Silver nanoparticles synthesized from the seaweed Sargassum polycystum and screening for their biological potential

Abstract

World-wide antimicrobial resistant is biggest threat in global health. It requires the urgent need of multisectoral action for the scientific community to achieve the sustainable development Goals. Due to their antimicrobial properties, silver nanoparticles are potential activates to pathogens, which explains their potential for multiple applications in various fields. In the present studies, we evaluate the antimicrobial properties of a Sargassum polycystum algal extract, an unrivaled green synthetic method for producing -defined shaped seaweed silver nanoparticles. To confirm their structure and size, some characterization techniques are used, such as Absorption spectrophotometer (UV–VIS), Fourier transforms infrared spectroscopy (FTIR), Scanning electron Microscope (SEM), Transmission electron microscopy (TEM) and X-Ray diffraction (XRD). Evaluate the antibacterial and anti-mycobacterial activity using silver nanoparticles. The toxicity study of this silver nanoparticle has been done with the help of zebrafish larva. The biological nanoparticle having good antimicrobial activity against Staphylococcus aureus, Micrococcus luteus, Pseudomonas fluorescens and Candida albicans and also it shows potent activity against MTB H37Rv, SHRE sensitive MTB Rifampicin resistant MTB around 98%. Seaweed nanoparticles had lower toxicity for the survival of the fish larvae. In comparison, other dosages will arrest the cell cycle and leads to death. The present finding revealed that these seaweeds nanoparticles have potential anti-mycobacterial activity against pathogens at low concentrations. This makes them a potent source of antibacterial and anti-TB agents

Full text

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Silver nanoparticles synthesized
from the seaweed Sargassum
polycystum and screening for their
biological potential
Rajasekar Thiurunavukkarau1,6*, Sabarika Shanmugam2,6, Kumaran Subramanian1*,
Priyadarshini Pandi3, Gangatharan Muralitharan2, Maryshamya Arokiarajan1,
Karthika Kasinathan2, Anbarasu Sivaraj1, Revathy Kalyanasundaram1,
Suliman Yousef AlOmar4 & Velmurugan Shanmugam5*
World-wide antimicrobial resistant is biggest threat in global health. It requires the urgent need of
multisectoral action for the scientic community to achieve the sustainable development Goals. Due
to their antimicrobial properties, silver nanoparticles are potential activates to pathogens, which
explains their potential for multiple applications in various elds. In the present studies, we evaluate
the antimicrobial properties of a Sargassum polycystum algal extract, an unrivaled green synthetic
method for producing -dened shaped seaweed silver nanoparticles. To conrm their structure and
size, some characterization techniques are used, such as Absorption spectrophotometer (UV–VIS),
Fourier transforms infrared spectroscopy (FTIR), Scanning electron Microscope (SEM), Transmission
electron microscopy (TEM) and X-Ray diraction (XRD). Evaluate the antibacterial and anti-
mycobacterial activity using silver nanoparticles. The toxicity study of this silver nanoparticle has been
done with the help of zebrash larva. The biological nanoparticle having good antimicrobial activity
against Staphylococcus aureus, Micrococcus luteus, Pseudomonas uorescens and Candida albicans
and also it shows potent activity against MTB H37Rv, SHRE sensitive MTB Rifampicin resistant
MTB around 98%. Seaweed nanoparticles had lower toxicity for the survival of the sh larvae. In
comparison, other dosages will arrest the cell cycle and leads to death. The present nding revealed
that these seaweeds nanoparticles have potential anti-mycobacterial activity against pathogens at
low concentrations. This makes them a potent source of antibacterial and anti-TB agents
Tuberculosis (TB) is an infectious disease and is one of the top 10 causes of the death globally1. Mycobacterium
tuberculosis (MTB) is a causative agent for TB which aects lungs and also other parts of body except hair and
nail. TB transmitted to other individuals through the aerosols expelled by infected people when they cough
and the symptoms are cough, fever, night sweat and weight loss2. In many Middle- and low-income countries,
it would be a main cause of mortality and morbidity. Multi drug resistant and extensively resistant TB are the
major challenge in eective control of the disease in many regions3. TB treatment was usually take a prolonged
time of the treatment of the TB patients with Anti-TB drugs4. e emergence of multidrug resistance (MDR)
bacteria has prompted the development of new antibacterial medicines.
Silver Nanoparticles have been broad-spectrum, strong antibacterial activity against a variety of pathogens
due to their small size and wide surface area. Low quantities of AgNPs can eectively destroy bacterial and viral
pathogens such as E. coli, Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans, Aspergillus niger,
HIV, and the hepatitis B virus (HBV)5. Hence, we are searching for a novel well activated compound with lesser
side eects. e synthesis of eco-friendly NPs is urgently needed to replace toxic chemicals in various elds.
OPEN
1Centre for Drug Discovery and Development, Col. Dr. Jeppiaar Research Park, Sathyabama Institute of Science
and Technology Jeppiaar Nagar, Rajiv Gandhi Road, Chennai 600 119, India. 2Departmentof Microbiology, Centre
of Excellence in Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tami Nadu, India. 3Department
of Biotechnology, Mohamed Sathak College of Arts and Science, Chennai, Tamil Nadu, India. 4Department of
Zoology, College of Science, Kind Saud University, Riyadh 11451, Kingdom of Saudi Arabia. 5Madda Walabu
University, Robe, Ethiopia. 6
These authors contributed equally: Rajasekar Thiurunavukkarau and Sabarika
Shanmugam. *email: microraja09@gmail.com; kumarun23@gmail.com; velkas.cas@gmail.com
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Biosynthesized Silver nanoparticles arecost-eective and eco-friendly biocompatible agent that possess the poten-
tial for biomedical and pharmaceutical applications6. Microorganisms such as mushroom, bacteria and algae,
as well as plant extracts, contain enzymes, alkaloids, terpenoids, and phenolic compounds that can be used as
stabilizers and capping agents during the biological synthesis of NPs7.
Nanoparticles has antimicrobial mechanism of action is said to follow one of three models: development of
oxidative stress, metal ion release, or non-oxidative processes8. ese three dierent mechanisms can all take
place at the same time. According to certain research, Ag Nanoparticles cause the bacterial membrane’s surface
electric charge to be neutralised and alter its permeability, which ultimately results in bacterial death9. Addition-
ally, the production of reactive oxygen species (ROS) impairs the antioxidant defence system and breaks down
the cell membrane mechanically. e main mechanisms behind the antibacterial actions of NPs, according to
current research, are as follows: (1) bacterial cell membrane disruption; (2) production of ROS; (3) cell membrane
penetration; and (4) development of intracellular antimicrobial eects, including interactions.
Due to their relatively moderate side eects, marine resources are currently being intensively examined for
antibacterial and anticancer medication candidates8. Marine algae, such as Chlorophyta (green), Phaeophyta
(brown), and Rhodophyta (red), are considered highly potent renewable living marine resources, and their pro-
duction of NPs has piqued interest. Polysaccharides, proteins, carbohydrates, vitamins, pigments, enzymes, and
secondary metabolites, among other organic substances found in algae, provide additional potential for their
function in the production of Silver nanoparticles by acting as natural reducing agents11.
Seaweeds are mostly used in industrial purpose, but they not yet extensively globally, seaweeds are exploited
as the raw material for various industrial products, but they are not yet extensively imposing for nanoparticle
biosynthesis. In this there are lesser number of studies are available on the synthesis of silver nanoparticle
by seaweed and their antibacterial and anti-proliferative12, antifungal13 and anticancer14. Many seaweeds have
potentiality to work against various disease rather some have been tested for clinical trial and next for medicine
preparation. Some seaweed is known against human normal and multidrug resistant pathogens, to nd out the
best potent seaweed for synthesis of Silver Nanoparticle and its potential against the human pathogens. ere are
more than 841 seaweeds have been reported from the Indian coast having good potential over human pathogen.
Chemical methods have adverse side eect on human and environment hence, we are going for a biological
synthesis of silver nanoparticle.
In this paper we evaluated Sargassum polycystumseaweeds aqueous extract for the preparation of silver
nanoparticle through green synthesis method. e spectroscopic techniques that have been used for the char-
acterized the silver nanoparticles, such as absorption spectrophotometer (UV–VIS), Fourier transforms infrared
spectroscopy (FTIR), scanning electron Microscope (SEM), transmission electron microscopy (TEM) X-Ray
diraction (XRD)and Dynamic light scattering (DLS). e eect of nanoparticle was analyzed and performed
antimicrobial and anti-TB activity against various microbial pathogens. e toxicity of the synthesized silver
nanoparticle was monitored through zebra sh larva.
Material and methods
Ethical statement. e experiment was conducted in line with the norms and regulations of the Institu-
tional Ethical Committee (IEC) of Sathyabama Institute of Science and Technology (1793/PO/REBI/S/2014/
CPCSEA) Chennai. All animal experimental protocols were approved by the Sathyabama Institute of Science
and Technology’s Institutional Animal Ethical Committee (IAEC). e ARRIVE guidelines (https:// arriv eguid
elines. org/ arrive- guide lines/ exper iment al- proce dures) were followed throughout the project.
Microbial strains. Escherichia coli (MTCC1687), Bacillus subtilis (MTCC441), Klebsiella pnemoniae
(MTCC4030), Staphylococcus epidermidis (MTCC435), Vibrio cholera (MTCC0139), Pseudomonas uores-
cens (MTCC664), Micrococcus luteus (MTCC4821), Staphylococcus aureus (MTCC 96), Serratia marcescens
(MTCC86), were used to test antimicrobial activity. M. tuberculosis H37Rv, SHRE sensitive M. tuberculosis and
Rifampicin resistant M. tuberculosis were used for anti-mycobacterial activity. All studies were conducted at
Centre for drug discovery and development, Sathyabama Institute of Science and Technology (Deemed to be
University), Chennai.
Collection of samples. Seaweeds (Sargassum polycystum, Acanthophora spicifera, and Ulva fasciata)are
collected during the June–Aug 2018 at low tide and 5–6mm dept from the coastal regions of Mandapam (South-
east coast of India). Samples are identied by Seaweed Expert Dr. P. Anantharaman Dean & Professor CAS
in Marine Biology Faculty of Marine Sciences, Annamalai University Parangipettai-608 502. Marine seaweeds
were characterized using Common Seaweeds and Seagrasses of India, Herbarium Vol.1 authenticated by Central
Marine Fisheries Research Institute, (Indian Council of Agricultural Research), Kochi-682 018, Kerala, lndia.
Seaweed samples were taken directly and quickly washed in seawater to remove any foreign particles, sands, or
epiphytes. It was then placed in an ice box and carried to the laboratory, where it was properly washed with run-
ning tap water and then distilled water to eliminate any leover adhesive particles, salt, or dust. e seaweeds
were then spread out on blotting paper to absorb any excess moisture. It was dried in a dark room for 3–4days.
Using a mixer grinder, the dried components were ground to a ne powder.
Preparation of seaweed extract. 1g of dried seaweed powder was placed in 250ml conical ask and
dissolved with 100ml of distilled water. e mixture was boiled in a water bath for 20min at 65°C. e crude
extract was ltered throughWhatman Grade 1 Qualitative Filter Paper. en the ltered extract was centrifuged
for 10min at 1800rpm. Further the supernatant was stored at 4°C and used for further studies.
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Biological synthesis of silver nanoparticles. 10mL of seaweed aqueous extract was added to 90mL
(0.1mM)AgNO3 solutions and stored at room temperature for 24–48h in the dark. Aer Incubation that sam-
ples were puried by centrifugation for 13,000rpm for 20min and remove the supernatant and dispersion of
silver nanoparticles pellet in HPLC grade distilled water to remove the unbound particles for the future charac-
terization of silver nanoparticles.
Characterization of the silvernanoparticles. Formation of silver nanoparticles was conrmed by
Ultraviolet–visible spectral analysis. e absorbance spectra were recorded using Ultraviolet–visible spectros-
copy (UV-1800 Shimadzu UV spectrophotometer) at a wavelength of 200–800nm. Fourier transforms infrared
spectroscopy (Shimadzu, Japan) to nd out the feasible functional group in the bioactive compounds of the
seaweeds extract. Transmission electron microscopy was used to examine the size and form of the Ag NPs
(TEM). e morphological properties of the silver nanoparticles were studied using a scanning electron micro-
scope (SEM), and the crystalline nature, quality, and crystallographic determination of the silver nanoparticles
were determined using an X-ray diractometer. To calculate the polydispersity index (PDI) of nanoparticles,
silver nanoparticles were examined in dynamic light scattering (DLS). Dynamic light scattering (DLS) (Zeta-
sizer NanoR Model S90; Malvern Instruments, UK) was used to measure the dispersion, homogeneity, and
nanoemulsion size in order to calculate the polydispersity index (PDI) of nanoemulsions. ree copies of each
measurement calculation were made. A laser with a wavelength of 780nm and a scattering angle of 90° was used
to conduct measurements using dynamic light scattering (DLS) in the range of 0.1–1000m at 25°C.
Antibacterial activity of silver nanoparticles. e antimicrobial activity of synthesized silver nanopar-
ticle was tested against various pathogenic bacteria such as, Escherichia coli, Klebsiella pneumonia, Bacillus subti-
lis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis, Vibrio cholera, Serratia marcescens by
agar well diusion method. Each bacterial culture was grown in nutrient broth medium for 18h at 37°C. en,
each grown cultures were swabbed on nutrient agar medium and the well were cut about 5mm using cork borer.
Each well was added with 50µl of synthesized silver nanoparticles and all plates were incubated at 37°C for 18h.
Aer incubation, the plates were observing and the inhibited clear zone was measured and calculated15. e
minimal inhibitory concentration (MIC) of the green synthesized silver nanoparticle was observed against each
pathogenic bacteria using dierent concentration of nanoparticles. Based on the silver nanoparticle screening
results, the bacterial pathogens such as, Serratiamarcescens, Bacillus cereus, Escherichia coli, Micrococcus luteus
are used for further study. Among the bacterial strain the MIC wasobserved ranging from 16 to 256μg/ml.
Antimycobacterial activity. Luciferase reporter phage (LRP) assay was used to screen for anti-TB activity
of synthesised silver nanoparticles mediated by seaweed. About 100 µL of M. tuberculosis H37Rv cell suspension
(McFarland Unit 2) was added into 350 µL of sterile middlebrook 7H9 broth (Himedia) containing 50 µL of 32
synthesised silver nanoparticles at concentration of 100µg/mL. en it was incubated at 37°C for 72h. About
50 µL of mycobacteriophage (phAE202) and 40 µL of 0.1M CaCl2 was added and incubated. Aer4 h at 37°C,
100 µL of D-luciferin substrate was added to cell-phage mixture and relative light unit (RLU) was measured
immediately at 10s integration time in luminometer (Lumat 9508, Berthold, Germany). e percentage of RLU
reduction was calculated by following formula:
Nanoparticles showed more than 50% RLU reduction were considered as having anti-mycobacterial activity.
e same experiment was followed for sensitive M. tuberculosis and Rifampicin M. tuberculosis strains16.
Toxicity evaluation of silver nanoparticles against zebra sh. Zebra sh embryos were purchased
from the zebra sh aquarium in Kanchipuram district. For toxicity studies, 15 healthy post hatched zebra sh
were transferred to the wells of a 24-well plate along with 1ml of embryo water (60mg of sea salt/l of ultrapure
water). Dierent concentrations of silver nanoparticle (5, 10, 25, 50 and100 μg ml−1) were added to the wells and
incubated for 72h at 28.5°C. Tests were performed in duplicate and repeated thrice (60 embryos per concentra-
tion). Mortality of the zebra sh was noted aer 24, 48 and 72h. e embryos that appeared opaque and white
in colour. e dead embryos were degraded without any distinguished characteristic, whereas the structures of
intact embryos were more visible distinguished characteristic by 48h which allowed a clear distinction between
the dead and alive. e mortality rate was observed and calculated. e embryos were photographed using an
inverted phase contrast microscope (Olympus ckx41).
Results
Synthesis of silver nanoparticles. In this study we have performed green synthesis of silver nanoparti-
cles. e three dierent seaweeds extract were mixed with silver nitrate and incubated at room temperature. e
colour changes were monitored and observed. e experiments were performed with triplicate reaction. For-
mation Yellowish-brown colour conrmed the nanoparticle formation. ere was no colour change in Control
silver nitrate solution.
Analysis of the silver nanoparticles using UV-spectrophotometer. e synthesized silver nano-
particles were conrmed by using UV-spectrophotometer. Further conrmation of the formation of the silver
nanoparticles was determined by UV–vis spectrophotometer. UV–Vis spectrum was showed in Figs.1, 2 and 3.
Control RLU
−
test RLU/control RLU
×
100.
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e signicant observance of Sargassum polycystum, Acanthophora spicifera, and Sargassum wightii were showed
at 422, 429 and 411nm respectively. is absorption band called as surface plasmon resonance (SRP).
Fourier transforms infrared (FT-IR) spectroscopy analysis. FT-IR spectroscopic analysis of Sargas-
sum polycystum silver nanoparticles showed in Fig.4. e analysis was evaluated and conrmed attachment of
the functional group of the silver nanoparticles.
X-Ray diraction analysis (XRD) and dynamic light scattering (DLS). e crystallite nature of the
synthesized silver nanoparticles was determined by XRD. Silver nanoparticles of Sargassum polycystum XRD
showed in Fig.4B. XRD peak values appeared at13.75, 27.45, 33.33,38.44. in the 2Ɵ range of 0° to 80° parallel to
the characteristic’s diraction of the (111), (200), (311) and (222). X-Ray diraction showed that a silver nano-
particle of the Sargassum polycystum was crystalline in structure. Silver nanoparticle particle size distribution
were determined using dynamic light scattering methods conrm the presence of silver nanoparticles. Silver
nanoparticles average size as Fig.5.
Scanning electron microscope (SEM) and transmission electron microscopy (TEM) imaging
analysis of the silver nanoparticles. SEM and TEM imaging analysis was performed synthesized Sargas-
sumpolycystum synthesized nanoparticles (Fig.6A,B). It’s clearly indicated that nanoparticles are mostly cluster
and spherical in shape and the size is less than 100nm. Its denoted that formation of S. polycystum synthesized
silver nanoparticles.
Antibacterial and anti-mycobacterial activities of three dierent silver nanoparticles. Sar-
gassum polycystum silver nanoparticles showed potential activities against Staphylococcus aureus showed a
maximum 36 mm clear zone of inhibition, followed by Micrococcus luteus (35mm), Pseudomonas uorescens
(25mm), Serratia marcescens, Klebsiella pnemoniae and Bacillus subtilis (18mm), Escherichiacoli, Staphylococcus
epidermidis and Candidaalbicans (17mm) and Vibrio cholera (15mm) of inhibition in the antibacterial activi-
ties. Acanthophoras picifera nanoparticle showed maximum inhibition against Staphylococcus aureus (30mm)
followed by Micrococcus luteus (32mm), Pseudomonas uorescens (26mm), Bacillus subtilis (18mm), Klebsiella
pnemoniae, Escherichia coli, Staphylococcus epidermidis, Serratia marcescens and Vibrio cholera (17 mm) and
Candida albicans (15mm). Acanthophora spicifera sliver nanoparticles showed maximum inhibition against
Micrococcus luteus and Staphylococcus aureus (36mm), followed by Pseudomonas uorescens (24mm), Can-
Figure1. Biosynthesis of silver nanoparticles. (a) Before incubation. (b) Aer 48h of Incubation and (c) UV–
visible spectrophotometry of silver nanoparticles synthesized from extract of Sargassum polycystum.
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dida albicans (20mm), Escherichia coli (18mm), Serratia marcescens (16mm), Klebsiella pnemoniae (16mm),
Bacillus subtilis (17mm), Staphylococcus epidermidis (17mm) andminimum inhibition against Vibrio cholera
(14mm). Sargassumwightii silver nanoparticle showed maximum inhibition against Micrococcus luteus (30mm)
followed by Staphylococcus aureus (27mm), Pseudomonas uorescens (20mm), Klebsiella pnemoniae (16mm),
Escherichia coli and Candida albicans (15mm), Staphylococcus epidermidis (14mm), Bacillus subtilis and mini-
mum inhibition found in Serratiamarcescens (12mm) and there is no inhibition found in Vibrio cholera (Fig.7).
Minimum inhibition concentration (MIC). Based on the antibacterial activities Bacillus subtilis, Micro-
coccus luteus, Staphylococcus aureus, and Serratiamarcescens were chosen for the minimum inhibition assay. Sar-
gassum polycystum silver nanoparticles showed maximum 47% of inhibition found at 16µg/ml against Escheri-
chia coli, followed by Micrococcus luteus 21% of inhibition, Serratiamarcescens 12% and Bacillus cereus 11%
inhibition. Sargassum polycystum silver nanoparticle showed 15% inhibition against Candida albicans at 32µg/
ml.
Anti-mycobacterial activities. e anti-mycobacterial activity of Sargassum polycystum showed prospec-
tive activities against mycobacterium tuberculosis. S. polycystum showed 99.38%, 94.79% and 82.44% of inhibi-
tion against MTBH37Rv, MTB all drug sensitive and MTD MTB respectively. Acanthophora spicifera showed
72.31%, 98.42%and 97.96% inhibition against MTBH37Rv, MTBalldrug sensitive and MTD MTB respectively.
Sargassum wightii showed 42.17%, 98.75% and 97.89% inhibition against MTBH37Rv, MTB all drug sensitive
and MTD MTB respectively (Fig.8).
Toxicity evaluation of silver nanoparticles. We have used ve dierent concentrations of silver nano-
particles ranging from 5, 10, 25, 50 and 100 mg were used for evaluate the toxicity of nanoparticles. ere is no
signicant mortality was observed at 5mg of the Silver nano particles of Sargassum polycystum showed a less
toxicity in zebra sh larvae (Fig.9).
Figure2. Biosynthesis of silver nanoparticles. (a) Before incubation. (b) Aer 48h of Incubation and (c) UV–
visible spectrophotometer of silver nanoparticles synthesized from extract of Acanthophora spicifera.
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Discussion
e chemical reduction process has been used to create silver nanoparticles with success. Visual observation
of the production of silver nanoparticles revealed colouring (yellowish). e sample’s development of a yellow-
ish colour is an indication that silver nanoparticle particlesdominated the synthesis process, which resulted in
colloidal nanoparticles17. In our present studies also showed that the three seaweeds extract showed a colour
change aer mixing mixed with silver nitrate. Aer incubation colour change was observed. e conrmation
in the experiments was of the formation Yellowish-brown colours. ere was no colour change in control silver
nitrate solution. Silver nanoparticles were conrmed by using UV-spectrophotometer.
It was noted that the colour of the solution changed from colourless to yellowish-brown with the addition of
marine macroalgae extracts, indicating the biogenesis of AgNPs. e aqueous medium’s obvious colorlessness
clearly suggests that extracellular reduction of Ag+ ions had not taken place. It is widely known that the generation
of smaller-sized NPs and reduction processes both depend greatly on the alkaline pH18. Secondary conrmations
of the formation of the silver nanoparticles were determined by UV–VIS spectrophotometer. It showed single
strong absorption at 422,429 and 411nm in UV–Vis spectrum of Sargassum polycystum, Acanthophora spicifera,
and Sargassum wightii respectively. is absorption band called as surface plasmon resonance (SRP). UV–Vis
spectra of AgNPs formed by the extracts of dierent algalspecies. Absorption peaks of AgNPs capped by U.
rigida, C. myrica, and G. foliifera appearedat 424nm, 409nm, and 415nm, respectively. ese types of UV–VIS
spectroscopy absorption peak obtained by Algotiml etal.19. ey reported that silver nanoparticles showed a
peak value at 422 and 425nm for Codiumcapitatum seaweeds of dried and fresh respectively. is type of sur-
face Plasmon vibration with characteristic peaks of silver nanoparticles was prepared by chemical reduction20.
e last ten years have seen a surge in interest in the eld of catalytic processing of algal biomass. Future
economic growth from algae should be signicant if ecient upstream and downstream processing can be cre-
ated. Algae are well known for their ability to transform into more pliable forms and to hyper-accumulate heavy
metal ions21. Due to their ability to reduce metal ions, the creation of nanoparticles from a variety of algal sources
has emerged as one of the most cutting-edge and current elds of biochemical study22. In the realm of materials
science, green synthesis has emerged as a dependable, long-lasting, and environmentally friendly method for
the synthesis of several nanomaterials, including metal oxides, hybrids, and bio-inspired materials23. Metallic
nanoparticles have fascinated scientists for more than a century and are now widely used in engineering and
the health sciences24.
Sivakumar etal. reported the silver nanoparticles from seaweed samples were found to have antibacterial
activity. e zone of inhibition in the plate demonstrated that silver nanoparticles made from seaweed samples.
Staphylococcus aureus was shown to be more vulnerable to silver nanoparticles than gram negative bacteria by
Figure3. Biosynthesis of silver nanoparticles. (a) Before incubation. (b) Aer 48h of Incubation and (c) UV–
visible spectrophotometer of silver nanoparticles synthesized from extract of Ulva fasciata.
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comparing the zones of inhibition between these bacteria (Salmonella typhi). Compared to those from T. conoides
and H. macroloba, silver nanoparticles from S. lamentosa had a noticeably higher activity against Staphylococ-
cusaureus. Silver nanoparticles from T. conoides and S. lamentosa have much stronger anti-Salmonella activity
than those from H. macroloba25. In our present studies also supported that silver nanoparticles from marine
seaweeds showed the potential activities human pathogens.
Fourier transforms infrared spectroscopy analysis of Sargassum polycystum silver nanoparticles showed in the
Table1. Fourier transforms infrared spectroscopy analysis evaluated the functional group of the silver nanoparti-
cles. Fourier transforms infrared spectroscopy (FTIR) spectrum was used to evaluate the functional or bio mol-
ecules in green synthesis of silver nanoparticles using Sargassum polycystum. e peaks 3888.62 cm−1 indicating
the presence of Alcohol (O–H), than 3395.99 (cm−1) indicating the presence of Stretching of Amide II (N–NH),
followed by 2921.93 (cm−1) indicating the Aklanes (–CH2–), 2129.99 (cm−1) demonstrating Alkyanes (–C–C)
Figure4. (A) Analysis of Silver nanoparticles using FTIR spectrum of Sargassum polycystum. (B) Analysis of
Silver nanoparticles using X-Ray Diraction of Sargassum polycystum.
Figure5. Analysis of Silver nanoparticles using DLS of Sargassum polycystum.
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Figure6. (A) Analysis of Silver nanoparticles using SEM of Sargassum polycystum. (B) Analysis of Silver
nanoparticles using TEM of Sargassum polycystum.
Figure7. Antimicrobial activity of silver nanoparticles (1. Sargassum polycystum, 2 Acanthophora spicifera, and
3 Ulva fasciata). 1. Bacillus subtilis, 2. Serratia marcescens, 3. Pseudomonas uorescens, 4. Staphylococcus aureus,
5. Escherichia coli, 6. Candida albicans, 7. Micrococcus luteus, 8. Vibrio cholerae, 9. Staphylococcus epidermidis,
10. Klebsiella pnemoniae.
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group, band was absorbed at 1582.96 (cm−1), is due to the presence of aromatic ring (C=C) Most of the marine
seaweeds contain high amount of phenolic and avonoids. is type of similar type of compounds reported in
the seaweeds of Caluerpataxifolia silver nanoparticles26.
en XRD used to conrm the particles as silver and know the structural information. X-ray diraction was
one of the important characterizes the silver nanoparticles of Sargassum polycystum. Silver nanoparticles crystal-
lite nature determined by XRD. XRD peak values appeared at 13.75, 27.45, 33.33, 38.44. in the 2Ɵ range of 0° to
Figure8. Anti-Mycobacterial activity against silver nanoparticle Seaweeds.
Figure9. Toxicity study of silver nanoparticles of Sargassum polycystum zebra sh embryo.
Table 1. Fourier transforms infrared spectroscopy analysis.
Peak values Functional groups
3888.62 (cm−1) Alcohol (O–H),
3395.99(cm−1) Amide II (N–NH)
2921.93 (cm−1) Aklanes (–CH2–)
1582.96 (cm−1) Aromatic ring (C=C)
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80° parallel to the characteristics diraction of the (111), (200), (311) and (222). X-Ray diraction showed that
a silver nanoparticle of the Sargassum polycystum was crystalline in structure.
Sivakumar etal.26 reported that silver nanoparticles of the Halimeda macroloba, Turbinaria conoides and
Spyridia lamentosa. XRD spectrum may be used to determine the precise nature of the silver particles that
were produced. e broadness of the X-ray diraction peaks at their bottoms suggests that the silver particles
are nanoscale. e X-ray diraction lines’ peak spreading at half their maximum intensity is caused by crystal-
lite size, attening and micro-strainsinside the diracting domains. e cubic silver’s crystalline planes’ unique
diraction peaks were revealed by XRD investigation (Ag). ere were peaks in all three samples, which were
noted at 111, 200, 220 and 311. It can be denoted that for the four samples, the values of were 28°, 32°, 41.5° and
49.5°. Analysis of Silver nanoparticles using SEM result showed that silver nanoparticles are less than 100nm in
size. Average diameter of the silver nanoparticles was between 10 and 85nm in size. TEM analysis of the silver
nanoparticles showed that Sargassum polycystum are mostly spherical in shape.
Researchers mostlyuse the TEM and SEM to show how dierent sizes of nanoparticles evolve. In order to
reduce charging artifacts and quick radiation damage to biomaterials during the imaging process, SEM imaging
requires a thorough preparation step that is frequently nished by metal coating. Additionally, for improved
identication, SEM and TEM are combined with an electron diraction (EDX) equipment. In order to prepare
samples, carbon copper grids are coated with a metal nanoparticle solution, dried, and then ready for measure-
ment. e crystalline structure of nano-metals will be revealed via an X-ray diraction (XRD) investigation.
EDS (energy-dispersive spectroscopy) is another tool that is frequently used to detect the presence of metal27.
EDS systems are oen incorporated with SEM or EPMA equipment. An EDS system consists of a sensitive x-ray
detector, a liquid nitrogen dewar for cooling, and soware for gathering and analysing energy spectra. ese
techniques make it simple for researchers to quantify the structural properties of nanomaterials.
We used 5 dierent concentrations of silver nanoparticles such as 5mg, 10mg, 25mg, 50mg and 100mg
used for the toxicity studies. In these studies, 5mg of the Silver nanoparticles of Sargassum polycystum showed
a less toxicity in zebra sh larvae (Supplementary TableS1).
Conclusion
Green synthesis of macro algae coated silver particle showed signicant antibacterial and anti-mycobacterial
activity. Among three dierent types seaweeds Sargassum polycystumcoated synthesized silver nanoparticles
showed the signicant activity against pathogens . e silver nanoparticles were characteristic with various
spectroscopic analyses. ere is no or less toxicity was observed against Zebra sh. It could act as alternative
agent for antibacterial and anti-mycobacterial activity.
Data availability
All the images given in the article are obtained based on experimental data. None of the images were reproduced
from other sources. e datasets used and/or analysed during the current study available from the correspond-
ing author on request.
Received: 19 January 2022; Accepted: 10 August 2022
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Author contributions
R.T. was supervision, editing and the entire manuscript was draed; S.S. were collected the sample and done the
experiment work. K.S. revising the manuscript and experimental guidance for carrying out the research work;
P.P. revising the manuscript. M.G. was done proofreading and corrections. M.A.: helping for experimental and
gure and data arrangement; K.K. were collected the sample and done the experiment work. A.S. revising the
manuscript and experimental guidance for carrying out the research work. R.K. Helping for experimental and
gure and data arrangement; S.Y.A. and V.S. revising the manuscript and proofreading. All authors read and
approved the manuscript.
Funding
is project was supported by the Researchers Supporting Project number (RSP-2021/35), King Saud University,
Riyadh, Saudi Arabia.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 022- 18379-2.
Correspondence and requests for materials should be addressed to R.T., K.S.orV.S.
Reprints and permissions information is available at www.nature.com/reprints.
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