 1:1 ratio. After 30 min incubation, the absorb-
ance was read at 517 nm. Different concentrations were
tested for each sample to get I­ C50 value which is defined as
the amount of antioxidant necessary to decrease the initial
DPPH ion by 50%. Ascorbic acid was used as a positive con-
trol and the values were compared with the crude extracts.
The % radical scavenging activity of the crude extracts was
calculated using the following formula: DPPH radical scav-
enging activity (%) = [(A0 − A1)/ ­(A0)] × 100. Where, ­A0 is
the Absorbance of control and ­A1 is the Absorbance of the
sample.
ABTS radical scavenging assay
The antioxidant activities of different extracts were evalu-
ated through the colorimetric method of ABTS radical
scavenging according to the method of Khan et al. [30]
with slight modification. Briefly, 7 mM ABTS solution
and 2.45 mM Potassium persulfate was added in 1:1 ratio
into water to prepare working solution. Extracts (50 μl) was
added with ABTS solution (950 μl) followed by incubation
at RT for 16 h in dark at 734 nm. I­ C50 values were tested
for each sample at each concentration. Ascorbic acid was
used as a positive control. The percentage of inhibition was
calculated using the following formula, ABTS scavenged
13

(%) = [(A0 − A1)/(A0)] × 100; where, ­A0 is the Absorbance
of control and ­A1 is the Absorbance of the sample.
Hydrogen peroxide scavenging activity
Extracts antioxidant activities were evaluated by the hydro-
gen peroxide scavenging activity according to the method
of Latos-Brozio and Masek [31] with slight modification.
Briefly, aliquot extracts at various concentrations was added
0.3 ml hydrogen peroxide solution (40 mM) and 1.2 ml
phosphate buffer (40 mM; pH 7.4) and vortexes well. After
10 min the absorbance was measured at 230 nm against a
blank solution (phosphate buffer). Different concentrations
were tested for each sample to get ­IC50 value. Ascorbic acid
was used as a positive control. The percentage of inhibi-
tion of the crude extracts was calculated using the follow-
ing formula, Hydrogen peroxide scavenged (%) = [(A0 − A1)/
(A0)] × 100. Where, ­A0 is the Absorbance of control and ­A1
is the Absorbance of sample solution.
Phosphomolybdenum assay
The antioxidant activity of different crude extracts was eval-
uated by the green Phosphomolybdenum complex formation
according to the method of Rafiquzzaman et al. [11] with
negligible modification. Briefly, 0.9 ml of working reagent
solution (0.6 M H ­ 2SO4, 28 mM Sodium phosphate, 4 mM
Ammonium molybdate) was mixed with 0.1 ml of dilute
extract solution. The samples were then placed in a boiling
water bath for 90 min at 95 °C and cool at room tempera-
ture. After cooling down, the absorbance of each sample was
measured at 695 nm. Blank was run using 0.9 ml of reagent
solution and 0.1 ml of the same solvent used for the sample
and incubated as for the other samples. Here ascorbic acid
was used as a positive control. Antioxidant activity was also
expressed as equivalents of ascorbic acid.
Reducing power assay
Antioxidant activity of different crude extracts reducing
power at various concentrations was evaluated using the
method of Monir et al. [32] with insignificant modification.
Briefly, 1.5 μl of extracts was mixed with 1.5 μl of phosphate
buffer (0.2 M, pH 6.6) and 1.5 μl of potassium hexacyano-
ferrate (1%, w/v). After incubation at 50 °C in a water bath
for 20 min, 1.5 μl of trichloroacetic acid solution (10%) was
added and centrifuge at 800×g for 10 min. The supernatant
was collected and mixed with 3 ml of DW and 200 μl of
ferric chloride solution (0.1%, w/v) and incubated at RT for
10 min for stable absorbance at 700 nm; greater absorbance
of the reaction mixture indicates greater reducing power.
Ascorbic acid was used as a positive control and the values
were compared with the crude extracts of seaweed.
13
M. K. A. Sobuj et al.
Statistical analysis
The obtained experimental data was analyzed through the
standard statistical procedure. Data were analyzed using
SPSS software (IBM Co., Chicago, IL). Analysis of variance
(ANOVA) and Duncan’s multiple range method were used
to compare solvents and samples. Values were expressed
as means ± standard deviations. Differences were consid-
ered significant at P < 0.05. All analyses were performed
in triplicate.
Results and discussion
The metabolic and physiological abilities of marine organ-
isms that allow them to persist in complex habitat types
provide a great potential for production of chemical com-
position (secondary metabolites), which are not found in
earthbound situations. Thus, marine algae and their organic
extracts are among the most extravagant wellsprings of
known and novel bioactive compounds [33]. However, a
few studies have been concerned on presences of phytocon-
stitute and bio-functional activities of seaweed resources in
Bangladesh. Therefore, it is significant to recognizing Bang-
ladeshi seaweed resources for better understanding of its bio-
functional activities as the concentration and availability of
phytochemicals of marine algae are largely varies accord-
ing to environmental condition, season, maturity, geographic
location and just as depth of immersion [34, 35]. However,
there are inadequate data available on available chemical
composition and antioxidant activity of seaweeds resources
in Bangladesh. Though, Rafiquzzaman et al. [11] explore
red algae H. musciformis collected from Bangladesh and
found a number of phytochemicals and promising antioxi-
dant properties. Therefore, in this study we identify several
bioactive chemical compositions, total phenolic content and
antioxidant activity of altered crude extracts of two com-
mercially important Bangladeshi seaweeds (S. coriifolium
and H. pannosa) along with correlation among TPC and
antioxidant activity was performed for better understanding
the function of phenols towards antioxidant activity.
Phytochemical screening
The nearness or nonappearance of phytoconstituents relies
on the dissolvable medium utilized for extraction and the
physiological property of the seaweeds. The essential bioac-
tive compounds can be screened in seaweed using frequent
methods retaining diverse solvents and conditions [36]. In
this study, we used methanol, ethanol and water extracts
for phytochemical screening which confirms the presence of
active chemical composition including terpenoids, saponin,
phlobatannin, cardiac glycosides, phenolics and flavonoids
Evaluation of bioactive chemical composition, phenolic, and antioxidant profiling of different…
among different extracts at varying concentration (Table 1).
Out of 36 tests, 30 were positive and the left over six were
negative. Among them phlobatannin were showed positive
result in water extract and negative result in ethanol and
methanol extracts. Phlobatannin is a tannin derivative and
tannin generally found in higher plants. This is an indication
that this compound can only be derived in water extract.
The occurrence of phlobatannin also indicates the diuretic
property of the seaweed. No previous study on the prelimi-
nary phytochemical screening of extracts from S. coriifo-
lium was found in the literature. However, Rafiquzzaman
et al. [11] identify several phytochemicals from red algae H.
musciformis collected from Bangladesh which is correlated
with the present finding. Other authors’ research [37, 38]
also identify several phytoconstituents from brown algae S.
wightii with other classes of seaweed from Gulf of Mannar
region which is verified by the present findings.
Fourier Transformed infrared (FT‑IR) spectroscopy
The different crude extracts (water, ethanol and methanol)
of S. coriifolium and H. pannosa showed different peaks
which confirmed the presence of different functional groups
in the range between 4000 and 500 cm−1. The results of
FT-IR analysis confirmed the presence of phenols, carbox-
ylic acids, aromatic, ketones, ethers, amides/amines and
sulfonates compounds at varying concentration (Fig. 1;
Table 2). Several peaks were identified in the single bond
area (900–4000 cm−1) of the different crude extracts of sea-
weeds. The peaks at 3459.8, 3458.6, 3425.7, 3310, 3385.2,
3398 cm−1 arises due to the O–H stretch of H-bonded
alcohols and phenols. Bands observed in the range of
2700–3300 cm−1 (2970, 2918.4, 2907.2, 2826, 2748.3,
2791.8) indicate the presence of O–H stretch of carboxylic
acids. The C=O stretch of amide were displayed in the pick
at 1673.4, 1660.5, 1629.7 cm−1. The C=O stretch of ketones
were also arises in the double bond area (1500–2000 cm−1).
The strong pick at 1026.3 cm−1 region arises due to the
C-O stretch of primary alcohol in case of S. coriifolium.
The C-H stretch of aromatics can be found in the peaks at
Table 1  Preliminary phytochemical analysis of crude extracts of S. coriifolium and H. pannosa
Name of the Species Name of the Extracts Terpenoids Saponin
S. coriifolium Methanol + +
Ethanol + +
Water + +
H. pannosa Methanol + +
Ethanol + +
Water − +
“ + ” = Present of constituent; “ − ” = Absence of constituent
945.7, 942.6, 881.7, 870.4 cm−1. The pick at 1557.7 and
1629.7 cm−1 was for the C=C stretch of aromatics in water
extracts. The functional group ether displayed C-O stretch
between the areas of 1000 to 1300 cm−1 of different crude
extracts of seaweeds. The sulfonates showed S=O stretch
in the area of 1300 to 1365 cm−1 and ­NO2 and ­SO2 stretch
in the area of 1100 to 1200 cm−1. Other authors’ research
[11, 16] also used FT-IR to identify several phytochemicals
such as phenols, carboxylic acids, ketones, ethers, aromatics,
amides and sulfonates from H. musciformis and S. wightii
respectively, which is interrelated with the present finding.
Though, Raubbin et al. [39] identify phenols, carboxylic
acids, alkanes, esters, aromatics, amines, phosphine from
ethyl acetate extract of H. flagelliformis. Also, Artemisia
et al. [40] identify several functional groups such as OH,
CH, S=O, C–O, C–S–O stretching and carbohydrate from
brown seaweeds. As they found alcohols, phosphine and
halogen compounds in different extracts which may be due
to the variation of solvent used for extraction and the origin
of the seaweed. The occurrence of certain fatty acids at dif-
ferent crude extracts has been revealed to display antioxidant
activity of each extracts.
Quantitative analysis of phytochemicals
Total phenolic content (TPC)
Extraction of phenolic compounds from plant materials or
from fruits largely depends on the solvent polarity (higher
in polar solvent) or solubility of phenolic compounds [41].
The concentration of total phenols in the crude extracts of
S. coriifolium and H. pannosa was determined using Folin-
Ciocalteu Phenol reagents and external calibration with Gal-
lic acid at a concentration of 7 mg/ml. Significant differences
were found in TPC among different solvent extracts, ranged
between 128.56 and 49.26 mg of GA/g (Table 3). Among
the three crude extracts of S. coriifolium methanol extracts
showed significantly highest amount (128.56 mg of GA/g)
of TPC followed by ethanol and water extract (106.43 and
78.67 mg of GA/g, respectively) (P < 0.05) (Table 3). And
Phlobatannin Cardiac Phenols Flavonoids
Glycosides
− + + +
− + + +
+ − + +
− + + +
− + + +
+ + + +
13

Fig. 1  Fourier transform infrared spectrum of different crude extracts of seaweed a S. coriifolium and b H. pannosa
in case of TPC of H. pannosa significantly highest amount
showed in methanol extracts (89.89 mg of GA/g) followed
by ethanol extract (84.12 mg of GA/g) and water extract
(49.26 mg of GA/g) (P < 0.05) (Table 3). Our present finding
of TPC is supported with the agreement of other authors’
research [15, 42, 43], who also reported that methanol as
solvent extracted highest amount of TPC compared to other
13
M. K. A. Sobuj et al.
extract. Similar results were also found by other authors [11]
in case of red seaweed collected from Bangladesh coast.
Chandini et al. [44] reported that water extract of brown
seaweed, Sargassum marginatum showed a phenolic content
of 24.61 mg GAE/g which is lower than the present finding.
This variation may be due to the variation of solvent, species
and origin of the seaweed.
Evaluation of bioactive chemical composition, phenolic, and antioxidant profiling of different…

Table 2  Summary of the functional groups of active components based on the peak value of Fourier transform infrared
Name of the Species Name of the Extracts Major Functional Groups
Phenol Carboxylic Ketones Ethers Aromatics Amides/ Sulfonates
acids Amines

S. coriifolium Methanol + + + + + + +
Ethanol + + + + + − −
Water + + + + + − +
H. pannosa Methanol + + + + + + +
Ethanol + + − + + − +
Water + + + + + + −

“ + ” = Present of constituent; “ − ” = Absence of constituent

Table 3  Total phenolic and Extracts S. coriifolium H. pannosa

flavonoid contents of different
crude extracts of S. coriifolium Total Phenolics (mg Total Favonoids (mg Total Phenolics (mg Total Favonoids
and H. pannosa of GA/g) quercetin/g) of GA/g) (mg quercetin/g)

Methanol 128.56 ± 0.59a 58.29 ± 1.19a 89.89 ± 1.13a 43.12 ± 0.98a

Ethanol 106.43 ± 0.28b 42.6 ± 2.15b 84.12 ± 2.09b 35.25 ± 1.65b
Water 78.67 ± 1.31c 31.75 ± 1.01c 49.26 ± 0.83c 25.48 ± 1.48c

Different superscript letter illustrate significantly different from each other. The level of significance is P <
0.05

Total flavonoid content (TFC)
Flavonoids are the most vital natural phenolic due to their
wide range of chemical and biological activities, including
antioxidant and free radical scavenging properties [45]. The
concentration of total flavonoid content in the crude extracts
was determined using the aluminum chloride method and
external calibration with quercetin at a concentration of
7 mg/ml. Amongst the three extracts of S. coriifolium metha-
nol extracts showed significantly highest amount (58.29 mg
of quercetin/g) of TFC followed by ethanol and water extract
(42.60 and 31.75 mg quercetin/g, respectively) (P < 0.05)
(Table 3). And for the crude extracts of H. pannosa metha-
nol extracts also showed significantly highest (P < 0.05)
amount (43.12 mg of quercetin/g) of TFC followed by
ethanol extract (35.25 mg of quercetin/g) and water extract
(25.48 mg of quercetin/g) (Table 3). Our present finding
of TFC is supported with the agreement of other authors
[11, 14, 46], who was also found that methanolic extracts
of brown and red seaweed contains 20.65 to 70.67, 42.5 and
31.9 to 131.3 mg of quercetin/g respectively.
Evaluation of total antioxidant capacity
DPPH assay
DPPH is a compound that retains a nitrogen free radical
and is readily destroyed by a free radical scavenger. The
DPPH radical in fact may be neutralized by either direct
reduction via single electron transfer (SET) or via hydrogen
atom transfer. The DPPH radical-scavenging capacities of
all crude extracts were showed extract and concentration
dependency and their activity varied significantly among
different extracts (P < 0.05). Here methanolic extracts of
S. coriifolium and H. pannosa showed significantly higher
(P < 0.05) percentage of inhibition (75.01, 71.20% respec-
tively) followed by ethanolic and water extracts (Fig. 2a).
Also, we observed that radical-scavenging capacities of
brown seaweed (S. coriifolium) are higher compared with
the red seaweed (H. pannosa) which is correlated with the
findings of Yan et al. [47] and Jiménez‐Escrig et al. [48]. Lit-
erature revealed that methanolic extract showed best DPPH
radical scavenging capacities compared to the other extracts
from some red and brown seaweed [11, 37, 49, 50]. Though,
Hidayati et al. [51] found highest antioxidant activity in
aquadest extract of Sargassum sp. which is conflicted our
present finding. These difference may be because the anti-
oxidant activity are varied, largely depends on the seaweed
species, location and as well as seasons [52]. According to
the ­IC50 values showed in Table 4, the DPPH· scavenging
ability of S. coriifolium exhibited the following extracts
order: Methanol (1.03) > Ethanol (1.42) > Water (2.67) and
for H. pannosa extracts order were Methanol (1.58) > Eth-
anol (2.17) > Water (3.04). The ­IC50 values of all crude
extracts exhibited lower DPPH radical scavenging effects
when compared with the positive control (i.e., ascorbic acid,

13
 M. K. A. Sobuj et al.

(a) 1 mg/ml 3 mg/ml

90
1 mg/ml 3 mg/ml (b) 5 mg/ml 7 mg/ml
5 mg/ml 7 mg/ml 80 cc
80 cc cc cc
cc cc
70 cd
cc

% DPPH scavenging
b
70 b

% ABTS Scavenging
b b b
cd cc 60 b
60 b cc b cc
b 50
50 a a b a b b
a a
a 40 a a a
40 a a a
30 30

20 20
10 10
0 0
Sc- M Sc-E Sc-W Hy-M Hy-E Hy-W Sc-M Sc-E Sc-W Hy-M Hy-E Hy-W
Different Extracts Different Extracts

1 mg/ml 3 mg/ml (d) 1 mg/ml 3 mg/ml

(c) 5 mg/ml 7 mg/ml
4.5
cc
5 mg/ml 7 mg/ml
80
cc cc 4 cd cc
b cc
70
% H2O2 Scavenging

cc 3.5 b b
b cc b cd
60 cd
b 3 a a
b b b
b a

OD at 695
50 cc cc 2.5
a a
a a
40 b a b
a 2
a a
30 a 1.5
20 1
10 0.5
0 0
Sc-M Sc-E Sc-W Hy-M Hy-E Hy-W Sc-M Sc-E Sc-W Hy-M Hy-E Hy-W
Different Extracts Different Extracts

3.5 (e) 1 mg/ml 3 mg/ml

5 mg/ml 7 mg/ml
3 cc
cc
cc cd
2.5 b b
b
OD at 700

2 b
a cd cc
a a
1.5 b a b
a a
1

0.5

0
Sc-M Sc-E Sc-W Hy-M Hy-E Hy-W
Different Extracts

Fig. 2  a 1,1-diphenyl-2-picrylhydrazyl Assay, b 2,2-azino-bis (3-eth- Assay of different crude extracts of S. coriifolium and H. pannosa.
ylbenzothiazoline-6-sulfonic acid) Assay, c Hydrogen Peroxide Scav- Sc- Sargassum coriifolium, Hy- Hypnea pannosa, M- Methanol, E-
enging Assay, d Phosphomolybdenum Assay and e Reducing Power Ethanol, W- Water

Table 4  IC50 (mg/ml) values obtained from different crude extracts from different antioxidant assays

Antioxidant assay Name of the species IC50 (mg/ml) values of different crude extracts
Ascorbic acid Methanol Ethanol Water

DPPH assay S. coriifolium 0.00297 ± 0.0005a 1.03 ± 0.07b 1.42 ± 0.05c 2.67 ± 0.09d
H. pannosa 1.58 ± 0.11b 2.17 ± 0.04c 3.04 ± 0.08d
ABTS radical scavenging assay S. coriifolium 0.160 ± 0.006a 1.55 ± 0.04b 2.19 ± 0.03c 3.67 ± 0.06d
H. pannosa 1.86 ± 0.10b 2.28 ± 0.09c 3.98 ± 0.11d
H2O2 scavenging assay S. coriifolium 0.0783 ± 0.004a 1.98 ± 0.06b 2.54 ± 0.07c 4.81 ± 0.05d
H. pannosa 1.92 ± 0.10b 2.57 ± 0.11c 4.66 ± 0.08d

Different superscript letter illustrate significantly different from each other. The level of significance is P < 0.05

13
Evaluation of bioactive chemical composition, phenolic, and antioxidant profiling of different…
­IC50 = 0.00297 mg/ml). Here the extracts containing high
levels of TPC were also effectual DPPH radical scavenger,
signifying that algal polyphenols may be the foremost con-
stituents accountable for the superior percentage of inhibi-
tion properties of the extracts.
ABTS radical scavenging assay
ABTS scavenging involves the direct production of blue/
green ABTS chromophore through the reaction between
ABTS and K ­ 2S2O8. The result reveals that the antioxidant
activity of the crude extracts of seaweed is in the signifi-
cantly increasing trend with the increasing concentration
of the seaweed extract (P < 0.05). Among the methanolic
extracts S. coriifolium recorded significantly higher ABTS
free radical scavenging activity (72.24%, ­IC50 = 1.55 mg/
ml) followed by H. pannosa (71.14%, ­IC50 = 1.86 mg/ml)
(Fig. 2b). However, in case of ethanol extracts (5 mg/ml)
H. pannosa showed significantly higher ABTS free radical
scavenging activity (65.27%, I­ C50 = 2.28 mg/ml) compared
to S. coriifolium (67.62%, I­ C50 = 2.19 mg/ml). Similar obser-
vation of antioxidant activities of the methanolic extract was
observed in the previous literature for seaweed [53, 54]. This
is because methanol extracts may have H-donating property
which can terminate the oxidation process by converting
free radicals to the stable forms. Though, Suganya et al.
[55] found highest antioxidant activity in ethanol extract of
Sargassum wightii which is opposed to our present finding.
This variance might be because of the varietal extraction
method, species variation and for location. According to the
­IC50 values showed in Table 4, the ABTS radical scaveng-
ing ability of S. coriifolium exhibited the following extracts
order: Methanol (1.55) > Ethanol (2.19) > Water (3.67) and
for H. pannosa extracts order were Methanol (1.86) > Etha-
nol (2.28) > Water (3.98). This order is also similar with the
extracts of phenolic and flavonoid content. The I­ C50 value
of the ascorbic acid (­ IC50 = 0.16 mg/ml) is showed higher
ABTS radical scavenging ability when compared with all
crude extracts.
Hydrogen peroxide scavenging activity
Hydrogen peroxide is a fragile oxidizing mediator which
can inactivate some enzymes directly, typically by oxidation
of essential thiol (-SH) groups. The ability of antioxidant
activities of different crude extracts of S. coriifolium and H.
pannosa were evaluated by the hydrogen peroxide scaveng-
ing activity according to the method of Latos-Brozio and
Masek [31]. All the raw seaweeds extracts were capable of
scavenging hydrogen peroxide in a concentration depend-
ent manner. Methanolic extracts showed significantly higher
(P < 0.05) scavenging activity of S. coriifolium (71.64%) and
H. pannosa (69.08%) followed by ethanol extracts and water
extracts (Fig. 2c). The antioxidant activity of different crude
extracts showed an increasing trend with increasing concen-
tration indicating the dose dependency of these properties.
According to the ­IC50 values showed in Table 4, the ­H2O2
scavenging ability of S. coriifolium exhibited the following
extracts order: Methanol (1.98) > Ethanol (2.54) > Water
(4.81) and for H. pannosa extracts order were Methanol
(1.92) > Ethanol (2.57) > Water (4.66) which is similar to
the DPPH and ABTS scavenging activity. The I­ C50 value of
the positive control (i.e., ascorbic acid, ­IC50 = 0.0783 mg/ml)
is showed higher H ­ 2O2 scavenging ability when compared
with all other crude extracts. Corpuz et al. [56] observed
­IC50 value of 2.27 mg/mL for Sargassum siliquosum which
is similar to our present finding but in different solvent
(dichloromethane). Also, Shireesha et al. [57] found 77.90%
hydrogen peroxide scavenging activity of methonolic extract
of Hypnea musciformis with ­IC50 value 417.58 µg/ml.
Phosphomolybdenum assay
The phosphomolybdenum method has been routinely used
to evaluate the antioxidant potential of extracts. In the pres-
ence of extracts, Mo (VI) is reduced to Mo (V) and forms
a green colored phosphomolybdenum V complex. Here S.
coriifolium showed significantly higher (P < 0.05) absorb-
ance ­(A695nm 3.959) compared to the absorbance of H. pan-
nosa ­(A695nm 3.716) (Fig. 2d). But in case of water extract
H. pannosa ­(A695nm 3.037) showed better absorbance than
S. coriifolium ­(A695nm 2.947). Antioxidant activity of differ-
ent crude extracts showed increasing trend with increasing
concentration and slight variation showed at 5 and 7 mg/
ml concentration. The antioxidant activity of different crude
extracts was determined using ascorbic acid as a reference
compound. The highest antioxidant activity was observed for
S. coriifolium was 28.08 mg of ASE/g in the crude metha-
nolic extract and for H. pannosa the data was 17.31 mg of
ASE/g. Previous research [11, 37, 45] have also mentioned
that crude extracts of brown and red seaweed can reduce
the phosphomolybdenum and the data were 8.2, 32.01 and
39.62 to 9.65 mg of ASE/g respectively, which is related to
our present observation.
Reducing power assay
The reducing power assay has been used to evaluate the
antioxidant activity of different crude extracts. Here S.
coriifolium showed significantly higher (P < 0.05) absorb-
ance ­(A700nm 0.76–2.85) compared to the absorbance of H.
pannosa ­(A700nm 0.85–2.69) (Fig. 2e). The reduction ability
of different crude extracts from the seaweed species was
determined using ascorbic acid as a reference compound.
Among the tested sample, the highest reducing power was
observed in the crude methanolic extract of S. coriifolium
13
 M. K. A. Sobuj et al.

and H. pannosa and the data were 938.52 and 861.38 mg of
ASE/g, respectively. Other researcher examined the reducing
power of ethyl acetate fraction from marine algae Polysipho-
nia urceolata and Rhodomela confervoides and the results
were 383 and 426 mg of ASE/g respectively [58, 59]. The
result might be different because of the differences of the
analytical method, variety among solvents and radicals or
reactive oxygen species that become target of the sample.
Correlations between total phenolic contents
and different antioxidant activity assays
A significant positive linear correlation between total
phenolic contents (TPC) and different radical scavenging
assays of seaweed extracts were recognized by Pearson
correlation analysis method [TPC-DPPH: R ­ 2 = 0.8144
(Fig. 3b), TPC-ABTS: ­R 2 = 0.637 (Fig. 3c), TFC-H 2O 2
scavenging activity: R ­ 2 = 0.6 (Fig. 3d), TPC-Phospho-
2
molybdenum: ­R = 0.714 (Fig. 3e), TPC-reducing ability:
­R2 = 0.529 (Fig. 3f)]. All of the antioxidant activities of
seaweed extracts were found to be positively correlated
with each other which clearly indicate that phenolic com-
pounds are mainly responsible for the antioxidant proper-
ties. Other scholars also documented a similar positive
correlation between TPC and antioxidant activities of sea-
weed extracts [11, 59, 60]. Also, negative correlation [55]
was found between TPC and antioxidant activity (lipid
peroxidation inhibition activity) which assay was not per-
formed in our present study. Our present finding clearly
recommended that the antioxidant activity registered by
crude extracts mainly methanolic may primarily be due to
the existence of polar phenolic compounds.

Fig. 3  Scatter plot diagrams 65 (a) 80

(b)
showing the correlation of total 60 Sc-M
phenolic content (mg of GA/g) Sc-M 75

% DPPH Scavenging
Total Flavonoid (mg

55
vis-à-vis a Total Flavonoid 70
Hy-M
50 Sc-E
quercetin/g)

Content (n = 6; ­R2 = 0.914),

45
b DPPH (n = 6; ­R2 = 0.8144), Hy-M Sc-E 65 Hy-E
c ABTS (n = 6, ­R2 = 0.637), 40
d ­H2O2 scavenging activity 35 Hy-E 60 Sc-W
Hy-W
(n = 6, ­R2 = 0.6), e Phosphomo- 30 Hy-W
Sc-W 55
lybdenum (n = 6, ­R2 = 0.714) 25
and f Reducing ability (n = 6, 20 50
­R2 = 0.529). Sc- Sargassum 45 65 85 105 125 145 45 65 85 105 125 145
coriifolium, Hy- Hypnea pan- Total Phenolic Content (mg of GA/g) Total Phenolic Content (mg of GA/g)
nosa, M- Methanol, E- Ethanol,
W- Water 80 (c) 77.5 (d)
75 Sc-M
72.5
Hy-M Sc-M
% ABTS Scavenging

Hy-M
% H2O2 Scavenging

70 67.5
Hy-E
65 Sc-E 62.5
60 57.5 Hy-E Sc-E

55 52.5
50 Sc-W 47.5 Hy-W
Hy-W Sc-W
45 42.5
40 37.5
40 90 140 45 95 145
Total Phenolic Content (mg of GA/g) Total Phenolic Content (mg of GA/g)

4.2 (e)
3.1 (f)
2.9 Hy-M
4 Sc-M
2.7 Sc-M
3.8 2.5 Hy-E
Hy-M
OD at 700

Sc-E
OD at 695

3.6 Sc-E 2.3

Hy-E
2.1
3.4
1.9
3.2
Hy-W 1.7 Sc-W
Sc-W Hy-W
3 1.5
2.8 1.3
45 65 85 105 125 145 45 65 85 105 125 145
Total Phenolic Content (mg of GA/g) Total Phenolic Content (mg of GA/g)

13
Evaluation of bioactive chemical composition, phenolic, and antioxidant profiling of different…
Correlations between total phenolic contents
and total flavonoids contents
A strong and positive linear correlation between total phe-
nolic contents (TPC) and total flavonoids contents (TFC) of
seaweed extracts were documented by Pearson correlation
analysis method [TPC-TFC: R ­ 2 = 0.914 (Fig. 3a)]. Our cur-
rent research findings were in agreement with the results of
Johnson et al. [51] and Farasat et al. [61] who also reported
a similar positive correlation between total phenolics and
flavonoids contents of different seaweed extracts. Also,
similar positive correlation has been reported by Chai and
Wong [62] in case of Turnera subulata plant. Considering
all these, the outcome of correlation analyses indicates that
high contents of total phenolics and flavonoids are important
determining factor for antioxidant activity in different crude
extracts.
Conclusion
From the results described here, it is evident that choice of
extraction solvent and type of seaweed significantly influ-
ence on the availability of functionally bioactive compound
as confirmed by in-vitro screening and FT-IR analysis. It has
also been found that there is significant correlation between
phenolic content of extracts and its antioxidant activity as
detected by different antioxidant assays including DPPH,
ABTS, hydrogen peroxide, phosphomolybdenum and reduc-
ing power assays. Overall, it can be said that both the sea-
weed could be used as potent source of natural antioxidant as
food supplement or functional feed. Further studies should
be performed on the identification, isolation and charac-
terization of active principles responsible for bioactivity by
using varietal solvents.
Funding This research was funded by Grants for Advanced Research
and Education, Ministry of Education, Bangladesh.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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