Food Chemistry Advances 4 (2024) 100565

Contents lists available at ScienceDirect

Food Chemistry Advances

journal homepage: www.elsevier.com/locate/focha

Qualitative and quantitative phytochemical analysis of brown seaweed

Sargassum polycystum collected from Bangladesh with its antioxidant
activity determination
Mohammad Khairul Alam Sobuj a, Morshed Shamim Shemul b, Md. Shoebul Islam c,
Md. Ariful Islam b, Shayla Sultana Mely d, Mehedi Hasan Ayon b, Shahittya Mitra Pranto b,
Mohammad Shafiqul Alam e, Mohammad Sarfuddin Bhuiyan f, S.M. Rafiquzzaman b, *
a
Marine Fisheries and Technology Station, Bangladesh Fisheries Research Institute, Cox’s Bazar 4700, Bangladesh
b
Department of Fisheries Biology and Aquatic Environment, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh
c
Shrimp Research Station, Bangladesh Fisheries Research Institute, Bagerhat 9300, Bangladesh
d
Bangladesh Fisheries Research Institute, Mymensingh 2201, Bangladesh
e
Department of Genetics and Fish Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh
f
On Farm Research Division, Bangladesh Agricultural Research Institute, Cox’s Bazar 4700, Bangladesh

A R T I C L E I N F O A B S T R A C T

Keywords: Seaweeds possess considerable potential for developing and enhancing pharmaceutical drugs because of their
Brown seaweed wide variety as abundant sources of numerous bioactive secondary metabolites, making them well-suited for
Sargassum polycystum developing and enhancing pharmaceutical drugs. This study evaluated the antioxidant, secondary metabolites,
Secondary metabolites
total phenolic content, and total flavonoid content of various crude extracts (methanol, ethanol) and their
FT-IR
fractions (50 % and 70 %) of Sargassum polycystum. Fourier transform infrared (FT-IR) analysis was also per­
Total phenolic content
Total flavonoid content formed to identify the active chemical composition of the crude extracts. The results showed that various extracts
contained varying concentrations of terpenoid, saponin, phlobatannin, cardiac glycosides, phenolic, and flavo­
noid. FT-IR analysis confirmed the presence of phenols, carboxylic acid, ketones, ethers, aromatics, amides, and
sulfonates. 100 % methanolic extract contained the maximum concentrations of total phenols (80.13 mg of GAE/
g) and total flavonoids (38.33 mg of quercetin/g). 100 % methanol extracts exhibited higher antioxidant activity
in the DPPH assay (78.32 %, IC50 = 1.59 mg/ml) and ABTS assay (69.82 %, IC50 = 2.39 mg/ml). The bio-
functional activity of crude extracts depended on their extract and concentration. The present study demon­
strates that the macroalgae S. polycystum contains noteworthy secondary metabolites and natural antioxidants,
indicating their potential utilization in various pharmacological and functional food applications.

1. Introduction Chapman (1980) and Okazaki (1971). Seaweeds are known to synthe­
size numerous chemical compounds, several of which serve as exclusive
Macroscopic marine algae, commonly referred to as seaweeds, sources for the production of agar, carrageenan, and alginate. The uti­
constitute a significant component of the ocean’s living resources. Sea­ lization of these substances has been documented in multiple applica­
weeds, also known as macroalgae, include a substantial and renewable tions, including human consumption, animal nutrition, plant
resource within the marine ecosystem, and their utilization has been fertilization, and the extraction of diverse chemicals with pharmaceu­
intertwined with human civilization since time immemorial. Previous tical properties, all without any observed adverse effects. Additionally,
studies have documented the historical utilization of seaweeds dating these substances have been employed in the production of biodiesel and
back to approximately 2500 years ago, as evidenced by the works of the management of wastewater (Nedumaran & Arulbalachandran,

* Corresponding author.
E-mail addresses: [email protected] (M.K.A. Sobuj), [email protected] (M.S. Shemul), [email protected] (Md.S. Islam),
[email protected] (Md.A. Islam), [email protected] (S.S. Mely), [email protected] (M.H. Ayon), [email protected]
(S.M. Pranto), [email protected] (M.S. Alam), [email protected] (M.S. Bhuiyan), [email protected] (S.M. Rafiquzzaman).

https://doi.org/10.1016/j.focha.2023.100565
Received 15 September 2023; Received in revised form 30 November 2023; Accepted 7 December 2023
Available online 14 December 2023
2772-753X/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M.K.A. Sobuj et al. Food Chemistry Advances 4 (2024) 100565

2015). The utilization of seaweeds has been extensively examined, antioxidant and phytochemical activity, total phenolic content, and total
demonstrating their significant contribution to sustainable development flavonoid content of various extracts and their fractions obtained from
and their potential as a resource for human well-being. crude extracts of S. polycystum, a plant species indigenous to Bangladesh.
Secondary metabolites, or phytochemicals, encompass a diverse
group of organic substances found in a wide range of species, including 2. Materials and methods
bacteria, fungi, plants, and algae. These substances do not directly affect
the physiological processes of growth, development, or reproduction in 2.1. Sample collection and preparation
these species. In contrast, these entities frequently assume significant
functions in safeguarding the organism against exogenous hazards, The samples were collected in September 2021 at Saint Martin’s Is­
including parasites, insects, and herbivorous organisms. Humans use land (92◦ 19ʹ21.28ʺE and 20◦ 37ʹ38.12ʺN) in the Bay of Bengal,
these phytochemicals as medicine, flavoring components, and recrea­ Bangladesh, specifically during the mature stage. The seaweed speci­
tional drugs. They also play an important role in mitigating oxidative mens were obtained from the exposed surfaces of rocks on St. Martin’s
stress in humans and other animals by binding with free radicals such as Island during the period of the lowest low tide. Several species that had
reactive oxygen and nitrogen species. The assessment of antimicrobial drifted or floated were also gathered from the coast. A sharp knife was
activities was regarded as a reliable measure of the seaweeds’ ability to used to gather the specimens to enhance precision. The samples
produce bioactive secondary metabolites. A plethora of literature exists collected were washed using clean salt water to eliminate debris and
documenting the discovery and characterization of various compounds other extraneous substances and transported in a temperature-
obtained from macroalgae, which exhibit a diverse array of biological controlled container at 4 ◦ C to the laboratory. The specimens under­
activities. These activities include antibacterial properties, as Berland went a rinsing process using distilled water to eliminate any presence of
et al. (1972) reported. Additionally, certain compounds derived from salt, sand, debris, or other forms of contamination. Subsequently, the
macroalgae have demonstrated antitumor effects, as evidenced by the seaweed underwent a freeze-drying process using a VaCo 2 freeze dryer
studies conducted by Espeche et al. (1984) and Yamamoto and Gaynor from Zirbus, UK, for two days to eliminate moisture. The dried seaweed
(2001). Moreover, these compounds have demonstrated anticoagulant was ground into a powder using a grinder machine. Subsequently, the
and anti-fouling characteristics. Several antimicrobial agents derived fine powder was separated using a sieve. The fine powder was made
from algae have been found, including chlorellin derivatives, acrylic using a mortar and pestle as well. The powder was stored in a refriger­
acid, halogenated aliphatic compounds, terpenes, sulphur-containing ator at a temperature of 4 ◦ C to facilitate further analysis.
heterocyclic compounds, and phenolic inhibitors (Espeche et al., 1984).
Flavonoids are the main phenolic compounds that are widely 2.2. Preparation of stock solution
distributed in fruits and vegetables in nature and possess a wide range of
biofunctional properties (Velioglu et al., 1998). Dietary flavonoids and A total of four grams of powdered S. polycystum was combined with
flavonoid-rich food products have unique roles in health promotion 100 ml of a solvent mixture consisting of methanol and ethanol. Solvents
through gut microbiota regulation (Li et al., 2023). The research were chosen based on the polarity index, as the polarity of any solvent
revealed that the interaction between dietary flavonoids and gut bac­ plays a significant role in the extraction of novel compounds from food
teria might collectively influence the generation of catabolites and me­ items (Naczk & Shahidi, 2006). The mixture was subjected to macera­
tabolites via many biological pathways (Chen & Yang, 2020). The tion using 70 % methanol, ethanol, and a 50 % mixture of methanol and
intestinal environment and gut microbiota are regulated by the pro­ ethanol to get an extract using solvent extraction. The specimen was
duction of various metabolites resulting from the fermentation of several subjected to a 24 h darkness period while being intermittently agitated
dietary flavonoids and other substances within the gastrointestinal tract. in an incubator shaker (CS-05C, Classic Scientific) to enhance the
In recent years’ metabolomics analyses have been widely applied in food extraction process. Following the incubation period, the solution un­
processing, food safety, food authenticity and quality, and food trace­ derwent initial filtration using cotton, followed by further filtration
ability (Li et al., 2021). Metabolomics methodology has a high capacity using Whatman filter paper No 4 (with a pore size of 20–25 µm) under
to evaluate chemical and organoleptic transformations that occur during aseptic conditions. The methanol and ethanol extracts were subse­
food production. Adding seaweed to any ordinary food items will result quently concentrated using a Rotary vacuum evaporator (LRE-702A,
in nutritionally balanced novel food items by enhancing the metabolic Labocon, UK) at a temperature of 36 ◦ C. The concentrated sample un­
profile after seaweed digestion (Kai et al., 2023). derwent additional drying by introducing nitrogen gas into the solution
Bangladesh now occupies about 1,18,813 sq. km. of marine area, using a Nitrogen Concentrator/Sample Evaporator (AT-EV-50, Athena
where up to 12 nautical miles from the shore is the territorial sea and Technology, India) at a temperature of 36 ◦ C, resulting in the complete
200 nautical miles from shore is an exclusive economic zone, which evaporation of the solvent. The dried crude extracts obtained from
opened up a huge opportunity to explore this untouched area for various solvents and their respective fractions were collected and sub­
different aquatic resources (Islam et al., 2022; Sobuj et al., 2023). Sea­ sequently produced as stock solutions and working solutions with a
weeds are one of the important resources of the Bay of Bengal, which are certain desired concentration. Lastly, these solutions were conserved for
almost completely unexplored. Sarkar et al. (2016) reported that the subsequent study.
Bangladeshi coast is home to around 193 kinds of seaweeds.
S. polycystum is a highly prevalent brown seaweed species commonly 2.3. Preliminary qualitative phytochemical screening
observed along the coastlines of Saint Martin’s Island, located in
Bangladesh. Sargassum, a genus of marine macroalgae classified under Qualitative phytochemical analyses were performed on stock solu­
Phaeophyceae, exhibits a global distribution across tropical and tions of various solvents and their fractions to determine the presence of
temperate oceans, encompassing over 400 species. S. polycystum is a distinct classes of active ingredients in S. polycystum. The procedures
significant species within the genus Sargassum, and a diverse array of employed for these analyses were reported in previous studies con­
biochemical constituents have been documented concerning this ducted by Sobuj et al. (2021a, 2021b). The investigations revealed the
particular species (Haque et al., 2009). Several studies have been con­ presence or absence of the chemicals in the examined crude extracts,
ducted on this particular species in various regions across the globe; eliciting general reactions.
however, none of these studies have specifically focused on investigating
the phytochemical contents of this species. This particular research 2.3.1. Test for terpenoids (Salkowski test)
serves as an initial stage in confirming the significance of a seaweed A volume of 5 ml of each extract was combined with 2 ml of chlo­
species as a valuable commercial resource. This study aims to assess the roform and 3 ml of concentrated H2SO4. The presence of terpenoids in

2
M.K.A. Sobuj et al. Food Chemistry Advances 4 (2024) 100565

the sample extracts is indicated by the production of a reddish-brown tube, followed by adding 0.2 mL of a solution containing 10 %
color at the interface (Harborne et al., 1998). aluminum chloride. Subsequently, a volume of 0.2 ml of a 1 M potassium
acetate solution was introduced into the test tube. After that, a volume of
2.3.2. Test for saponin (frothing test) 5.6 ml of distilled water was added to the test tube. Then, the test tube
About 3 ml of each extract was treated with 3 ml of distilled water, was incubated at ambient temperature for 30 min. The absorbance was
and the sample was shaken vigorously for a stable, persistent froth for measured at a wavelength of 420 nm with a spectrophotometer (T80+
about 1 min. The frothing was added with three drops of olive oil and UV/VIS Spectrophotometer, UK). Blank for each solvent was run using
shaken vigorously, then observed for emulsion formation, indicating the the same procedure but exchanging the plant extract with an equal
presence of saponins in the extracts (Trease & Pharmacognsy, 1989). volume of solvent. The concentration of total flavonoids was calculated
as mg of quercetin equivalent according to the calibration curve. The
2.3.3. Test for phlobatannins calibration equation for Quercetin was Y = 0.0072X ̶ 0.016 (R2 =
A volume of 3 ml of extract was combined with a small quantity of 1 0.9969).
% hydrochloric acid and then monitored for precipitation. The crude
extract was found to include phlobatannin, as indicated by the positive 2.5. Evaluation of total antioxidant capacity
results observed by the formation of a red precipitate (Rafiquzzaman
et al., 2016). Total antioxidant activities of different crude extracts and their
fractions of S. polycystum were observed using the following methods.
2.3.4. Test for cardiac glycosides (Keller-Killani test)
A total volume of 5 ml for each extract was combined with 2 ml of 2.5.1. DPPH (1, 1 diphenyl 2- picryl hydrazyl) assay
glacial acetic acid solution, which included the addition of one drop of The radical scavenging activity in various extracts was assessed using
ferric chloride solution. Additionally, 1 ml of concentrated sulfuric acid the DPPH assay, as described by Sobuj et al. (2021a), with slight ad­
(H2SO4) was introduced into the solution. The observation of a brown justments. The extract solution was prepared at 1, 3, 5, and 7 mg/ml
ring formed at the interface indicates the existence of deoxy sugar, a concentrations. The extract solution was thoroughly mixed with 0.15
distinctive feature of cardenolides. According to Edeoga et al. (2005), it mM DPPH solution (prepared in ethanol) with a 1:1 ratio and the
is possible for a violet ring to manifest below the brown ring in the resulting reaction mixture was incubated at ambient temperature for 30
H2SO4 layer. Additionally, in the acetic acid layer, a greenish ring may min. The absorbance of the mixture was measured at a wavelength of
develop just above the brown ring, which will progressively disperse 517 nm using a spectrophotometer (T80+ UV/VIS Spectrophotometer,
throughout the entirety of this layer. UK) after 30 min. The IC50 values were determined for each sample,
corresponding to the antioxidant concentration necessary to achieve a
2.3.5. Fourier-Transformed infrared (FT-IR) spectroscopy 50 % reduction in the initial DPPH ion concentration. Ascorbic acid was
Active secondary metabolite chemicals in S. polycystum were inves­ used as a positive control, and the values were compared with the crude
tigated using FT-IR spectroscopy (Perkin Elmer Spectrum 2) on dried extracts. The percentage of radical scavenging activity of the crude ex­
crude extracts. The FT-IR analysis technique employs infrared radiation tracts was determined using the following formula:
to examine the test samples and ascertain their chemical characteristics.
The sample’s active constituents exhibit light absorption within the DPPH radical scavenging activity (%) = [(A0 − A1 ) / (A0 )] × 100
infrared range of the electromagnetic spectrum, which is precisely
Where, A0 is the absorbance of control, which is the absorbance of DPPH
associated with the bonds found in the sample.
radicals + methanol, and A1 is the absorbance of the sample, which is
the absorbance of DPPH radicals + sample extract/standard.
2.4. Quantitative analysis of phytochemicals
2.5.2. ABTS radical scavenging assay
2.4.1. Total phenolic content (TPC)
The antioxidant activities of different extracts were evaluated by the
The quantification of total phenols in the crude extracts was con­
ABTS radical scavenging activity according to the method of Rafi­
ducted by employing Folin-Ciocalteu Phenol reagents and external
quzzaman et al. (2016) with minor modifications. Briefly, 7 mM ABTS
calibration with Gallic acid, as described by Sobuj et al. (2021b), with
solution and 2.45 mM Potassium persulfate were added in a 1:1 ratio
minor adjustments. In summary, 0.5 ml of an extract solution with a 5
into water to prepare a working solution. Extracts (50 μl) were added at
mg/ml concentration was transferred into a test tube. Subsequently, a
various concentrations (1, 3, 5 and 7 mg/ml) in different test tubes with
volume of 0.1 ml of the Folin–Ciocalteu working reagent solution was
ABTS solution (950 μl), followed by incubation at RT for 16 h in the
introduced into the test tube, followed by thoroughly mixing the con­
dark. The absorbance of each sample was measured at 734 nm using a
tents. The solution was kept undisturbed for 15 min at ambient tem­
spectrophotometer (T80+ UV/VIS Spectrophotometer, UK). Ascorbic
perature. Following a time interval of 15 min, a volume of 2.5 ml of a
acid was used as a positive control. The IC50 values were determined for
saturated solution of sodium carbonate (with a concentration of 75 g/L)
each sample, representing the concentration of antioxidants required to
was introduced into the test tube. Subsequently, the combination was
reduce the initial ABTS ion by 50 %. The percentage of inhibition of the
permitted to remain undisturbed for 30 min at ambient temperature.
crude extracts was calculated using the following formula:
The absorbance measurement was taken at a wavelength of 760 nm
(T80+ UV/VIS Spectrophotometer, UK). The quantification of total ABTS scavenged (%) = [(A0 − A1 ) / (A0 )] × 100
phenolics was determined by converting the measured values to milli­
grams of Gallic acid equivalent using the Gallic acid calibration curve. where, A0 is the absorbance of control, which is the absorbance of ABTS
The calibration equation used for Gallic acid was Y = 0.0121X + 0.0404 radicals + solvent, and A1 is the absorbance of the sample, which is the
(R2 = 0.9861). absorbance of ABTS radicals + sample extract.

2.4.2. Total flavonoid content (TFC) 2.6. Statistical analysis

The total flavonoid levels of S. polycystum were measured using the
aluminum chloride colorimetric method, as outlined by Chang et al. The obtained experimental data were analyzed using the conven­
(2002), with minor adjustments. In summary, a 1 ml aliquot of an tional statistical method. Data were analyzed using SPSS software (IBM
extract solution with a 5 mg/ml concentration was transferred into a test Co., Chicago, IL). Solvents and samples were compared using analysis of
tube. A volume of 3 ml of methanol was then introduced into the test variance (ANOVA) and Duncan’s multiple range method. Values were

3
M.K.A. Sobuj et al. Food Chemistry Advances 4 (2024) 100565

expressed as means ± standard deviations. Differences were considered have verified. 70 % methanol extracts of two species of Gracilaria genus
significant at p < 0.05. Each analysis was conducted in triplicate. (G. corticata and G. edulis) were subjected to several phytochemicals
tests by Arulkumar et al. (2018). 70 % ethanol extracts of two red sea­
3. Results and discussion weeds Pterocladiella capillacea and Osmundaria obtusiloba were subjected
to several phytochemical tests by De Alencar et al. (2016) which con­
3.1. Qualitative phytochemical screening forms with the present finding.

The metabolic and physiological capacities of marine creatures 3.2. FT-IR analysis
enable them to thrive in diverse habitats, hence offering significant
prospects for the synthesis of secondary metabolites that are absent in According to Versari et al. (2010), utilizing FT-IR technology enables
terrestrial ecosystems. Marine algae and their organic extracts have been efficient and consistent analysis of food samples, offering
identified as highly abundant sources of known and novel bioactive non-destructive and speedy results. This method requires minimal
chemicals (Blunt et al., 2006). The levels and accessibility of phyto­ sample preparation and relies on identifying peak values within the
chemicals in marine algae exhibit significant variations based on factors infrared radiation region. The functional group of the active components
such as climatic conditions, seasonal changes, age, geographical loca­ was determined using the FT-IR spectrum, which involved analyzing the
tion, and depth of immersion (Marinho-Soriano et al., 2006). The peak value in the infrared radiation area. The utilization of this method
presence or absence of phytoconstituents in seaweeds is contingent upon is predominantly observed in examining polysaccharides in seaweeds,
the solvent medium employed for extraction and the physiological with limited literature available on the analysis of polyphenols (Lim
characteristics of the seaweeds. Ganesan et al. (2008) demonstrated that et al., 2002; Duan et al., 2006). S. polycystum crude extracts (ethanol and
identifying crucial bioactive chemicals in seaweed can be achieved by methanol) and fractions were analyzed using FT-IR, and the functional
utilizing several solvents and conditions. This study aimed to analyze the groups of the components were separated based on their peak ratios. The
presence of six phytochemicals, namely terpenoid, saponin, phloba­ results of the FT-IR analysis confirmed the presence of phenols, car­
tannin, cardiac glycosides, phenolics, and flavonoids, in two crude ex­ boxylic acids, aromatics, ketones, ethers, and amide compounds, which
tracts (methanol and ethanol) with two distinct fractions (50 % and 70 are presented in Fig. 1 and Table 2.
%). The presence of active secondary metabolites (phytochemicals) was The crude extracts of S. polycystum showed different peaks, con­
noted in each extract, albeit in varied quantities. Out of 36 tests con­ firming the presence of various functional groups in the range between
ducted, 28 yielded positive results, while the remaining eight produced 500 and 4000 cm− 1. FT-IR analysis confirmed the presence of phenols,
negative results. Among the many extracts analyzed, it was observed carboxylic acids, ketones, ethers, aromatics, and amides in different
that methanol and its fractions (70 % and 50 %) exhibited the highest extracts (Table 2). The peaks arise at 3295.6, 3306.0, 3326.6, 3330.0,
number of compounds (15), including terpenoids, saponins, phloba­ 3364.6, and 3407.5 cm− 1 regions because of the O –H stretch of phenols
tannins, cardiac glycosides, phenolics, and flavonoids. Following and alcohols with H-bonds. The existence of carboxylic acids can be
methanol, ethanol and its fractions demonstrated the presence of 13 inferred from the observation of bands within the spectral range of 2700
compounds. The phytochemical properties of the extracts from the six – 3300 cm− 1. Bands observed between this range correspond to the O –
components under investigation are presented in Table 1. H stretching vibrations of carboxylic acids. The C = O stretching vi­
Phlobatannin is present in 100 % and 70 % methanolic extract and bration of ketones is generally observed in the double bond region (1500
absent in other fractions. Phlobatannin is a tannin derivative, and tannin – 2000 cm− 1). The peaks observed at 1802.7, 1854.8, 1855.2, 1889.7,
is generally found in higher plants. This indicates that this compound 1924.8, and 1978.6 cm− 1 correspond to the C = O stretching vibrations
can only be derived from the methanol extract of S. polycystum. The of ketone compounds. Furthermore, the C = O stretching vibration of
presence of phlobatannin also indicates the diuretic property of this amide is generally observed within the spectral range of 1630 – 1680
seaweed. Terpoind shows positive results in all the fractions; only in the cm− 1. The peaks observed at 1634.9, 1635.0, 1635.3, 1640.4, 1652.2,
case of 50 % ethanol, it shows negative results. Cardiac glycoside is and 1652.3 cm− 1 correspond to the C = O stretching vibrations of amide
present in all the extracts. Saponin is present in pure ethanol, 70 % compounds. The C –O stretch of the ether functional group was seen
ethanol, and pure methanol extract and absent in the other extracts. throughout the spectral range of 1000 to 1300 cm− 1 in various crude
Phenolics and flavonoid compounds showed positive results in all of the seaweed extracts. The peaks observed at 1015.4, 1015.9, 1025.3,
extracts. Methanol and its fractions show a maximum number of com­ 1056.7, 1076.9, and 1088.6 cm− 1 correspond to the C –O stretching
pounds than ethanol. vibrations of ether compounds. The C –H bend of the aromatic group
Our results suggest that methanol is a better solvent for consistently was seen throughout the spectral range of 1660 to 2000 cm− 1 in various
extracting bioactive compounds from brown seaweeds. No previous crude seaweed extracts. The sulfonates exhibited a characteristic S = O
study on the preliminary phytochemical screening of extracts from stretching vibration in the spectral range of 1300 to 1365 cm− 1, while
S. polycystum was found in the literature. However, Sobuj et al. (2021b) the stretching vibrations of NO2 and SO2 were observed in the spectral
identify several phytochemicals from brown algae S. coriifolium range of 1100 to 1200 cm− 1. The peak arises at 1363.2, 1363.1, and
collected from Bangladesh, which is correlated with the present finding. 1363.2 cm− 1 in 70 % ethanol, 50 % ethanol, and 70 % methanol ex­
Jeeva et al. (2012) and Mansuya et al. (2010) also identified several tracts, respectively, because of the presence of S = O stretching vibra­
phytoconstituents from brown algae S. wightii with other classes of tions of sulfonates.
seaweed from the Gulf of Mannar region, which the present findings Sobuj et al. (2021a) and Sobuj et al. (2021b) used FT-IR to identify

Table 1
Preliminary qualitative phytochemical analysis of solvents and their fractions of different crude extracts of S. polycystum.
Solvents Terpenoid Saponin Phlobatannin Cardiac Glycoside Phenols Flavonoids

100% Ethanol + + − + + +
70% Ethanol + + − + + +
50% Ethanol − − − + + +
100% Methanol + + + + + +
70% Methanol + − + + + +
50% Methanol + − − + + +

“+” = presence of constituent; “− ” = absence of constituent.

4
M.K.A. Sobuj et al. Food Chemistry Advances 4 (2024) 100565

Fig. 1. FT-IR spectrum of (A) Ethanol and (B) Methanol solvents and their fractions of S. polycystum.

5
M.K.A. Sobuj et al. Food Chemistry Advances 4 (2024) 100565

Table 2
Summary of the functional groups of active components of solvents and their fractions based on the peak value of Fourier transform infrared spectroscopy.
Extracts Major functional groups
Phenol Carboxylic Ketones Ethers Aromatics Amides Sulfonates
acids

100% Ethanol + + + + + + −
70% Ethanol + + + + + + +
50% Ethanol + + + + + + +
100% Methanol + + + + + + −
70% Methanol + + + + + + +
50% Methanol + + + + + + −

“+” = presence of constituent; “− ” = absence of constituent.

several phytochemicals from brown and red algae, which is consistent respectively. These values were lower than the present study’s findings,
with the present findings. 70 % methanol extracts of two species of which also concluded that methanolic extracts exhibited superior
Gracilaria genus (G. corticata and G. edulis) were subjected to FT-IR properties to other extraction methods. According to Chandini et al.
analysis by Arulkumar et al. (2018) and identified several functional (2008), the phenolic content of brown seaweed extracts was determined
groups. Rafiquzzaman et al. (2016) reported several phytochemicals to be 24.61 mg of GAE/g of seaweed extract. This value is comparatively
from red algae H. musciformis, which are interrelated with the present lower than the current study’s findings. These factors could be attributed
finding. Marimuthu et al. (2012) also identified several functional to the solubility and provenance of the seaweed.
groups such as amides, alcohols, phenols, and phosphorous compounds
in crude powder of S. wightii using FT-IR analysis, which is almost similar 3.3.2. Total flavonoid content (TFC)
to the present findings. However, they found alcohols and halogen The total flavonoid content in the crude extracts of S. polycystum was
compounds in different extracts, which might be due to the variation of quantified by the aluminum chloride method using quercetin as a
solvent used for extraction and the origin of the seaweed. standard for external calibration. The study revealed significant varia­
tions in total flavonoid content (TFC) among different solvent extracts.
Within the many ethanol extracts, it ranges between 32.63 ± 0.7 to
3.3. Quantitative analysis of phytochemicals 27.63 ± 0.3 mg of quercetin/g, where the highest value was observed in
the case of 100 % ethanol (p < 0.05) (Table 3). In the case of methanol,
3.3.1. Total phenolic content (TPC) the value ranges between 38.33 ± 0.3 to 29.41 ± 0.8 mg of quercetin/g,
Phenolics are a class of chemical compounds characterized by a with the significantly highest value observed in 100 % methanol (p <
hydroxyl group directly bonded to an aromatic hydrocarbon moiety. The 0.05) (Table 3). The sample that carries more water has shown less
phenolic chemicals inside are the primary contributors to the antioxi­ value. 50 % ethanol and 50 % methanol values were lower (27.63 ± 0.4
dant activity of various seaweeds. The total phenols in the crude extracts mg of quercetin/g and 29.41 ± 0.8 mg of quercetin/g) than 70 %
of S. polycystum were quantified by utilizing Folin-Ciocalteu Phenol re­ methanol and 70 % ethanol. This finding is supported by the concur­
agents and external calibration involving Gallic acid. In the case of rence of Rajauria et al. (2013) and Dellai et al. (2013). In their study,
ethanol, the range of total phenolic content varies from 76.49 ± 0.9 to Rajauria et al. (2010) employed methanol extracts with 100 % and 60 %
50.13 ± 0.2 mg of GA/g (Table 3), and the significant highest was found concentrations in H. elongate. The flavonoid content in their extracts was
in 100 % ethanol, which is 76.49 ± 0.9 mg of GA/g (p < 0.05). In the measured to be 109.8 ± 2.68 mg QE/g and 44.0 ± 1.47 mg QE/g,
case of methanol, the range varies from 80.13 ± 0.5 to 49.07 ± 0.2 mg respectively. In their study, Dellai et al. (2013) employed a solution
of GA/g (Table 3), and the significantly highest was found in 100 % consisting of 80 % ethanol and reported a total flavonoid content (TFC)
methanol, which is 80.13 ± 0.5 mg of GAE/g (p < 0.05). The findings value of 26.80 ± 4.35 mg of quercetin/g. In their study, Cox et al. (2010)
presented in our study are supported by the concurrence of De Alencar reported the presence of 42.5 mg of quercetin per gram in extracts
et al. (2016), Arulkumar et al. (2018), and Sobuj et al. (2021b). De derived from brown seaweed sourced from Ireland. According to Dellai
Alencar et al. (2016) employed a 70 % ethanol solution to extract et al. (2013), flavonoids exhibit diverse chemical and biological activ­
compounds from P. capillacea and O. obtusiloba in their study. The ities, making them highly dynamic natural phenolic compounds. These
phenolic content of these extracts was determined to be 15.23 and 30.54 activities include antioxidant capabilities and the ability to scavenge
mg of GAE/g, respectively, which is comparatively lower than the free radicals.
phenolic content observed in the current investigation. In a study con­
ducted by Arulkumar et al. (2018), a 70 % methanol solution was
3.4. Total antioxidant capacity
employed to extract G. corticata and G. edulis. The resulting phenolic
values were 40.0 ± 0.35 mg of GAE/g and 34.0 ± 0.21 mg of GAE/g,
Evaluation of total antioxidant activities of different crude extracts
and their fractions of S. polycystum was observed using the DPPH and
Table 3
ABTS radical scavenging assay.
Total phenolic and flavonoid contents of different solvents and their fractions of
S. polycystum (mean ± SD).
3.4.1. DPPH assay
Extracts Total Phenolics (mg of GA/ Total Flavonoids (mg of DPPH is a chemical compound characterized by a nitrogen-free
g) quercetin/g)
radical, which can be readily quenched by a material exhibiting scav­
100% Ethanol 76.49 ± 0.9b 32.63 ± 0.7c enging properties towards free radicals. The dependence of the DPPH
70% Ethanol 58.80 ± 0.6c 30.13 ± 0.3d
radical-scavenging capacities of S. polycystum on the extract and con­
50% Ethanol 50.13 ± 0.2e 27.63 ± 0.4e
100% 80.13 ± 0.5a 38.33 ± 0.3a centration was observed, and significant variation in their activity was
Methanol seen among different extracts (p < 0.05). Here, 100 % methanol extract
70% Methanol 55.09 ± 0.5d 34.86 ± 0.2b showed the highest percentage of inhibition (78.32 %, IC50 = 1.59 mg/
50% Methanol 49.07 ± 0.2f 29.41 ± 0.8d ml), followed by 70 % methanol extract (65.29 %, IC50 = 2.77 mg/ml),
Different superscript letters illustrate significantly different from each other. The 100 % ethanol extract (60.81 %, IC50 = 4.05 mg/ml), 70 % ethanol
level of significance is p < 0.05. extract (54.44 %, IC50 = 5.46 mg/ml), 50 % ethanol extract (52.49 %,

6
M.K.A. Sobuj et al. Food Chemistry Advances 4 (2024) 100565

IC50 = 5.75 mg/ml), and 50 % methanol extract (49.72 %, IC50 = 6.58 the positive control (i.e., ascorbic acid, IC50 = 0.181 ± 0.005 mg/ml). A
mg/ml) (Fig. 2A, Table 4). Compared to the positive control (i.e., similar observation of antioxidant activities of the methanolic extract
ascorbic acid, IC50 = 0.00282 ± 0.0004 mg/ml), the IC50 values of all was observed in the previous literature for seaweed (Sobuj et al., 2021a,
crude extracts showed lower DPPH radical scavenging effects (Table 4). 2021b). This is because methanol extracts may have H-donating prop­
The extracts that included a higher percentage of TPC and TFC were also erties, which can terminate the oxidation process by converting free
influential in scavenging DPPH radicals. This suggests that polyphenols radicals to stable forms. Wu et al. (2022) found the highest antioxidant
or flavonoids present in the algae extracts may be the main ingredients activity in the aqueous acetone extract of S. polycystum, which conflicted
responsible for the extracts’ strong inhibitory characteristics. Sobuj et al. with our present finding. The observed variance may be attributed to
(2021a) have previously documented an approach similar to the one factors such as the method employed for varietal extraction and
described in this study. Rattaya et al. (2015) also observed that meth­ geographical location.
anolic extract exhibited the highest antioxidative activity compared to
other extracts. However, Johnson et al. (2019) found the most increased 4. Conclusion
antioxidant activity in the acetone extract of Sargassum sp., contra­
dicting our present funding. This variance might be because of the The findings of the current study suggest that S. polycystum has the
varietal extraction method, species variation and location. potential to serve as a valuable source of bioactive chemicals containing
powerful functional groups. Phytochemical screening and Fourier
3.4.2. ABTS radical scavenging assay Transform Infrared (FT-IR) approaches were employed to identify the
The ABTS scavenging process involves the formation of a blue/green presence of several secondary metabolites. The phytochemical activities
ABTS chromophore through the direct reaction between ABTS and exhibited by various solvents of S. polycystum demonstrate a correlation
K2S2O8. The results of this study demonstrate a direct relationship be­ with the total phenolic and flavonoid contents found in the extracts. The
tween the concentration of the crude extract and its antioxidant activity. 100 % methanol extracts showed higher concentrations of phenolics,
Fig. 2B shows that the 100 % methanol extracts exhibited the highest flavonoids, and antioxidant activity, suggesting that methanol was more
percentage of activity (69.82 %) compared to the other extracts. The 70 effective for extracting the bioactive phytochemicals than the other
% methanol extract demonstrated the second-highest percentage of ac­ solvents. Nevertheless, due to its poisonous nature, methanol is not
tivity (65.37 %), whereas the 50 % methanol extract showcased the commonly used as a solvent to extract phytochemicals intended for
lowest activity (45.66 %). As shown in Table 4, the IC50 values of ABTS human consumption. Instead, alternative solvents such as ethanol are
radical scavenging ability of S. polycystum exhibited the following preferred, as they possess lower toxicity levels. These solvents can
extract order: 100 % methanol (2.39) > 70 % methanol (3.30) > 100 % extract specific phytochemicals that cannot be effectively extracted
ethanol (4.02) > 70 % ethanol (4.96) > 50 % ethanol (5.70) > 50 % using water. The phytochemical content and activity were affected by
methanol (7.22). The IC50 values of all crude extracts and their fractions the polarity or dielectric constant of the solvent. The pharmacological
exhibited lower ABTS radical scavenging effects when compared with activity and identification of bioactive substances in S. polycystum

Fig. 2. (A) 1,1-diphenyl-2-picrylhydrazyl assay and (B) 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assay of different crude extracts and their fractions of
S. polycystum. M- Methanol, E- Ethanol.

7
M.K.A. Sobuj et al. Food Chemistry Advances 4 (2024) 100565

Table 4
IC50 (mg/ml) values obtained from different crude extracts and their fractions of S. polycystum from different antioxidant assays (mean ± SD).
Antioxidant assay IC50 (mg/ml) values of different crude extracts and their fractions
Ascorbic acid 100% Methanol 70% Methanol 50% Methanol 100% Ethanol 70% Ethanol 50% Ethanol

DPPH assay 0.00282 ± 0.0004a 1.59 ± 0.08b 2.77 ± 0.07c 6.58 ± 0.04g 4.05 ± 0.05d 5.46 ± 0.04e 5.75 ± 0.06f
ABTS radical scavenging assay 0.181 ± 0.005a 2.39 ± 0.05b 3.30 ± 0.06c 7.22 ± 0.11g 4.02 ± 0.07d 4.96 ± 0.10e 5.70 ± 0.09f

Each superscript letter represents a distinct and substantial difference from one another. The significance level is p < 0.05.

provide strong scientific support for many conventional medicinal as­ Chandini, S. K., Ganesan, P., & Bhaskar, N. (2008). In vitro antioxidant activities of three
selected brown seaweeds of India. Food Chemistry, 107(2), 707–713. https://doi.org/
sertions. The results of this study reveal the possibility of incorporating
10.1016/j.foodchem.2007.08.081
seaweed extracts into food products to improve their quality and Chen, T., & Yang, C. S. (2020). Biological fates of tea polyphenols and their interactions
nutritional value. Additional research is needed to identify, isolate, and with microbiota in the gastrointestinal tract: Implications on health effects. Critical
characterize the bioactive compounds possessing antibacterial proper­ Reviews in Food Science and Nutrition, 60(16), 2691–2709. https://doi.org/10.1080/
10408398.2019.1654430
ties using different solvents. Cox, S., Abu-Ghannam, N., & Gupta, S. (2010). An assessment of the antioxidant and
antimicrobial activity of six species of edible Irish seaweeds. International Food
Research Journal, 17(1), 205–220. https://doi.org/10.21427/D7HC92
CRediT authorship contribution statement
Dellai, A., Laajili, S., Le Morvan, V., Robert, J., & Bouraoui, A. (2013). Antiproliferative
activity and phenolics of the Mediterranean seaweed Laurencia obusta. Industrial
Mohammad Khairul Alam Sobuj: Formal analysis, Data curation, Crops and Products, 47, 252–255. https://doi.org/10.1016/j.indcrop.2013.03.014
Methodology, Writing – original draft, Writing – review & editing. Duan, X. J., Zhang, W. W., Li, X. M., & Wang, B. G. (2006). Evaluation of antioxidant
property of extract and fractions obtained from a red alga, Polysiphonia urceolata.
Morshed Shamim Shemul: Formal analysis, Methodology, Writing – Food Chemistry, 95(1), 37–43. https://doi.org/10.1016/j.foodchem.2004.12.015
original draft. Md. Shoebul Islam: Data curation, Methodology, Edeoga, H. O., Okwu, D. E., & Mbaebie, B. O. (2005). Phytochemical constituents of some
Writing – review & editing. Md. Ariful Islam: Writing – review & Nigerian medicinal plants. African Journal of Biotechnology, 4(7), 685–688. https://
doi.org/10.5897/AJB2005.000-3127
editing. Shayla Sultana Mely: Writing – review & editing. Mehedi Espeche, M. E., Fraile, E. R., & Mayer, A. M. (1984). Screening of Argentine marine algae
Hasan Ayon: Formal analysis. Shahittya Mitra Pranto: Formal anal­ for antimicrobial activity. In Eleventh international seaweed symposium (pp. 525–528).
ysis. Mohammad Shafiqul Alam: Writing – review & editing. Springer. https://doi.org/10.1007/978-94-009-6560-7_107.
Ganesan, P., Kumar, C. S., & Bhaskar, N. (2008). Antioxidant properties of methanol
Mohammad Sarfuddin Bhuiyan: Writing – review & editing. S.M. extract and its solvent fractions obtained from selected Indian red seaweeds.
Rafiquzzaman: Conceptualization, Methodology, Supervision, Writing Bioresource Technology, 99(8), 2717–2723. https://doi.org/10.1016/j.
– review & editing. biortech.2007.07.005
Harborne, A. J. (1998). Phytochemical methods. London: Chapman and hall.
Haque, K. F., Chy, S. Y., Akter, S., Wahab, M. A., & Nath, K. K. (2009). Collection,
Declaration of Competing Interest identification and biochemical analyses of different seaweeds from Saint Martin’s
island. Bangladesh Journal of Agricultural Research, 34(1), 59–65. https://doi.org/
10.3329/bjar.v34i1.5754
We wish to confirm that there are no known conflicts of interest Islam, M. S., Sobuj, M. K. A., Islam, H. M. R., Hosain, M. E., & Rashid, M. H. (2022).
associated with this publication. Present status of Seaweed resources in Bangladesh: A review on the diversity, culture
methods and utilisation. Bangladesh Journal of Zoology, 50, 283–307. https://doi.
org/10.3329/bjz.v50i3.65537
Data availability Jeeva, S., Marimuthu, J., Domettila, C., Anantham, B., & Mahesh, M. (2012). Preliminary
phytochemical studies on some selected seaweeds from Gulf of Mannar, India. Asian
Data will be made available on request. Pacific Journal of Tropical Biomedicine, 2(1), S30–S33. https://doi.org/10.1016/
S2221-1691(12)60125-7
Johnson, M., Kanimozhi, S. A., Malar, T. R. J. J., Shibila, T., Freitas, P. R., Tintino, S. R.,
et al. (2019). The antioxidative effects of bioactive products from Sargassum
Acknowledgment polycystum C. Agardh and Sargassum duplicatum J. Agardh against inflammation and
other pathological issues. Complementary Therapies in Medicine, 46, 19–23. https://
doi.org/10.1016/j.ctim.2019.06.014
This research was funded by the Grants for Advanced Research in Kai, Y., Liu, Y., Li, H., & Yang, H. (2023). Wakame replacement alters the metabolic
Education, Ministry of Education, Bangladesh. profile of wheat noodles after in vitro digestion. Food Research International, 164,
Article 112394. https://doi.org/10.1016/j.foodres.2022.112394
Li, S., Tian, Y., Jiang, P., Lin, Y., Liu, X., & Yang, H. (2021). Recent advances in the
References application of metabolomics for food safety control and food quality analyses.
Critical Reviews in Food Science and Nutrition, 61(9), 1448–1469. https://doi.org/
De Alencar, D. B., De Carvalho, F. C. T., Rebouças, R. H., Dos Santos, D. R., dos Santos 10.1080/10408398.2020.1761287
Pires-Cavalcante, K. M., de Lima, R. L., et al. (2016). Bioactive extracts of red Li, Z., Ren, Z., Zhao, L., Chen, L., Yu, Y., Wang, D., et al. (2023). Unique roles in health
seaweeds Pterocladiella capillacea and Osmundaria obtusiloba (Floridophyceae: promotion of dietary flavonoids through gut microbiota regulation: Current
Rhodophyta) with antioxidant and bacterial agglutination potential. Asian Pacific understanding and future perspectives. Food Chemistry, 399, Article 133959. https://
Journal of Tropical Medicine, 9(4), 372–379. https://doi.org/10.1016/j. doi.org/10.1016/j.foodchem.2022.133959
apjtm.2016.03.015 Lim, S. N., Cheung, P. C. K., Ooi, V. E. C., & Ang, P. O. (2002). Evaluation of antioxidative
Arulkumar, A., Rosemary, T., Paramasivam, S., & Rajendran, R. B. (2018). activity of extracts from a duan brown seaweed, Sargassum siliquastrum. Journal of
Phytochemical composition, in vitro antioxidant, antibacterial potential and GC–MS Agricultural and Food Chemistry, 50(13), 3862–3866. https://doi.org/10.1021/
analysis of red seaweeds (Gracilaria corticata and Gracilaria edulis) from Palk Bay, jf020096b
India. Biocatalysis and Agricultural Biotechnology, 15, 63–71. https://doi.org/ Mansuya, P., Aruna, P., Sridhar, S., Kumar, J. S., & Babu, S. (2010). Antibacterial activity
10.1016/j.bcab.2018.05.008 and qualitative phytochemical analysis of selected seaweeds from Gulf of Mannar
Blunt, J. W., Copp, B. R., Munro, M. H. G., Northcote, P. T., & Prinsep, M. R. (2006). region. Journal of Experimental Sciences, 1(8), 23–26.
Marine natural products. Natural Products Reports, 23, 26–78. https://doi.org/ Marimuthu, J., Essakimuthu, P., Narayanan, J., Anantham, B., Tharmaraj, R. J. J. M., &
10.1039/B502792F Arumugam, S. (2012). Phytochemical characterization of brown seaweed Sargassum
Berland, B. R., Bonin, D. J., Cornu, A. L., Maestrini, S. Y., & Marino, J. P. (1972). The wightii. Asian Pacific Journal of Tropical Disease, 2, S109–S113. https://doi.org/
antibacterial substances of the marine alga Stichochrysis immobilis (chrysophyta) 12. 10.1016/S2222-1808(12)60134-0
Journal of Phycology, 8(4), 383–392. https://doi.org/10.1111/j.1529-8817.1972. Marinho-Soriano, E., Fonseca, P. C., Carneiro, M. A. A., & Moreira, W. S. C. (2006).
tb04052.x Seasonal variation in the chemical composition of two tropical seaweeds. Bioresource
Chang, C. C., Yang, M. H., Wen, H. M., & Chern, J. C. (2002). Estimation of total Technology, 97(18), 2402–2406. https://doi.org/10.1016/j.biortech.2005.10.014
flavonoid content in propolis by two complementary colorimetric methods. Journal Naczk, M., & Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: Occurrence,
of Food and Drug Analysis, 10(3), 178–182. https://doi.org/10.38212/2224- extraction and analysis. Journal of Pharmaceutical and Biomedical Analysis, 41(5),
6614.2748 1523–1542. https://doi.org/10.1016/j.jpba.2006.04.002
Chapman, V. J., & Chapman, D. J. (1980). Algin and alginates. Seaweeds and their uses Nedumaran, T., & Arulbalachandran, D. (2015). Seaweeds: A promising source for
(pp. 194–225). Dordrecht: Springer. https://doi.org/10.1007/978-94-009-5806-7_6. sustainable development. Environmental sustainability (pp. 65–88). https://doi.org/
pp. 10.1007/978-81-322-2056-5_4

8
M.K.A. Sobuj et al. Food Chemistry Advances 4 (2024) 100565

Okazaki, A. (1971). Seaweeds and their uses in Japan. Tokyo: Tokai University Press. collected from Bangladesh. Scientific Reports, 11(1), 19082. https://doi.org/
Rafiquzzaman, S. M., Ahmad, M. U., Lee, J. M., Kim, E. Y., Kim, Y. O., Kim, D. G., et al. 10.1038/s41598-021-98461-3
(2016). Phytochemical composition and antioxidant activity of edible red alga Sobuj, M. K. A., Islam, M. A., Haque, M. A., Islam, M. M., Alam, M. J., &
Hypnea musciformis from Bangladesh. Journal of Food Processing and Preservation, 40 Rafiquzzaman, S. M. (2021b). Evaluation of bioactive chemical composition,
(5), 1074–1083. https://doi.org/10.1111/jfpp.12688 phenolic, and antioxidant profiling of different crude extracts of Sargassum
Rajauria, G., Jaiswal, A. K., Abu-Gannam, N., & Gupta, S. (2013). Antimicrobial, coriifolium and Hypnea pannosa seaweeds. Journal of Food Measurement and
antioxidant and free radical-scavenging capacity of brown seaweed Himanthalia Characterization, 15, 1653–1665. https://doi.org/10.1007/s11694-020-00758-w
elongata from western coast of Ireland. Journal of Food Biochemistry, 37(3), 322–335. Sobuj, M. K. A., Islam, M. M., Rahman, S., & Mahmud, Y. (2023). Cultivation and product
https://doi.org/10.1111/j.1745-4514.2012.00663.x development study of commercially important seaweeds in south-eastern coast of
Rajauria, G., Jaiswal, A. K., Abu-Ghannam, N., & Gupta, S. (2010). Effect of Bangladesh. Food Safety - New Insights. https://doi.org/10.5772/intechopen.111937
hydrothermal processing on colour, antioxidant and free radical scavenging Trease, G.E., & Pharmacognsy, E.W. (1989). Brailliar tiridel can Macmillian publishers.
capacities of edible Irish brown seaweeds. International Journal of Food Science and London, UK.
Technology, 45(12), 2485–2493. https://doi.org/10.1111/j.1365-2621.2010.02449. Velioglu, Y., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and total
x phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural
Rattaya, S., Benjakul, S., & Prodpran, T. (2015). Extraction, antioxidative, and and Food Chemistry, 46(10), 4113–4117. https://doi.org/10.1021/jf9801973
antimicrobial activities of brown seaweed extracts, Turbinaria ornata and Sargassum Versari, A., Parpinello, G. P., Scazzina, F., & Del Rio, D. (2010). Prediction of total
polycystum, grown in Thailand. International Aquatic Research, 7, 1–16. https://doi. antioxidant capacity of red wine by Fourier transform infrared spectroscopy. Food
org/10.1007/s40071-014-0085-3 Control, 21(5), 786–789. https://doi.org/10.1016/j.foodcont.2009.11.001
Sarkar, M. S. I., Kamal, M., Hasan, M. M., & Hossain, M. I. (2016). Present status of Wu, Y., Gao, H., Wang, Y., Peng, Z., Guo, Z., Ma, Y., et al. (2022). Effects of different
naturally occurring seaweed flora and their utilization in Bangladesh. Research in extraction methods on contents, profiles, and antioxidant abilities of free and bound
Agriculture Livestock and Fisheries, 3(1), 203–216. https://doi.org/10.3329/ralf. phenolics of Sargassum polycystum from the South China Sea. Journal of Food Science,
v3i1.27879 87(3), 968–981. https://doi.org/10.1111/1750-3841.16051
Sobuj, M. K. A., Islam, M. A., Islam, M. S., Islam, M. M., Mahmud, Y., & Yamamoto, Y., & Gaynor, R. B. (2001). Therapeutic potential of inhibition of the NF-κB
Rafiquzzaman, S. M. (2021a). Effect of solvents on bioactive compounds and pathway in the treatment of inflammation and cancer. The Journal of Clinical
antioxidant activity of Padina tetrastromatica and Gracilaria tenuistipitata seaweeds Investigation, 107(2), 135–142. https://doi.org/10.1172/JCI11914