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Green Synthesis of Silver Nanoparticles from
Salvia
Aethiopis
L. and their Antioxidant Activity
Esma Nur Gecer ( esmanurgecer@hotmail.com )
Tokat Gaziosmanpaşa University: Tokat Gaziosmanpasa Universitesi
Research Article
Keywords: Salvia aethiopis L., nanoparticles, antioxidant activity, natural products, spectroscopy.
Posted Date: April 30th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-456844/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Salvia aethiopis
L. was heated in distilled water for 2 hours. After ltration, water extract was treated with
silver nitrate for 2 hours at 60°C to yield the silver nanoparticles (Sa-AgNPs). The structure of silver
nanoparticles was elucidated by spectroscopic methods such as Ultraviolet-visible (UV-Vis), Fourier
transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and Scanning electron microscope
(SEM). The maximum absorption in UV-Vis spectrum was observed at 508 nm. XRD pattern at (
2
θ) 38.1°,
44.3°, 64.4°, and 77.4° degrees can be assigned to the (111), (200), (220) and (311) Bragg’s reections of
the face-centered cubic crystalline structure. The average size of Sa-AgNPs was found as 74.09 nm by
SEM analysis. The characteristic hydroxyl vibration signal appeared at 3222 cm
− 1
. Antioxidant activity of
extract and Sa-AgNPs were carried out using DPPH
•
, ABTS
•+
FRAP assay. The Sa-AgNPs revealed the
considerable ABTS
•+
scavenging effect with the value of 4.93 (IC
50
, µg/mL) compared to BHT (IC
50
,
µg/mL, 8.34). However, Sa-AgNPs displayed the lower DPPH
•
activity (IC
50
, µg/mL, 24.37) than that of the
standard BHT (IC
50
, µg/mL, 9.67). The reducing power activity of Sa-AgNPs was found as 4.52 (µmol
TE/mg extract) while the standard BHT value was 488 (µmol TE/mg extract).
1 Introduction
Nanotechnology is a science that has developed rapidly in recent years and has a wide range of
applications [1]. Nanoparticles are basic building blocks with different properties due to their large
surface area/volume ratio. In recent years, nanoparticles have formed the basis of modern materials
science [2]. Silver nanoparticles (AgNPs) gain great interest in biology, biomedical, drug delivery, medicine,
agriculture, food industries, textile industries, and electronics [3].
Several synthetic routes have been developed to produce AgNPs including electrochemical, radiation
technique [4], and photochemical [5]. However, these methods lead to environmental contamination, toxic
residue, and high cost. Therefore, natural products gain great interest in the synthesis of nanoparticles
due to their eco-friendly, low cost, high eciency, and scale-up properties [6, 7].
Natural products such as plant extract, microorganisms, algae, oilcake, vegetable waste, seaweed,
enzymes, arthropods have been used for the production of AgNPs. It has been accepted that plant-based
materials are the promising substrate for the AgNPs synthesis due to the corresponding advantages [8].
The biological effects of AgNPs depend on some crucial factors such as surface chemistry, size, shape,
particle morphology, particle composition, coating/capping, agglomeration, and dissolution rate, particle
reactivity in solution, the eciency of ion release, cell type, the type of reducing agent [9]. AgNPs
synthesised from plants were reported to show signicant biological activities such as antioxidant [10],
antibacterial [11], anticancer [12, 13], antifungal [14], antiviral [15], anti-inammatory [16].
Silver nanoparticles were synthesised from
Salvia leucantha
that revealed considerable antibacterial
activity [17].
Salvia ocinalis
is well known of
Salvia
genus. Silver nanoparticles synthesised from
S.
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ocinalis
was reported to display signicant antibacterial [18], antiplasmodial [19], antioxidant and anti-
inammatory [20], antileishmanial effects [21].
Free radicals are called reactive oxygen species including hydroxyl (OH
•
), peroxyl (ROO
•
), superoxide
(O
2
•
), peroxinitrite (
•
ONOO
−
) radicals that were produced throughout oxidation within the mammalian
body [22]. The human body has many protection systems against oxidative stress. The natural
antioxidants become insucient in some situations and then, the excess radicals can damage to cell
membrane resulting in diseases [23]. Therefore, food including antioxidants should be consumed to cope
with this situation. An antioxidant is dened as a substance that inhibits the oxidation of the substrate
[24]. Accordingly, phenolic compounds are produced from the secondary metabolism of plants and are
considered natural antioxidants because they protect many organs from oxidation [25]. There has been
an increase in the use of natural antioxidants due to the benets provided by the aromatic herbs of
extracts, essential oils, and spices [26]. Herbal-based products contain phenolic phytochemicals, one of
the most powerful antioxidants, and contribute to body defense against oxidative damage. These
compounds protect against deterioration and provide antioxidant substances to the human body
because of their consumption[27–29].
Salvia
L. species have been used since ancient times due to their antioxidant, natural preservative, spice,
aromatic substance, and medicinal properties [30].
Salvia
is an important genus belonging to the
Lamiaceae (formerly Labiatae) family. Around the world, 1000 species of
Salvia
are used as herbal tea
and avoring, as well as in the cosmetics and pharmaceutical industries.
Salvia
species have been used
in the treatment of colds, coughs, toothache, gastrointestinal problems, coronary heart disease,
hepatochirosis, hepatitis, cerebrovascular disease, chronic renal failure, dysmenorrhea, and neurasthenic
insomnia as traditional medicine [31]. In addition,
Salvia
herbs are known to have a wide variety of
pharmacological activities such as antimicrobial, antioxidant, anti-inammatory, anticancer,
hypoglycemic, hypolipidemic, antinociceptive, memory-enhancing effects.
Salvia
genus is rich in
polyphenols, especially phenolic acid, and avonoids [32, 33].
In this study,
Salvia aethiopis
mediated synthesis of silver nanoparticles was achieved and antioxidant
activity of corresponding Sa-AgNPs was carried out.
2 Materials And Methods
2.1 Chemicals
Butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), silver nitrate (AgNO
3
), 1,1-diphenyl-2-
picryl-hydrazyl (DPPH), and solvents with analytical grade were purchased from Fluka and Sigma-Aldrich
Chemicals.
2.2 Plant materials
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Salvia aethiopis
L. was obtained from Tokat Gaziosmanpasa University Aromatic and Medicinal Plant
Field.
2.3 Synthesis of silver nanoparticles
Salvia aethiopis
leaves were powdered by grinder and powder material (50 g) was heated with distilled
water (200 mL) at 50°C for 2 hours. After ltration with Whatman lter paper, silver nitrate distilled water
solution (0.037 mM, 200 mL) was added to the extract solution slowly. The reaction mixture was heated
at 60°C for 2 hours. The color change from yellow to brown was observed. After completion of the
reaction, Sa-AgNPs were obtained by repeated centrifugation at 5000 rpm for 20 minutes then washed
thoroughly with distilled water. The Sa-AgNps were dried by lyophilisation [7].
2.4 Characterization of silver nanoparticles
The UV-vis spectra were recorded on Hitachi U-2900 spectrophotometer. The maximum absorption was
detected at 508 nm. XRD measurement was carried out on an Empyrean, Malvern Panalytical
diffractometer, the operation voltage of 45 kV at a 40-mA current strength. The crystallographic structure
of Sa-AgNPs was determined by the XRD pattern. The diffracted intensity was carried out in the region of
2θ from 20º to 90º at 0.02º/ min. The particle size was calculated by dynamic light scattering (DLS) on a
Delsa Nano C instrument. The Sa-AgNPs properties were determined by Scanning Electron Microscope
(SEM) on Quanta Feg450. EDAX detector and EDX were used to determine the elemental analysis. quanta
450 FEG was used for surface and point analysis.
2.5 Antioxidant activity
2.5.1 DPPH
•
free radical scavenging assay
DPPH
•
free radical scavenging effect of
S. aethiopis
mediated silver nanoparticles and the extract was
carried out according to the procedure described in the literature [29]. DPPH
•
radical (1.0 mL, 0.26 mM)
was treated with the Sa-AgNPs (3.0–30 µg/mL) at room temperature (rt) for 20 minutes. During the
reduction, the solution color fades, and the absorbance decreases. BHT, BHA and Trolox were used as
standard compounds. The equation was used for the calculation of DPPH
•
scavenging effect (1)
DPPH
•
scavenging effect (%) = [(A
1
– A
2
) / A
1
] × 100 (1)
A
1
is the absorbance of the control and A
2
is the absorbance of the sample.
2.5.2 ABTS
•+
radical cation activity
ABTS
•+
radical cation assay is based on the ability of antioxidants to reduce ABTS
•+
(blue/green) to
ABTS
− 2
(colorless). ABTS radical cation solution was produced by the reacting of 7.0 mM ABTS with
K
2
S
2
O
8
(2.45 mM) at a ratio of 2/1 (v/v), the mixture could stand in the dark at rt for 12 h. After adjusting
pH by treatment of ABTS
•+
solution with phosphate buffer (0.1 mM, pH 7.4), Sa-AgNPs were treated with
ABTS
•+
(1.0 mL) at several concentrations (3.0–30 µg/mL). The absorbance measurement was executed
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at 734 nm and ABTS concentration was calculated by the calibration curve. ABTS
•+
effect was calculated
by the given Eq.(2):
ABTS
•+
scavenging effect (%) = [(A
1
– A
2
) / A
1
] × 100 (2)
in which, A
1
is ABTS
•+
initial concentration and A
2
is ABTS
•+
remaining concentration in the sample [30].
2.5.3 Reducing power
Reducing power of extract and Sa-AgNPs were measured according to previously published method.[22]
Reducing power was calculated from the calibration curve of ascorbic acid and presented as µg/ml of
extract or silver nanoparticles. The samples (extract and Sa-AgNPs) were mixed with 200 mM of sodium
phosphate and volume was adjusted to 1.25 mL with water, followed by the addition of potassium
ferricyanide, K
3
Fe(CN)
6
(1.25 mL, 1%). Later, the mixture was incubated for 20 min at 50°C, and then 1.25
mL of 10% trichloroacetic acid was added and then thoroughly vortexed. An aliquot (1.0 mL) was mixed
with water (1.0 mL) and ferric chloride (0.5 mL, 0.1%) and then vortexed. The absorbance was measured
at 700 nm against a blank using a spectrophotometer [31]. The high absorbance value revealed the high
reducing activity.
2.6 Statistical analysis
GraphtPad Prism software (version 8.0.1), one-way ANOVA with Tukey’s multiple comparisons test was
used for statistical analysis. The results were stated as mean values ± standard deviation (P < 0.05).
3 Results And Discussion
3.1 Synthesis of silver nanoparticles
The silver nanoparticles were synthesized using
Salvia aethiopis
leaves. The plant material was heated in
distilled water, after removal of the solid part, the extract solution was treated with the silver nitrate
solution. The secondary metabolites in the water solution reduced the Ag
+
to Ag
0
. Afterward, Ag atoms
were capped and stabilized by secondary metabolites that the plant synthesized. The color change of the
reaction mixture from dark yellow to dark brown conrmed the formation of Sa-AgNPs (Fig.1).
In the reaction mechanism (Fig.2), the silver ions were reduced by bioactive compounds that oxidized.
After the stage of ion reduction, clustering, and growth of nanoparticles, the silver nanoparticles formed.
Since
Salvia
species include luteolin, the reaction mechanism was showed for this compound.
3.2 UV-Vis spectral analysis
The maximum absorption at 508 nm at UV-vis spectrum revealed the formation of the silver
nanoparticles (Fig.1). The UV-Vis spectroscopy is mostly used for the identication of silver
nanoparticles. Free electrons in metal nanoparticles yield a surface plasmon resonance absorption band.
