Thalassas: An International Journal of Marine Sciences (2024) 40:659–668

https://doi.org/10.1007/s41208-023-00649-z

RESEARCH

Chemical Composition of Spheciospongia Aff. Mastoidea Sponge from

the Red Sea and Uses of Its Polysaccharides in the Biosynthesis of
Silver Nanoparticles with Antimicrobial and Anticancer Activity
Rasha MA Eltanany1 · Ahmed H. I. Faraag2 · Hassan Y Ebrahim3 · Mohammed I. Y. Elmallah1 ·
Mohamed S. Abdelfattah1,4

Received: 26 September 2022 / Revised: 26 October 2023 / Accepted: 28 December 2023 / Published online: 5 January 2024
© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2024

Abstract
Here, we studied the chemical composition of Spheciospongia aff. Mastoidea sponge collected from the Red Sea. The
chemical profile of the n-hexane fraction was studied using GC-MS and revealed the presence of 11 compounds. The
most abundant compounds were hexadecanoic acid methyl ester (49.93%), 9-octadecenoic acid methyl ester (22.13%), and
other minor products. Additionally, three compounds were isolated from the ethyl acetate and n-butanol fractions of Sphe-
ciospongia aff. Mastoidea and identified as β-sitosterol, cholesterol, and allantoin, respectively. The chemical structures of
the isolated compounds were identified by different spectroscopic methods, including mass and NMR spectroscopy. Crude
polysaccharides (CPs) were also extracted from the aqueous extract of the collected sponge, and HPLC-RID characterized
their monosaccharides. We developed a biological method for synthesizing silver nanoparticles using the reducing power
of CPs. The biosynthesized AgNPs were confirmed using UV-Vis spectroscopy, Fourier transform infrared spectroscopy
(FTIR), Transmission electron microscopy (TEM), X-ray diffraction (XRD), and Zeta potential. The nanoparticles had
a spherical shape in the TEM image, with an average size of 18.21 to 36.92 nm and zeta potential values of -27.3 mV.
The biosynthesized AgNPs-CPs showed enhanced antibacterial activity against several pathogens compared to CPs with
no remarkable activity. Moreover, the AgNPs-CPs exhibited a cytotoxic effect against breast adenocarcinoma cell lines
(MCF-7), liver cancer cell lines (HepG-2), prostate cancer cell lines (PC-3), adenocarcinomas alveolar basal epithelial
cells (A549), and colorectal carcinoma cell lines (HCT116) with IC50 values of 5.60, 13.0, 2.62, 46.3, and 29.20 µg/ml,
respectively.

Keywords Spheciospongia Aff. Mastoidea · Fatty acids · Polysaccharides · Biosynthesis · Silver nanoparticles ·
Antimicrobial · Anticancer activity

Introduction

The Red Sea in the Arab region is an exciting ecosystem

characterized by high salinity and temperature and has a
large bio-diverse coral reef system (Triantafyllou et al.
Mohamed S. Abdelfattah
mabdelfattah@science.helwan.edu.eg 2014; Fine et al. 2019; Kleinhaus et al. 2020). In these reefs,
sponges are critical structural components that play impor-
1
Chemistry Department, Faculty of Science, Helwan tant functional roles in the marine ecosystem, such as acting
University, Cairo 11795, Egypt as efficient filter feeders, playing crucial roles in carbon and
2
Botany and Microbiology Department, Faculty of Science, nitrogen cycles in coral reef ecosystems, and exerting con-
Helwan University, Cairo 11795, Egypt trol on plankton communities (Pawlik and McMurray 2020).
3
Faculty of Pharmacy, Helwan University, Ain Helwan, Cairo, Chemists found that sponges possess a cockatiel of chemi-
Egypt cal compounds with unique pharmacological properties and
4
Natural Products Research Unit (NPRU), Faculty of Science, diverse biological activities (Shanmugam and Vairamani
Helwan University, Cairo 11795, Egypt

13
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2016; Abdelaleem et al. 2020). Among such compounds
are polysaccharides, which play an important role in the
life of a sponge (Vilanova et al. 2009). Polysaccharides, in
general, are a substantial class of natural compounds that
have advantageous features in biological systems, including
biodegradability, biocompatibility, and a lower toxic effect
(Plucinski et al. 2021). In the case of the marine sponge,
polysaccharides display a conventional type of cell-cell
adhesion based on carbohydrate-carbohydrate interaction.
As per the literature, different sponges synthesize polysac-
charides with a high diversity of sugar moieties (Zierer and
Mourão 2000; Maia et al. 2016). They have various biologi-
cal activities, such as immunological, anti-blood coagula-
tion, anticancer, anti-human immunodeficiency virus (HIV),
and hypoglycemic activities (Marques et al. 2016).
Synthesis of metal nanoparticles using biological meth-
ods drew the attention of scientists during the last three
decades, as these procedures provide a safe, easy prepara-
tion, low-cost, and eco-friendly approach (Roy et al. 2019;
Soltys et al. 2021). In these techniques, the metal nanoparti-
cles (i.e., silver, gold, zinc, titanium, copper. etc.) have been
synthesized using different biological sources such as bac-
teria, fungi, algae, and plants (Shah et al. 2015). As per the
literature, natural polysaccharides are a promising source
for biosynthesizing metal nanoparticles due to their role as
stabilizing and reducing agents (Wang et al. 2017; Shavandi
et al. 2019; Yosri et al. 2021). Throughout this process,
polysaccharides have the potential to bind with metal ions
and metal nanoparticles via non-covalent bonding. Subse-
quently, the order of free energy is changed, which enables
the metal nanoparticles to achieve stabilization, morphologi-
cal control, and kinetic growth (Harish et al. 2022). The bio-
synthesized nanoparticles have many medical applications
as antimicrobial and anticancer agents (Suba et al. 2022).
Among the nanometals, silver nanoparticles (AgNPs) have
been recognized as an effective antibacterial agent capable
of combating Gram-negative and Gram-positive bacteria
that cause illnesses in vitro and in vivo (Bruna et al. 2021).
It displays a synergistic effect against different pathogens
when combined with organic compounds and/or antibiotics
(Aabed and Mohammed 2021). Moreover, silver nanoparti-
cles (AgNPs) synthesized by green methods have cytotoxic
effects against different types of cancer, such as breast, lung,
and skin cancer (Morais et al. 2020).
The use of marine species to produce metal nanoparti-
cles is still relatively uncommon in comparison to the use
of terrestrial organisms, particularly plants (Ponnuchamy
and Jacob 2016). The potential use of marine sponges in
the preparation of metal nanoparticles is also unexplored, as
a few investigations have been conducted to use the aque-
ous extract of marine sponges as reducing agents to pre-
pare silver and gold nanoparticles (Inbakandan et al. 2010,
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Thalassas: An International Journal of Marine Sciences (2024) 40:659–668
2012; Hamed et al. 2017; Al-Khalaf et al. 2021). The Red
Sea sponge Spheciospongia aff. Mastoidea, which belongs
to the family of Demospongiae, is found abundantly along
the Egyptian coast. It is characterized by different second-
ary metabolites such as terpenoids and steroids (Salmoun et
al. 2007; Whitson et al. 2008; Kalinin et al. 2012). Because
little research has been done on Spheciospongia aff. Mas-
toidea collected from the Red Sea, we studied its chemi-
cal composition and used its polysaccharides as a reducing
agent in the biosynthesis of silver nanoparticles (AgNPs).
The produced nanoparticles were characterized using UV,
X-ray diffraction, TEM, zeta potential, and FTIR. The in
vitro antimicrobial and anticancer activity of AgNPs-CPs
was also studied.
Materials and Methods
Collection of Sponge Sample
The marine sponge was collected by Scuba diving at Ras
Mohammed Protectorate (27° 43′ 20″ N, 34° 15′ 14″ E),
Sharm El-Sheikh, South of Sinai, Egypt. It was identified
as Spheciospongia aff. Mastoidea by Prof. Rob WM van
Soest at the Netherlands Centre for Biodiversity Naturalis,
Leiden, Netherlands. The collected sponge is a member
of the order Clionaida (Class Demospongiae, Subclass
Heteroscleromorpha, Genus Spheciospongia, and Species
Spheciospongia aff. mastoidea)(van Soest et al. 2012). The
sample was cleaned with distilled water to remove any sand
or other debris.
Fatty Acids Quantitative Determination
The wet sponge sample (400 g) was cut into small pieces
and extracted by macerating overnight in 70% methanol
(3 × 1 L). The extract was concentrated under a vacuum,
and the resulting mixture was extracted with n-hexane
(3 × 0.5 L), followed by ethyl acetate (3 × 0.5 L), and finally
with n-butanol (3 × 0.5 L). The hexane fraction (0.9 g) was
refluxed for seven hours with 0.5 N alcoholic KOH (70 ml)
in a boiling water bath for saponification. The products were
extracted with diethyl ether, and the remaining aqueous
alkaline solution was acidified with HCl and extracted sev-
eral times with diethyl ether. To prepare fatty acids methyl
ester, the combined ethereal extracts were evaporated to
dryness and then dissolved in 50 ml MeOH acidified with
0.3 ml of H2SO4 and refluxed for about three hours. The
fatty acid methyl esters (FAME) produced were extracted
several times with ether. The ethereal extract was evapo-
rated, and the residue was subjected to GC-MS analysis
on a Hewlett-Packard 6890 GC equipped with an HP-5MS
Thalassas: An International Journal of Marine Sciences (2024) 40:659–668
capillary column (30 m × 0.32 mm × 0.2 μm film thickness)
and MS spectrometric detector (Ghandourah et al. 2021).
Helium was used as a carrier gas at a column head pressure
of 60 kPa. The column oven temperature cycle was 50 °C
for 10 min, then 50 to 310 °C for 60 min, then 310 °C for
20 min. The fatty acid methyl esters (FAME) were identified
by comparing their retention times and mass fragmentation
patterns with Wiley’s chemical database.
Isolation of Compounds
The ethyl acetate extract (1.1 g) was fractionated using
silica gel column chromatography (3 × 60 cm) through a
stepwise gradient solvent system of increasing polarity
starting (DCM: MeOH = 100:0, 98:2, 96:4, 92:6, 90:10,
80:20, 50:50, 0:100) to give five fractions (I-V). Fraction
II (99.3 mg) was purified by Sephadex LH-20 (2 × 35 cm,
DCM: MeOH, 3: 2) to get β-sitosterol (1, 5.60 mg) as a
white solid with Rf = 0.60 (DCM/5%MeOH). Fraction III
(150.9 mg) was dissolved in methanol and kept overnight.
A white precipitate was produced and recognized as cho-
lesterol (2, 50.20 mg) with Rf = 0.41 (DCM/5% MeOH).
The butanol extract (2.1 g) was fractionated using Sephadex
LH-20 (2 × 35 cm, MeOH) to obtain four fractions. Com-
pound 3 was separated as a white solid from fraction III and
identified as allantoin with Rf = 0.46 (n-butanol: acetic acid:
water = 3: 1: 1).
Extraction of Polysaccharides
Around 100 g of a sponge sample was cut into small pieces,
ground, and mixed with distilled water (10% w/v) in a soni-
cated water bath for 20 min at room temperature. The result-
ing homogenate was centrifuged at 15,300 rpm for 35 min.
The residues were discarded, and the supernatant consisted
of crude polysaccharides (CPs). For deproteinization of sug-
ars, the solution was treated with 5% trichloroacetic acid
(TCA) and kept in the refrigerator for 10 min at 4 °C. The
sample was centrifuged for 15 min at 4500 rpm, and the
precipitate was rejected to obtain the deproteinized solution.
An equal volume of ethanol was added (ca. 100 mL) to the
deproteinized CPs and kept overnight at -20 °C. The pre-
cipitate was collected, washed with acetone, and dried in an
oven at 50 °C.
Acid Hydrolysis of CPs
The crude polysaccharides were hydrolyzed before being
analyzed via high-performance liquid chromatography
(HPLC). The hydrolysis was performed by refluxing 0.5 g
of CPs with 30 ml of 2 N sulfuric acid for 24 h. The hydro-
lysized sample was evaporated under a vacuum using a
661
rotary evaporator to yield a residue, which was dried, and
extracted with hot distilled pyridine. The pyridine extract
was evaporated to dryness, and the residues were preserved
at 4ºC for HPLC identification (Ren et al. 2019).
HPLC-RID Analysis of CPs Sugar Content
The CPs extract and the standard samples were filtered
through a 0.45 μm membrane and injected in a 20 µL. The
analysis was conducted using HPLC, Shimadzu Class-VPV
5.03 (Kyoto, Japan), equipped with a refractive index RID-
10 A Shimadzu detector, LC-16ADVP binary pump, PL Hi-
Plex Pb column, and a heater set at 80ºC. The mobile phase
was 0.01% reagent grade calcium chloride prepared with
deionized water, while the flow rate was 0.6 mL min-1. The
analysis of the filtrate sugars was performed according to
previous literature (Chen et al. 2013).
Synthesis and Characterization of Silver
Nanoparticles (AgNPs) Using CPs
A 10 ml sample of polysaccharides extract was adjusted to
pH 8.0 with 0.05 M sodium hydroxide, and 1 ml of 0.1 M
silver nitrate solution (AgNO3) was added. The temperature
of the reaction mixture was 40ºC, and it stirred continuously
for two hours. The formation of AgNPs was seen by chang-
ing the color of the reaction mixture from colorless to yel-
low and then to brown.
Characterization of Silver Nanoparticles (AgNPs)
The biosynthesized AgNPs were characterized using meth-
ods described in the literature (Chakraborty et al. 2021;
Basavarajappa et al. 2022). A UV-Vis spectrophotometer
analysis (T80 UV/vis spectrometer, PG Instrumentals) was
used to observe the synthesized AgNPs at 300–800 nm
wavelengths. The Bruker D8 Discover X-ray powder dif-
fractometer (XRD) at a wavelength of 1.5406 A0 was used
to inspect the phase and constitution of the nanoparticles
(Essa et al. 2021). The JEOL-JEM-2100 high-resolution
transmission electron microscopy (TEM) was used to inves-
tigate the size and morphology of AgNPs. The zeta poten-
tial was measured by Zetasizer® Nano ZS, Malvern PCS
Instruments, UK to determine the surface charges on syn-
thesized AgNPs. The FTIR characterization was carried out
to identify functional groups found in the aqueous sponge
crude extract and their biosynthesized AgNPs in the wave
number range of 4000–400 cm− 1 (FTIR Perkin-Elmer with
ATR technique).
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Antimicrobial Activity
Antimicrobial activity of CPs and AgNPs-CPs was evalu-
ated using an agar well-diffusion assay according to the
Clinical and Laboratory Standards Institute (Kiehlbauch et
al. 2000). Briefly, 100 µl of bacterial suspension contain-
ing 1 × 105 cells of each reference bacteria (Escherichia
coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027,
Staphylococcus aureus ATCC 25,923, Streptococcus fae-
calis ATCC 8043 and Candida albicans ATCC 10,231)
was spread individually on the surface of Muller Hinton
agar plates, and 0.8 cm diameter wells were made. Each
well received 50 µl of tested samples with a concentration
of 100 µg/ml, and then the plates were kept in a refrigera-
tor for 30 min to allow the test samples to diffuse into the
agar medium. The plates were then incubated for 24 h at
37ºC. The antimicrobial susceptibility of each extract was
determined by measuring the diameter of the developed
inhibition zones in mm. The activity was compared to that
of ciprofloxacin, and diluted DMSO was used as a negative
control.
Anticancer Activity
The in vitro anticancer activity of the CPs and their bio-
synthesized AgNPs was evaluated using the MTT method
(Elmallah et al. 2017). Five human cancer cell lines, includ-
ing the breast adenocarcinoma cell line (MCF-7), liver
cancer cell line (HepG-2), prostate cancer cell line (PC-3),
Fig. 1 GC/MS spectrum of n-hexane fraction of Spheciospongia aff. Mastoidea
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Thalassas: An International Journal of Marine Sciences (2024) 40:659–668
adenocarcinomic alveolar basal epithelial cells (A549), and
the colorectal carcinoma cell line (HCT116), were used in
our study. All cancer cells were obtained from the Ameri-
can Type Culture Collection. Dulbecco’s modified Eagle’s
medium was used to cultivate cancer cells. Briefly, cells
were seeded in 96-well plates (4 × 104 cells per well) with
100 µl of Dulbecco’s modified Eagle’s medium. The plates
were incubated at 37 °C for 48 h in 5% CO2. The CPs and
AgNPs-CPs, at various concentrations of 100; 50; 25; 12.5;
6.25; 3.12 and 1.56 µg/ml, were added to each well and
incubated for 24 h. The media was discarded, and 10 µl of
MTT solution (5 mg/ml) was added. A microplate reader
read the absorbance at 630 nm, and the IC50 values were
calculated. Doxorubicin was utilized as a positive control at
concentrations of 2.0; 1.0; 0.5; 0.25, and 0.1 µg/ml, whereas
DMSO was employed as a negative control.
Results and Discussion
Fatty Acids Compositions
The fatty acids profile of Spheciospongia aff. Mastoidea
was estimated by GC/MS (Fig. 1and Table 1) and revealed
11 major compounds. Metabolites were identified based on
retention parameters and mass spectrometric fragmenta-
tion of their methyl (ethyl) esters using archive and stan-
dard derivatives data. The most abundant compounds are
hexadecanoic acid methyl ester (49.93%), followed by
Thalassas: An International Journal of Marine Sciences (2024) 40:659–668 663

Table 1 Fatty acids compositions (Relative %) of the investigated Table 2 Constituents of sugars of polysaccharides extracted from
Spheciospongia aff. Mastoidea Spheciospongia aff. Mastoidea sponge
No Name Molec- Molecular Rt Area Monosaccharaides %
ular formula (min) (%) Glucuronic acid 39.72
weight Mannose 4.52
1 12-Methyltridecanoic 242 C15H30O2 31.52 2.13 Galactose 39.14
acid methyl ester (13:0)
Glucose 8.98
2 4,8,12-Trimethyltrideca- 270 C17H34O2 32.50 6.45
Xylose 7.64
noic acid methyl ester
13:0)
3 11-Hexadecenoic acid 268 C17H32O2 35.28 0.87 The β-sitosterol was previously isolated from the Red
methyl ester (16:1) Sea sponge Smenospongia (Neupane 2017). It was isolated
4 Hexadecanoic acid 270 C17H34O2 35.75 49.93 for the first time from Spheciospongia aff. Mastoidea. Com-
methyl ester (16:0) pound 3 was obtained from the n-butanol fraction using
5 5,9-Octadecadienoic acid 294 C19H34O2 38.70 1.93 Sephadex LH-20 (MeOH) and identified by mass spectros-
methyl ester (18:2)
copy, 1D and 2D NMR (Figs. S3-S7) as allantoin. It was
6 5,8,11,14,17-Eicosapen- 302 C20H30O2 38.77 0.32
taenoic acid (20:5) confirmed by comparing the spectral data with the literature
7 6,9-Octadecadienoic acid 294 C19H34O2 38.94 0.46 (Cao and Hahn 2023). Allantoin (3) was previously isolated
methyl ester (18:2) from invertebrates, including the marine sponge Tethya
8 9­Octadecenoic acid 296 C19H36O2 39.08 22.13 aurantia (Selamoglu 2018). It was isolated from the butanol
methyl ester (18:1) fraction of Spheciospongia aff. Mastoidea for the first time.
9 6-Octadecenoic acid 296 C19H36O2 39.15 0.42
methyl ester (18:1)
10 5,8,11,14­Eicosatetraenoic 318 C21H34O 2 41.88 1.36
Extraction and Characterization of Polysaccharides
acid methyl ester (20:4)
11 5,9-Hexacosadienoic acid 642 C42H46N2O4 51.48 0.45 The polysaccharides were ultrasonically extracted at room
methyl ester (26:2) temperature from Spheciospongia aff. Mastoidea uses water
as a green solvent. The supernatants containing the crude
9­octadecenoic acid methyl ester (22.13%), 4,8,12-trimeth- polysaccharides (CPs) were deproteinized with 5% trichlo-
yltridecanoic acid methyl ester (6.45%), and 12-methyltri- roacetic acid (TCA) and precipitated with ethanol to get a
decanoic acid methyl ester (2.13%). Sponges are a unique yield of 2.5%. It was reported that polysaccharides contain
animal in terms of the diversity of their fatty acids, with up to 3% of the sponge-dry weight and differ from species
generally high levels of long-chain (i.e., C14–C25), a high to species (Esteves et al. 2011). The HPLC-RID identi-
degree of unsaturation, and a high incidence of branched fied the sugar monomers obtained from the crude extract
and odd carbon-atom fatty acids (Řezanka and Sigler 2009; of Spheciospongia aff. Mastoidea. The CPs extract mainly
Koopmans et al. 2011). comprised glucuronic acid (39.72%), mannose (4.52%),
galactose (39.14%), glucose (8.98%), and xylose (7.64%)
Isolation and Structure Elucidation of Compounds (Table 2). According to the best of our knowledge, this is
1–3 the first report on the sugar quantification of Spheciospon-
gia aff. Mastoidea marine by HPLC.
The ethyl acetate extract was loaded on silica gel column
chromatography and eluted with CH2Cl2/MeOH to get five Biosynthesis and Characterization of Silver
fractions. Compounds 1 and 2 were isolated as white solids Nanoparticles
and identified by NMR spectroscopy, as well as by compari-
son with authentic samples, as β-sitosterol and cholesterol, Synthesis of silver nanoparticles by using the solution
respectively (Fig. 2). of crude polysaccharides (CPs) of the marine sponge

Fig. 2 Chemical structures of β-sitosterol (1), cholesterol (2) and allantoin (3) from Spheciospongia aff Mastoidea

13
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Spheciospongia aff Mastoidea is described here. The forma-
tion of AgNPs was achieved by mixing the CPs and silver
nitrate solution (0.1 M) at 40˚C under stirring conditions
for two hours. A color change of the reaction mixture from
colorless to yellow and then to brown indicated the synthe-
sis of silver nanoparticles. The reaction was also monitored
through a UV–vis spectrophotometer in the range of 300–
800 nm (Fig. 3a). The AgNPs synthesized by CPs exhibited
Fig. 3 Characterization of AgNPs: (a) UV spectrum, (b) X-ray diffraction pattern, (c) TEM image, (e) Zeta potential and (e) FTIR spectra. The red
lines are the CPs, and the blue lines are AgNPs-CPs
13
Thalassas: An International Journal of Marine Sciences (2024) 40:659–668
a characteristic peak at 437.6 nm. Previous studies reported
that the silver ions absorb between 430 and 440 nm due to
their surface plasmon resonance (SPR) (Chung et al. 2016).
To study the morphological character of the synthesized
AgNPs, X-ray diffraction (XRD), and transmission electron
microscopy (TEM) were used. The crystalline nature of the
AgNPs, was determined by XRD analysis (Fig. 3b). The
diffractogram showed four diffraction peaks at 2θ values of
Thalassas: An International Journal of Marine Sciences (2024) 40:659–668
Table 3 Antimicrobial activity of CPS and AgNPs-PS
Sample Inhibition zone diameter (mm)
Bacterial species
E. P. Aeruginosa S. S.
Coli faecalis aureus
AgNPs-CPs NA 18 16 19
Ciprofloxacin 14 17 12 NA
CPs is not active (NA)
33.40, 44.98, 65.01 77.93 degrees corresponding to (hkl)
values (111), (200), (220), and (311), the peaks agreed with
the standard powder diffraction card of JCPDS, silver file
No. 04-0783. The peak corresponding to the (111) plane
is more intense than the other planes, indicating that the
nanoparticles are abundant in (111) plane. The visual sharp-
ness of the XRD peaks can be attributed to the stabiliza-
tion of biosynthesized AgNPs by the aqueous extract of the
marine sponge Spheciospongia aff. Mastoidea (Rudrappa
et al. 2022). The TEM image of the nanoparticles (Fig. 3c)
showed a spherical shape with an average size of 18.21 to
36.92 nm. Zeta potential was recorded to understand the sta-
bility of nanoparticles in the aqueous extract of the marine
sponge Spheciospongia aff. Mastoidea. It has a value of
-27.3 mV, indicating that the AgNPs’ surfaces are nega-
tively charged (Fig. 3d). The FT-IR was recorded to identify
the possible functional groups for reducing and stabiliz-
ing the silver ions (Fig. 3e). The spectrum of CPs showed
peaks at 3357 cm− 1 corresponding to O-H stretching. The
band at 1630.20 cm− 1 corresponded to the carbonyl group,
and the band at 1349 cm− 1 corresponded to C-O stretch-
ing. The bands between 1140 and 673 cm− 1 can be used
to describe polysaccharides. After the formation of AgNPs,
some bands (i.e., the O-H group at 3357 cm− 1 and carbonyl
at 1630.20 cm− 1) dropped their intensities, indicating that
they were consumed in the bioreduction process during the
formation of AgNPs.
These results are in good agreement with those of Hamed
et al. who synthesized AgNPs using the crude extract of the
marine sponge Axinella sinoxia, where the size of AgNPs
was 23 to 38 nm with a spherical and centered cubic struc-
ture (Hamed et al. 2017).
Antimicrobial Activity of Conjugate Silver
Nanoparticles and Polysaccharides
The antimicrobial activity of CPs and AgNPs-CPs was
determined against different human pathogens (Table 3)
Table 4 Cytotoxic activity of CPs Sample MTT assay IC50 (µg/ml)
and AgNPs-CPs of Spheciospon- MCF-7
gia aff. Mastoidea
CPs 29.01 ± 0.82
AgNPs-CPs 5.60 ± 0.34
Doxorubicin 0.71
665
using ciprofloxacin as a positive control. The antimicrobial
activity was determined from the inhibition zone diameters
Fungi
around the discs. The result showed that AgNPs-CPs have
C. albi-
cans inhibitory activity against pathogenic bacteria and fungi
19 compared to CPs. Among the pathogens, S. aureus and C.
NA albicans were more susceptible to AgNPs-CPs (19 mm), fol-
lowed by P. aeruginosa (18 mm), and S. faecalis (16 mm),
whereas E. coli was less susceptible to AgNPs-CPs. Cipro-
floxacin did not exhibit any antimicrobial activity against
S. aureus and C. albicans, as shown by the “NA” entries
in Table 3. The minimum inhibitory concentration (MIC)
of AgNPs-CPs against S. faecalis, S. aureus, P. aeruginosa,
and C. albicans is 5 µg/ml.
These results indicate that AgNPs-CPs can serve as
an antibacterial agent against a variety of microorgan-
isms, which could be useful in treating infections caused
by diverse microorganisms. The mechanism of action of
AgNPs-CPs is thought to involve the release of silver ions,
which can disrupt bacterial cell membranes and interfere
with cellular processes (Mikhailova 2020). Notably, factors
like particle size, shape, and surface charge, as well as the
study’s test conditions, may have an impact on the antimi-
crobial activity of AgNPs-CPs. More research is needed to
investigate the potential of the biosynthesized AgNPs-CPs
as antibacterial agents, including in vivo investigations to
assess their safety and efficacy.
Anticancer Activity of the Conjugated Silver
Nanoparticles and Polysaccharides
The anticancer activity of the CPs of the marine sponge
Spheciospongia aff Mastoidea and their synthesized silver
nanoparticles was tested against MCF-7, HepG-2, PC-3,
A549, and HCT116 cell lines (Table 4). The viability results
were evaluated using the MTT colorimetric assay (Abdel-
fattah et al. 2011; Ghasemi et al. 2021). Various concen-
trations of CPs and AgNPs-CPs were used to treat cancer
cells, including 0.4, 1.63, 6.25, 25.0, 50.0, and 100.0 µg/
ml, where Table S1 displays the percentage of cell viabil-
ity. Doxorubicin was used as a positive control because it
inhibits topoisomerase II, which is essential for cancer cell
division and proliferation (Matias-Barrios et al. 2021). The
IC50 values were calculated using various concentrations
of CPs and AgNPs-CPs. Table 4 shows that crude polysac-
charides (CPs) have the highest anticancer activity against
the breast adenocarcinoma cell line (MCF-7) compared to
HepG-2 PC-3 A549 HCT116
74.72 ± 0.74 36.61 ± 0.52 83.32 ± 0.60 64.88 ± 0.93
13.0 ± 0.79 2.62 ± 0.16 46.30 ± 2.81 29.20 ± 1.77
0.81 0.75 0.42 0.56
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the other cancer cells. The biosynthesized AgNPs-CPs were
more active than CPs against all the evaluated cell lines.
Moreover, the AgNPs-CPs have promising anticancer activ-
ity against the prostate cancer cell line (PC-3) with an IC50
value of 2.62 µg/ml.
Al-Khalaf et al. (Al-Khalaf et al. 2021) reported the anti-
cancer activity of the biosynthesized AgNPs against MCF-
7, MDB-231, and MCF-10 A cancer cells using the Red
Sea Sponge Phyllospongia lamellosa. There are a number
of plausible mechanisms through which AgNPs exert their
anticancer effects (Mikhailova 2020).
It was discovered that AgNPs produced Ag+ ions, which
have a strong affinity for cancer cell membrane proteins.
These Ag+ ions then interact with DNA and RNA causing
alterations in chromosomal structure (Chakraborty et al.
2021).
Conclusion
This study indicated that the marine sponge Spheciospon-
gia aff. Mastoidea from the Red Sea is rich in saturated
and unsaturated fatty acids, such as hexadecanoic acid and
9-octadecenoic acid, respectively, and has several steroidal
compounds, including β-sitosterol, cholesterol, and the het-
erocyclic allantoin. It also described a simple and safe green
method for synthesizing AgNPs using crude polysaccharides
(CPs) from Spheciospongia aff. Mastoidea. The biosynthe-
sized AgNPs were confirmed using UV-Vis spectroscopy,
and the TEM analysis showed that they were spherical in
shape and had an average size of 18.21 to 36.92 nm. The
XRD pattern revealed the crystal structure of AgNPs, and a
negative charge on their surfaces was confirmed by a Zeta
potential of -27.3 mV. The CPs responsible for the reduc-
tion, stabilization, and capping of AgNPs were identified in
the FTIR. The biosynthesized AgNPs-CPs exhibited signifi-
cant antimicrobial and anticancer activity against different
cancer cells, with efficacy against prostate cancer cell lines
(PC-3). Therefore, this research work gives evidence that
the polysaccharides of marine sponges are capable of syn-
thesizing AgNPs and may serve as a good source of anti-
bacterial and anticancer agents that can be produced using
eco-friendly methods. However, more extensive biology
research will be undertaken in future studies based on the
encouraging anticancer activity, and additional research is
needed to biosynthesize more metal nanoparticles using
various marine sponge species and to investigate their cel-
lular as well as molecular therapeutic mechanisms.
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/s41208-
023-00649-z.
13
Thalassas: An International Journal of Marine Sciences (2024) 40:659–668
Acknowledgements The authors thank the Department of Chemistry,
Faculty of Science, Helwan University for their valuable assistance.
Author Contributions Sample collections and anticancer assay:
R.M.A. Eltanany; Biosynthesis of nanoparticles and antimicrobial as-
say: A.H.I. Faraag; Analysis and writing the drafted paper: H.Y. Ebra-
him, M.I.Y. Elmallah, and M. S. Abdelfattah.
Funding There has been no funding received.
Data Availability Data are available on request.
Declarations
Ethical Approval not applicable.
Competing Interests The authors declare no competing interests.
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