Applied Nanoscience

https://doi.org/10.1007/s13204-019-01059-5

ORIGINAL ARTICLE

Phytochemical investigation and phytosynthesis of eco‑friendly stable

bioactive gold and silver nanoparticles using petal extract of saffron
(Crocus sativus L.) and study of their antimicrobial activities
Omid Azizian‑Shermeh1   · Moharam Valizadeh1 · Mozhgan Taherizadeh2 · Maryam Beigomi3

Received: 28 December 2018 / Accepted: 18 May 2019

© King Abdulaziz City for Science and Technology 2019

Abstract
In this study, aqueous extract of saffron petal was used to synthesize gold nanoparticles (GNPs) and silver nanoparticles
(SNPs). The antioxidant activity, phenol, flavonoids, anthocyanin, and carotenoids were measured in methanol, ethanol, and
water extracts to assess the reducing potential. After extracting, for the phytosynthesis of GNPs and SNPs, the extract was
added to the solutions of gold (III) and silver nitrate at a concentration of 1 mM. The effective parameters for the synthesis,
i.e., pH of reaction solution, extract volume, metal ion concentration, and reaction time, were optimized by UV–Vis spec-
troscopy technique, and the nanoparticles (NPs) were characterized by transmission electron microscopy (TEM) and X-ray
diffraction (XRD). Antimicrobial activities of extracts, GNPs, and SNPs were investigated against Staphylococcus aureus,
Bacillus cereus, Escherichia coli, Aspergillus, and Candida albicans. The results shown that the aqueous extract although
exhibiting less antioxidant activity than other extracts, but have a high potential for reducing and stabilizing of metal ions
to synthesis NPs. In addition, the NPs had a uniform spherical shape with an average of 17–22 nm for GNPs and 10–14 nm
for SNPs. These sizes are useful for inhibiting many bacteria, so that according to the results of this study, phytosynthesized
nanoparticles could inhibit all bacteria that had used, and this inhibitory effect on SNPs was greater than GNPs. According
to the results of this study, plants are high potential for the synthesis of metal NPs, which can be used as an antibiotic to
inhibit many pathogenic bacteria and fungi.

Keywords  Saffron · Gold nanoparticles · Silver nanoparticles · Antioxidant activity · Antimicrobial activity

Introduction
Due to the ability to produce secondary metabolites such as
terpene compounds, polyphenols, and alkaloid, the medi-
cal herbs have has been one of the most important sources
of food and medicine in preventing and treating illnesses
(Ghasemi et al. 2011). Antioxidants are compounds that
significantly linger the oxidation of the substrate or prevent
it. These are biologically active compounds that protect
* Omid Azizian‑Shermeh
omid_aziziyan@yahoo.com
1
Medicinal and Ornamental Plant Research Center, University
of Sistan and Baluchestan, Zahedan, Iran
2
Department of Chemistry, Faculty of Basic Sciences,
Kerman Branch, Islamic Azad University, Kerman, Iran
3
Food and Drug Deputy, Zahedan University of Medical
Sciences, Zahedan, Iran
the body from damage caused by active species that lead
to diseases (Zaveri 2006). Antioxidant compounds such as
phenolic compounds inhibit free radicals (Silva et al. 2004).
The presence of these free radicals can cause damage to
cells and tissues due to the tendency to react with other
molecules of the body, leading to diseases such as diabetes,
cancer, arthritis, heart disease, anxiety, and premature aging
(Nagendrappa 2005). Phenolic compounds are common
secondary metabolites in plants that not only have physi-
ological functions in plants, but also have a positive effect
on human health. These compounds also provide important
colorants, flavors and aromas of fresh fruits, vegetables, and
herbs, which, in addition to playing the antioxidant role,
have anti-mutation or anti-cancer, anti-inflammatory, and
antimicrobial properties (Eberhardt et al. 2000). Flavonoids
are a group of phenolic compounds with the ability to inhibit
free radicals and inhibit oxidative enzymes to incur pain kill-
ing and anti-inflammatory properties. These combinations,
just as phenolic compounds, are considered to be antioxidant

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compounds (Zhou et al. 2000). In other words, the antioxi-
dant effects of plants are partly attributed to the presence
of phenolic and flavonoids combinations which occur in
all parts of the plant with different proportions (Wolniak
et al. 2007). Extensive studies have shown the antioxidant,
antimicrobial, and anti-fungal effects of plants to be due to
phytochemical and secondary compounds such as phenolic,
flavonoids, and carotenoids ones. In the last few decades, the
preparation and study of NPs have attracted the attention of
scientists in various fields of applied sciences (McNeil and
Leukoc 2005). Nanotechnology means the study of materials
at the atomic, molecular, and macromolecular scale, which
leads to the manipulation of material construction units and
the transforming them into a nanoscale scale (1–100 nm)
(Wang et al. 2012). The production of NPs using chemical
methods not only imposes high costs, but also has many
environmental contaminations and the physical methods
used to produce NPs, not only are difficult, but also are
low-efficiency processes (Mohanpuria et al. 2008). In addi-
tion, in physical and chemical methods, the existence of a
small amount of NPs’ synthesis precursors on the surface
of NPs poisons the human body. The disadvantages men-
tioned above have created a new approach to nanotechnology
called “green chemistry”. Green chemistry is a non-toxic and
environmentally friendly method for the synthesis of NPs, in
which biological, instead of physical and chemical, methods
are used for the synthesis of metal NPs. One of the most
important goals of green chemistry is to obtain NPs, which
have the highest compatibility with human body. Recently,
the use of biological methods [use of fungi (Gajbhiye et al.
2009), bacteria (Shahverdi et al. 2007), biomass and plant
extracts (Shankar et al. 2004)] have been brought into atten-
tion due to being simple, low-cost, highly efficient, non-
toxic, and environmentally friendly. In biological synthesis
of NPs, the use of plant extracts, in addition to the above-
mentioned reasons, is of paramount importance due to acces-
sibility, completeness of the reaction, short reaction time, the
production of NPs with different shapes and in a uniform
manner, and particle size homogeneity (Ahmed et al. 2016).
Metallic NPs are designed specifically for appropriate elec-
tron, catalytic, and optical properties, and due to the pres-
ence of these properties, they are used in different fields such
as sensors and catalysts manufacturing. Metallic NPs such as
gold, silver, copper, etc. have been widely studied for their
unique properties, such as: surface plasmon resonance, opti-
cal properties, and antimicrobial activity (Song et al. 2006).
Meanwhile, GNPs and SNPs have attracted the attention of
researchers more than other metal NPs. Non- cytotoxicity
of gold NPs for healthy tissues, ease of production, nucleus
size, surface-to-volume ratio, transmission and adjustment
of drug delivery processes, and high antimicrobial activity
of SNPs make these NPs a suitable candidate for therapeu-
tic and diagnostic applications (Faghri Zenooz and Solouti
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Applied Nanoscience
2011). Several studies have reported the synthesis of GNPs
and SNPs using plant extracts. In studies on the synthesis
of GNPs and SNPs by plants, the production of these NPs
has been reported from ginger (Praveen Kumar et al. 2011),
Hibiscus rosa sinensis (Vijayaraghavana et al. 2011), Che-
nopodium album (Dwivedi et al. 2010), Neurospora crassa
(Castro-Longoria et al. 2011), and Thevetia peruviana Juss
(Oluwaniyi et al. 2016). Saffron is known, scientifically, as
Crocus sativus L. and belongs to the iris family. Saffron is
a valuable herb that is also known as the Red Gold. This
plant has many uses in various pharmaceutical, food, and
textile industries (Hadizadeh et al. 2010). Unfortunately, in
Iran, only the stigma of the saffron plant is used, and the
other of its parts (petals and flags) is considered as agri-
cultural wastes and, despite the high volume of production,
is disposed. Therefore, in this study, the phenolic, flavo-
noid, anthocyanin content and antioxidant, and antimicro-
bial activities of various extracts of petals of saffron were
studied. Then, using its aqueous extract as a reducing and
stabilizing agent, the synthesis of GNPs and SNPs and their
antimicrobial activities were investigated.
Experimental
Collection of plant
The fresh petals of saffron were collected from farmland
in Kashmar in Iran in September 2017. After separating
various parts (flowers and flags) and placing in a room at
room temperature and away from direct sunlight, the petals
were dried and then completely grinded by an electric mill.
Chemical materials and microorganisms
All the chemical materials used in this study were pre-
pared with high purity. Reagents for folin, gallic acid,
sodium carbonate, iron sulfate, sodium acetate, sodium
sulfate, 2,4,6-tripyridyl-s-triazine (TPTZ), ethanol,
methanol and dimethyl sulfoxide, sodium hydroxide,
hydrochloric acid, and silver nitrate were bought from
Merck, 2,2-diphenyl-2-picryl hydrazide (DPPH), butyl-
ated hydroxytoluene (BHT), gold salt (­ HAuCl 4·3H 2O),
and quercetin were bought from Sigma-Aldrich, and the
microorganisms used, including Staphylococcus aureus
(PTTC 1112), Bacillus cereus (PTTC 1154), Escherichia
coli (PTTC 1399), and Aspergillus niger (PTTC 5012) and
Candida albicans (PTTC 5027) were supplied by the Ira-
nian Research Organization for Science and Technology
(IROST). Double-distilled water was used as solvent and
for washing.
Applied Nanoscience
Preparation of extracts
The extraction was performed by maceration method (Tru-
sheva et al. 2007). To prepare the extracts for phytochemical
studies, we added 10 g of dried powdered petals of saffron to
100 ml of different solvents (methanol, ethanol, and double-
distilled water) and the solution was agitated for 24 h on a
shaker. The obtained samples were filtered by the What-
man 42, and every extracts were centrifuged at 10,000 rpm
for 20 min to completely remove the suspended particles.
The solvents were then allowed to evaporate. To prepare
the extract for the synthesis of gold and silver NPs, 1 g of
powder was stirred with 100 ml of distilled water and heated
for 30 min at 50 °C (heater). After cooling, the filtration
process was performed according to the previous step. Due
to the high light, heat, and oxygen sensitivity of the extracts
obtained for phytochemical studies and the synthesis of gold
and silver NPs, they were enclosed and refrigerated at 4 °C
for analysis.
Evaluation of antioxidant activity by DPPH
The antioxidant activities of the extracts were evaluated
by the radical scavenging capacity measuring method by
the 2-2,diphenyl-1-picryl-hydrazide (DPPH) stable radical
(Einali et al. 2018). At first, a concentration of 1000 μg/
ml was prepared from all extracts and was added to 1 ml
DPPH methanolic solution at a concentration of 0.1 mM.
After 30 min at room temperature, the absorbance of the
samples was read at 517 nm in front of blank. The inhibition
percentage of free radicals was calculated using the follow-
ing formula:
( )
%IP = Acontrol − Asample ∕Acontrol × 100,
where %IP is the inhibition percentage of free radicals
(percentage of antioxidant inhibition against free radicals),
Acontrol is the control absorbance (containing 1 ml of metha-
nol in 1 ml of the DPPH solution), and ASample is the sample
absorbance (containing various volumes of plant extract
(antioxidant), methanol, and DPPH solution).
In addition, butylated hydroxytoluene (BHT) and ascorbic
acid were used as positive control.
Evaluation of the ferric (III) ion‑reducing antioxidant
power (FRAP)
To measure the ferric (III) ion-reducing antioxidant power,
the method by Benzie and Strain was used with some modifi-
cations (Sadeghi et al. 2015). 1.8 ml of fresh FRAP solution
was added to 200 μl of extracts (freshly FRAP solution was
prepared by dissolving 10:1:10 ml of acetate buffer, TPTZ
solution, and ­FeCl3.6H2O solution before the test). The above
mixture was placed at 37 °C for 5 min. Then, the absorbance
of the solutions was read by the UV–Vis spectrophotometer at
595 nm. Reducing activity of the extracts was calculated using
standard curve in mM of ferric (III) ion/mg of sample dry
weight. Butylated hydroxytoluene (BHT) and ascorbic acid
were used as positive control.
Determination of the total phenolic content
Total phenol content was measured using Folin–Ciocalteu rea-
gent. 0.5 ml of each extracts was added to 2.5 ml of Folin–Cio-
calteu reagent %10, and then, after 5 min, 2 ml of sodium car-
bonate solution 5% were added to this solution. The solution
was placed for 30 min in a dark place at room temperature.
Subsequently, the absorbance of the samples was read by the
UV–Vis spectroscopy at 765 nm (Einali et al. 2018). Gallic
acid was used as the standard for drawing the calibration curve,
and the total phenol content was reported based on the equiva-
lent of mg of gallic acid per g of the extract.
Determining of the total flavonoids’ content
To measure the total flavonoid content, aluminum chloride
colorimetric method was used (Chang et al. 2003). 500 μl
of each extracts were individually mixed with 1.5 ml of
ethanol, 0.1 ml of aluminum chloride (10% ethanol), 0.1 ml
of potassium acetate (1 M), and 2.8 ml of double-distilled
water. Then, the solutions were placed at room temperature
for 30 min. The absorbance of solutions was read at 415 nm
with a UV–Vis spectrophotometer. Quercetin was used to
draw the standard curve, and at the end, the total flavonoid
content was reported in terms of mg of quercetin per g of
extracts.
Determination of total anthocyanin content
To measure the total anthocyanin content, 0.02 g of herba-
ceous dry tissue was grinded with 4 ml of methanol chloride
1% solution in a porcelain mortar. The solution was stored in
the refrigerator for 24 h. Then, the solution was centrifuged
for 10 min at 13,000 rpm. The supernatant was removed and
the absorbance of the solutions was measured to be 530 and
657 nm wavelengths against the control (Mita et al. 1997).
A methanol hydrochloric acid 1% solution was used as the
control. Anthocyanin content was calculated using the fol-
lowing relation:
(
A = A530 − 0.25 A657 .
)
Determination of total carotenoid content
To measure the total carotenoid, 0.05 g of fresh sample with
5 ml of acetone was homogenized in a porcelain mortar in
13

an ice bath. Then, 1 g of non-aqueous sodium sulfate was
added to the homogeneous product and filtered using filter
paper 42. The solution was diluted with acetone to a vol-
ume of 10 ml and centrifuged for 10 min at 2600 rpm. The
supernatant phase was removed and the absorbance of the
solution was measured at 662, 645, and 470 nm wavelengths.
Acetone was used as the control. The amount of carotenoids
for each extract was calculated using the following formulas
(Lichtenthaler 1987):
Ca = 11.24 A662 − 2.04 A645
Cb = 20.13 A645 − 4.19 A662
Ct = A470 − 1.9 Ca − 63.14 Cb ∕214,
where Ca is the chlorophyll A, Cb is the chlorophyll B, and
Ct is the total carotenoids.
Phytosynthesis of gold and silver nanoparticles
First, a solution of gold (III) was prepared from its salt
­(HAuCl4·3H2O) and a solution of silver nitrate was prepared
from its salt ­(AgNO3) at a concentration of 1 mM. Then,
2 ml of aqueous extract of petals of saffron was added to
4 ml of gold (III) and silver nitrate solutions. The pH values
of the solutions obtained by the pH meter were read, respec-
tively, as 4.63 and 5.54 in gold (III) and silver nitrate solu-
tions. In a few minutes and after observing the color change
from dark pink to (purple for the solution containing gold
(III) and brown for the solution containing silver nitrate.
Optimization of effective parameters to synthesis
of nanoparticles
To obtain NPs of suitable morphology and size, the factors
affecting to synthesis of the GNPs and SNPs were investi-
gated and optimized such as: pH of the reaction mixture,
volume of extract, metal ion concentration, and reaction time
(Azizian Shermeh et al. 2017).
pH of solution
To optimize the pH of the reaction, 6 solution series were
produced including 2 ml of the extract and 4 ml of gold (III)
solution at a concentration of 1 mM (pH 2, 3, 4, 5, 6, and
7) to synthesize the GNPs, and 5 series of solutions were
produced containing 2 ml of the extract and 4 ml of silver
nitrate solution at 1 mM concentrations (pH 2, 4, 6, 8, and
10) for the synthesis of SNPs. In addition, the absorption of
spectra of all of them was investigated with UV–Vis spec-
trophotometry and the optimum pH was selected. To adjust
the pH of the solution, either of the solutions of NaOH or
HCl was used at a concentration of 0.1 molar.
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Applied Nanoscience
Volume of extract
To optimize the amount of extracts, the amount of 0.5–4 ml
of the extract was added to 4 ml of the gold (III) solution
at the concentration of 1 mM for the synthesis of GNPs,
and 1–4 ml of the extract was added to 4 ml of silver nitrate
solution at the concentration of 1 mM for the synthesis of
SNPs, and the reaction pH was adjusted to the optimum
pH. For all solutions, the UV–Vis spectrophotometry was
performed separately, and finally, the optimum volume of
extract was selected.
The concentration of gold (III) and silver nitrate
To investigate the effect of concentration of metal ion and
optimize them, an optimum amount of extracts was added
to 4 ml of different concentrations of gold (III) solution (0.5,
1, 1.5, 2, and 2.5 mM) and silver nitrate (0.5, 1, 2, 3, 4, and
5 mM) and the reaction pH was adjusted to optimal pH.
UV–Vis spectrophotometry of the solutions was investigated
and the optimal concentration was selected.
Reaction time
To optimize the reaction time and study of the stability of
NPs, a solution was prepared at different times (from the
start of mixing up to 6 h after making the sample with a
time interval of 1 h for GNPs and up to 5 h for SNPs) and
was investigated with UV–Vis spectroscopy, and then, the
optimal time was selected.
Separation and purification of gold and silver
nanoparticles
The NPs’ solution obtained was purified and separated by
repeated centrifugation at 10,000 rpm for 15 min. The cen-
trifugation process was repeated 2–3 times to ensure the
removal of any adsorbed substances on the surface of the
GNPs and SNPs.
Characterization of synthesized gold and silver
nanoparticles
The bio-reduction of the ­Ag+–Ag0 and ­Au3+–Au0 was moni-
tored using (Jenway-6715) UV–visible spectrophotometer. The
scanning range employed was 200–800 nm with a resolution
of 1 nm. The crystallinity of the GNPs and SNPs was exam-
ined using X-ray diffraction (D8-Advance, Bruker) meter with
monochromatic CuK a radiation (h = 1.54 Å) operating at a
voltage of 40 kV and a current of 30 mA at room tempera-
ture. The intensity data for the SNPs and GNPs were collected
over a 2θ range of 10°–80°. Transmission Electron Microscope
(TEM) was used to characterize the NPs’ surface morphology
Applied Nanoscience
and measure the size and the shape. Transmission electron
microscopy (TEM) measurements were performed on a (Zeiss-
EM10C-80 kV) transmission electron microscope at 80 kV.
Investigation of antimicrobial activity
Microbial specimens were reduced using Luria–Bertani and
Sabouraud dextrose culture media and by standardized meth-
ods. For making microbial suspensions, inoculation was made
separately for 24-h culture of each microorganism in vials
containing 3 ml of Mueller–Hinton broth. A suspension was
prepared with half-McFerland opacity.
Antimicrobial and anti-fungal effects of petals of saffron
extracts were investigated by disc-diffusion method from wells
in agar (Azizian Shermeh et al. 2017). For this purpose, 100 μl
of suspensions of each microbe were placed on a plate con-
taining Mueller–Hinton agar for bacteria and Sabouraud dex-
trose agar for fungi and mass-cultured by sterile swabs in three
directions. Then, at each of the cultivated plates, wells were
formed with a diameter of about 6 mm with 2 cm distances.
In each well, by a sampler, 50 μl was poured of each of the
sample dilutions prepared. Antibacterial antibiotics ampicillin,
gentamicin, and the anti-fungal antibiotic clotrimazole were
used as positive controls and the DMSO solution as a nega-
tive control. Finally, bacterial culture media were incubated at
37 °C for 24 h and the fungal cultures were incubated at 28 °C
for 48 h. After 24–48 h, the microbial cultures were investi-
gated in terms of formation or non-formation of the inhibition
zones, the diameters of which were measured and reported in
millimeters. To investigate the antimicrobial and anti-fungal
effects of NPs, disc-diffusion method from the well into agar
was used (Azizian Shermeh et al. 2017). The dilution series
used to determine the inhibition zone was 10, 20, 40, 60, and
100 μg/ml, and a solution of 50 μg/ml of gold (III) and silver
nitrate and antibacterial antibiotic gentamicin and anti-fungal
antibiotic clotrimazole were considered as positive controls.
Statistical analysis
All phytochemical experiments were performed in three rep-
lications in a completely randomized design. The results were
analyzed using the SPSS (V.16) software. Mean significance
comparison was performed using Duncan multi-grain test at
5% probability level. Microsoft Excel 2010 software was also
used to draw charts and diagrams.
Results and discussion
Phytochemical studies
Antioxidant activity
To make a consistent comparison, the concentration of all
of the extracts was selected as 1000 μg/ml for all experi-
ments. The proper methodology and solvent play a sig-
nificant role for the extraction of secondary compounds
(Moure et al. 2001). Studies have shown that methanol and
ethanol solvents penetrate inside plant cells to extract more
natural compounds including more phenols and flavonoids,
and this amount is less for solvents such as water, ethyl
acetate, and chloroform (Khorasani Esmaeili et al. 2015).
The results of this study show that methanol solvent plays
an important role in the extraction of phenolic and flavo-
noid secondary compounds, and these compounds have
resulted in its high antioxidant properties. The results of
reducing power of DPPH radical and iron (III) ions by
the extracts of petals of saffron showed that in both meth-
ods, DPPH and FRAP, methanolic extracts had the high-
est ­(IC50 = 32.16 ± 3.24 µg/ml and 48.91 ± 3.16 mM ­Fe2+/
mg sample) and aqueous extract (27.89 ± 2.15 mM ­Fe2+/
mg sample and I­ C50 = 88.69 ± 6.11 µg/ml) had the least
antioxidant and reducing powers and the ethanolic extract
was in the second level and its antioxidant strength was
slightly less than the methanolic extract and more than the
aqueous extract (39.12 ± 2.19 mM ­Fe2+/mg sample and
­I C 50 = 58.91 ± 5.84 µg/ml). To calculate the ­I C 50 value,
the calibration curve of the inhibition power (% IP) was
plotted in μg/ml, and after obtaining the line equation and
replacing the number 50 in the horizontal axis, the ­IC50
value was calculated on the vertical axis. The ­IC50 value
refers to concentrations of extracts that lead to 50% inhi-
bition of radicals. In the FRAP method, the antioxidant
activity was measured based on the ability to reduce fer-
ric and ferrous ions and the results were reported in mM
of the ferrous onto the mg of the extracts. Because of the
effects of antioxidants in preventing diseases and food
corruption due to radicals, the role and effects of antioxi-
dants have been taken into consideration by researchers
and medical society, and studies of antioxidant capacity
are among the most commonly studied issues in recent
years. Often, to validate the results of a study, two or more
methods are used to determine the antioxidant activity of
the samples. A strong correlation between the antioxidant
capacities on similar substances by different methods may
demonstrate the correctness of the test (Huang et al. 2005).
Studies have shown that the DPPH method is one of the
simple, inexpensive, and easy methods that can be used to
evaluate the antioxidant capacity of samples, especially
13

for the herbal ones (Sanchez et al. 2007), but not only the
FRAP method is simple, it is not sample specific and is
widely used to validate other antioxidant power assess-
ment methods (Ghiselli et al. 2000). So far, extensive stud-
ies have been carried out on the antioxidant properties
of the petals of saffron (Goli et al. 2012). In addition, its
strong anti-cancerous effect is dependent on its antioxidant
properties (Bhandari 2015). Research has shown that the
amount of antioxidant is less in petals of saffron than its
stigma (Karimi et al. 2010).
Total phenol, flavonoids, anthocyanins, and carotenoids
content
The phenolic and flavonoid contents of all extracts of saf-
fron petals were measured using spectrophotometer appa-
ratus and by the methods of Folin–Ciocalteu reagent and
colorimetric of aluminum chloride, respectively. In meas-
uring anthocyanin level, only saffron petal-dried powder
was used. Saffron fresh petal tissue was used to measure
total carotenoid levels. The findings showed that, like the
results of antioxidant power measurements, methanol
extracts have the highest total phenol and flavonoid con-
tent (35.11 ± 3.16 mg GAE/mg sample and 20.15 ± 2.96
mgQUE/mg sample) and the aqueous extracts had the low-
est phenol and flavonoid content (17.67 ± 3.84 mg GAE/mg
sample and (9.86 ± 1.12 mgQUE/mg sample). For ethanolic
extract, these values were measured as (28.55 ± 3.01 mg
GAE/mg sample and 21.15 ± 2.95 mg GAE/mg sample). In
addition, the order of phenol and flavonoids contents was
similar in that of the antioxidant power evaluated by DPPH
and FRAP. The results of the measurement of total antho-
cyanin and carotenoids were calculated in (mg/g dry weight
of tissue) and (mg/fresh weight), respectively. The results
Fig. 1  UV–Vis spectrophoto-
metric spectrum of petals aque-
ous extract of Crocus sativus L.
and GNPs and SNPs solution
13
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indicated that saffron petals had 31.01 ± 2.56 of anthocyanin
and 16.18 ± 1.12 of carotenoids. Carotenoids are pigments
that play an important role in protecting plants from oxida-
tive processes that can react as an antioxidant with a single
oxygen and radical peroxide (Stahl and Sies 2003). Research
has shown that petals of saffron have high levels of antho-
cyanins, glycosides, and flavonoids (Gil and kaber 2002).
Kaempferol are the major flavonoid compounds in saffron
petals (84%) (Goupy et al. 2013). Research has shown that
petals of saffron have flavonoids, anthocyanins, and tannins,
but no alkaloid and saponin compounds have been observed
(Babaei et al. 2014).
Phytosynthesis and optimization of parameters affecting
to synthesis of gold and silver nanoparticles
In the initial synthesis of GNPs and SNPs by saffron petals,
it was that observed by mixing the extract with gold and sil-
ver salt at the initial acidity, the color of the solution changed
from dark pink to purple and brown, respectively, which
indicated the successful synthesis of GNPs and SNPs. In
addition, after spectrum of the desired solutions was taken,
GNPs had the highest absorption rates in the wavelength
range of 600–500 nm and SNPs in the range of 450–400 nm,
which is related to the surface plasmon resonance (SPR) of
GNPs and SNPs (Fig. 1). Figure 1 shows that the extract in
the desired range does not contain any peaks, and therefore,
the presence of peaks in the synthesized nanoparticles could
be another reason for the successful synthesis of these NPs.
Afterwards, to obtain more consistent shapes and smaller
size of NPs, the following parameters affecting the synthesis
of GNPs and SNPs were studied: pH of the reaction solution,
extract volume, concentration of gold (III) and silver nitrate
salt, and reaction time.
Applied Nanoscience
pH of the reaction solution
After mixing the extract and gold (III) and silver nitrate
at the initial pH of the solution (pH = 4.63 for GNPs and
pH = 5.54 for SNPs), color change was observed in a short
time. Therefore, to investigate the effect of the important
pH factor on the synthesis of silver nanoparticles, solu-
tions with higher and lower pHs than the initial pH were
prepared and UV–Vis spectrophotometry spectrum of each
solution was separately extracted (Fig. 2). According to
Fig. 2, the absorption increased and then decreased with
increasing pH up to 6 for GNPs and up to 8 for SNPs. The
maximum absorption of GNPs and SNPs was observed
at pH 6 and pH 8, respectively. As a result, this pH was
selected as the optimum acidity. It appears that at higher
pHs, the gold and silver ions are hydrolyzed to give rise
to the stable species of ions of gold and silver hydroxides,
which ultimately prevents the entry of these ions into a
biological reduction reaction (Gardea-Torresdey et  al.
1999).
Fig. 2  UV–Vis spectrophotometric spectrum in different pHs. a Gold nanoparticles (GNPs), b silver nanoparticles (SNPs)
Fig. 3  UV–Vis spectrophotometric spectrum in different volumes of extract. a Gold nanoparticles (GNPs), b silver nanoparticles (SNPs)
pH is one of the most important factors affecting the syn-
thesis of NPs (Waghmar et al. 2014). Previously, there have
been reports on the effect of pH on the formation of NPs.
Reports indicate that pH does not have a significant effect on
the shape of NPs, but affects their size significantly (Arm-
endariz et al. 2004).
Volume of extract
To determine the optimum extract amount, 0.5–4  ml of
extract were added to 4 ml of gold salt solution at a concen-
tration of 1 mM and 1–4 ml of the extract was added to 4 ml
of silver nitrate salt solution at a concentration of 1 mM and
the reaction acidity was adjusted to the optimum pH. From
all the solutions obtained, the 4 spectrophotometry was per-
formed separately (Fig. 3). With the gradual increase in the
amount of the extract, the amount of absorption of SNPs
and GNPs increased significantly. However, after reaching
a volume of 3 ml of extract for GNPs and 2 ml of extract
for SNPs, there was a decrease in absorbance. As a result,
13

the amount of 3 and 2 ml of extract was selected as the opti-
mized volume of extract for synthesis of GNPs and SNPs,
respectively.
In the synthesis of NPs by plant’s extracts, the extract
plays the role of reduction of metal ions and also stabilizes
these NPs (Philip 2010) (Scheme 1). Various secondary
compounds, enzymes, proteins, or other reducing agents
(antioxidant compounds) play a major role in the production
of metallic NPs by plants (Yulizar et al. 2017). The results
the phytochemical compounds of this study also emphasized
the presence of compounds effective on the synthesis of NPs
(antioxidant compounds, phenols, flavonoids, etc.). Reports
indicate that with increasing the amount of extracts, the
amount of natural active ingredients in the plant is increased
and more NPs are produced and the absorption is increased
(Prabha Dubeya et al. 2010). The findings regarding the cho-
sen volume of the extracts in this study also confirm these
reports. Studies have also shown that at concentrations
below the optimum level, the reduction and stabilization of
the NPs did not fully occur and the NPs were obtained in
Scheme 1  Mechanism of change the silver and gold ions to silver and
gold nanoparticles by saffron aqueous extract
Fig. 4  UV–Vis spectrophotometric spectrum in different concentrations of ­HAuCl4·3H2O and ­AgNO3. a Gold nanoparticles (GNPs), b silver
nanoparticles (SNPs)
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less amounts and with a bulkier size. In addition, by an addi-
tion exceeding the optimum amount, the stabilizing particles
accumulate further on themselves, which causes a defective
stabilization and coarser particles. Studies have shown that
an increase in the NPs size, the absorption rate is decreased,
and conversely, with increasing the particle distribution, the
width of the spectra increases (Azizian Shermeh et al. 2017).
Concentration of gold (III) and silver nitrate
To study the effect of the concentration of gold and silver
salt on the synthesis of gold and silver nanoparticles, solu-
tions of different concentrations, i.e., (0.5, 1, 1.5, 2, and
2.5 mM) and (0.5, 1, 2, 3, 4, 5), respectively, were prepared
and all solutions underwent investigated with UV–Vis spec-
trophotometry (Fig. 4).
Considering Fig. 4, with the gradual increase in the con-
centration of salts, the absorption of NPs increased. This
increasing trend continued for gold salt up to a concentration
of 2 mM and for silver nitrate salt up to a concentration of
4 mM. However, at a higher concentration, a decrease in the
absorption rate was observed. As a result, the concentra-
tion of 2 mM for the ­HAuCl4·3H2O and 4 mM for ­AgNO3
was selected as the optimum concentration. Reports indicate
that increased absorption under an increased concentration
of metal ions is due to the fact that ions are more exposed
to reduction, resulting in more NPs (Dwivedi et al. 2010).
In a similar study, absorption increased significantly with
increasing concentrations of 1–3 mM in metal salts (Vinod
et al. 2011). In addition, the decrease in absorption under
the excessive increase in the concentration of metal ions can
be due to the bonding of NPs and the synthesis of NPs of
a slightly larger size (Mock et al. 2002). The results of this
study were consistent with similar articles.
Applied Nanoscience
Time of reaction
By examining the effects of the time of proximity of gold
(III) and silver nitrate solutions with extract of saffron pet-
als on the synthesis of GNPs and SNPs, it was found that
with increasing interaction time, the adsorption for GNPs is
slightly increased, but this increase is not significant (Fig. 5).
Such that no significant change in absorption was
observed after 1 h, which proved the stability of the NPs. As
a result, the duration of 1 h was chosen as the optimal time.
However, for SNPs, with increasing the time up to 5 h, the
absorption rate of silver SNPs raised. Time is also one of the
factors affecting the synthesis and stability of NPs. In addi-
tion, the results showed that the NPs are very stability during
of this times. In a similar study, in a time-dependent reac-
tion in the synthesis of NPs, it has been reported that with
increasing time from the start of the reaction to 120 min,
after 60 min, there is no significant change in the absorption
Fig. 5  UV–Vis spectrophotometric spectrum in different times. a Gold nanoparticles (GNPs), b silver nanoparticles (SNPs)
Fig. 6  TEM images of phytosynthesized nanoparticles. a Gold nanoparticles (GNPs), b silver nanoparticles (SNPs)
value associated with the NPs. Therefore, 60 min was a good
time (Ramteke et al. 2013).
The transmission electron microscopy images of gold
and silver nanoparticles
The distribution of the shape and size GNPs and SNPs by
TEM image was investigated. Figure 6 shows the image of
TEM of the synthesized GNPs and SNPs with all optimized
conditions. The image shows that the resulting NPs are
almost spherical and have a moderate size for GNPs between
17 and 22 nm and for SNPs of 10-14 nm.
Investigation of X‑ray diffraction spectrum
XRD technique was used for further investigation of the
crystalline structure of the synthesized GNPs and SNPs.
Using the Debye–Scherer formula (D = 0.9λ/β cos θ), the
13

mean crystallinity of the NPs was obtained. According to
this formula, β is the peak widths along half of the maximum
height, λ is the wavelength of the X-ray equaling 1.54 Å, θ
is the angle between the open radiation beam (2θ = 0–80°),
and D is the crystalline grain size in nanometers. GNPs and
SNPs showed sharp peaks in regions 2θ = 38.17, 44.37,
64.56, 77.55, 81.70 and 2θ = 38.11, 44.28, 67.43, 77.45,
respectively, which are related to the crystalline structure of
GNPs and SNPs with Miller’s indices (111), (200), (220),
(311), (222) and (111), (200), (220), (311), respectively
(Fig. 7). The presence of sharp peaks in patterns shows a
high degree of crystallinity for NPs. Peak (111) is sharper
than other peaks resulting in more crystalline GNPs and
SNPs in this direction. The average size of the synthe-
sized crystalline granules was estimated by calculating the
Fig. 7  XRD images of phytosynthesized nanoparticles. a Gold nanoparticles (GNPs), b silver nanoparticles (SNPs)
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Debye–Scherer formula 16–28 and 12–25 nm for SNPs and
GNPs, respectively.
Antimicrobial activity of extracts and gold and silver
nanoparticles
The results of the various concentrations of the extracts
with a well-diffusion method are presented in Table 1. The
results of antimicrobial activity of the extract showed that
the extracts have a significant bactericidal effect on all bac-
teria, which increases by increasing the concentration of
the extracts. In addition, the methanolic extract had a more
inhibitory effect than other extracts, but this effect was less
in the aqueous extract. By comparing the mean diameter
of the methanolic extract inhibition region, it can be said
Applied Nanoscience

Table 1  Diameter of zone microbial insufficiency of the petal extracts of Crocus sativus L. using agar well diffusion (millimeters)
Samples Microorganism Concentration (µg/ml) Antibiotics Solvent
25 50 100 200 400 Ampicillin Gentamicin Clotrimazole DMSO

Methanol extract S. aureus 9 12 15 18 20 28 27 – 0

B. cereus 8 10 13 15 17 27 26 – 0
E. coli 5 7 9 13 16 27 25 – 0
A. niger 7 10 13 15 18 – – 27 0
C. albicans 5 5 9 12 14 – – 28 0
Ethanol extract S. aureus 7 10 12 16 17 27 27 – 0
B. cereus 6 11 10 13 15 26 26 – 0
E. coli 5 5 9 10 13 25 24 – 0
A. niger 5 7 10 12 15 – – 26 0
C. albicans 4 5 8 10 13 – – 25 0
Aqueous extract S. aureus 5 8 10 13 15 26 25 – 0
B. cereus 5 7 10 12 14 24 25 – 0
E. coli 3 5 8 10 12 25 26 – 0
A. niger 0 3 7 11 12 – – 26 0
C. albicans 0 3 6 10 11 – – 25 0

that this extract has more potential to inhibit Staphylococcus
aureus (20 mm inhibition zone diameter) and Aspergillus
niger (18 mm) and the least effect was on Escherichia coli
(diameter of the inhibition 16 mm) and Candida albicans
(14 mm). The results of ethanolic extract showed that this
extract was successful in inhibiting all bacteria and fungi,
so the strongest antibacterial and anti-fungal effects were
on Staphylococcus aureus (16 mm) and Aspergillus niger
(20 mm). The inhibitory effect of all extracts was concentra-
tion dependent. The aqueous extract, while in the third place,
has been able to inhibit Staphylococcus aureus (13 mm) and
Aspergillus niger (7 mm).
The results of antimicrobial activity of GNPs and SNPs
on the microorganisms studied, and a comparison with the
antibiotics used are presented in Table 2. Antimicrobial
activity of GNPs and SNPs was much more than the petal
extract of saffron and this effect has been impressive, so
that at low concentrations, it also prevented the growth of
bacteria and fungi. In addition, the antimicrobial activity of
the synthesized GNPs and SNPs was far more than the solu-
tion containing gold and silver salts. The results showed that
silver and GNPs had the highest effect on Staphylococcus
aureus (25 and 20 mm inhibition zone diameter) and Asper-
gillus niger (diameter 22 and 17 mm) and had the least effect
on E. coli (diameter 20 and 14 Mm) and Candida albicans
(diameter 19 and 14 mm).
Many studies have been performed to determine the
effects of NPs on the bacteria. In addition, various researches
on the probable reactions between the NPs and macromol-
ecule of living creatures show that the difference between
the microorganism negative charge and the metal NPs posi-
tive charge acts as an absorptive electromagnet between
the bacteria and the NPs and attaches the NPs to the cell
surface and kills the cell (Lin and Xing 2007). In addition,
the formation of colonies, bacteria cell growth, and forma-
tion of bacterial compacted biofilm matrices lead to infec-
tions, and the NPs prevent the formation of these defensive
agents of the bacteria against the immune system of the host
cell (Jones et al. 2006) (Schrand et al. 2010). Many studies
have been performed on the antibacterial effects of metal
NPs. Some research results also indicate that the GNPs are
less effective on Escherichia coli (Cho et al. 2005). Fur-
ther studies have shown that the concentration of GNPs and
SNPs used is effective on antimicrobial activity of bacte-
ria (Sondi and Salopek-Sondi 2004). In an analysis of the
effects of GNPs, SNPs, and ZnNPs on pathogenic bacteria,
it was reported that the antimicrobial effect of gold GNPs
is far less than ZnNPs and SnpS (Nazari et al. 2012). Other
researchers in a similar study stated that the antimicrobial
effect of GNPs in low concentrations was not observed on
any of the isolates, but GNPs may have less effect on Heli-
cobacter pylori than other NPs (Gopinath et al. 2016). In
another study, it has been shown that the effects of SNPs on
E. coli are greater than S. aureus (Kim et al. 2007). Anti-
microbial activity of snpS on different bacteria such as E.
coli, P. aeruginosa, and S. aureus has shown that although
the activity of NPs is concentration dependent, it is more
significant for Gram-negative bacteria than Gram-positive
ones (Shrivastava et al. 2007) (Guzman et al. 2012). In addi-
tion, the synthesized GNPs had efficacy against a Gram-neg-
ative bacteria (Escherichia coli), a Gram-positive bacteria
(Staphylococcus aureus), and a mold (Aspergillus niger).
The maximum conversion was 66%. (Das and Shuvasmita
2018). In other research, the antimicrobial evaluation result

13
 Applied Nanoscience

Table 2  Diameter of zone microbial insufficiency of GNPs and SNPs using agar well diffusion (millimeters)
Samples Microorganism Concentration (µg/ml) Antibiotics Solvent
25 50 100 200 400 Ampicillin Gentamicin Clotrimazole DMSO

SNPs S. aureus 12 15 18 21 25 29 28 – 0
B. cereus 10 13 17 20 23 28 26 – 0
E. coli 8 12 15 17 20 28 27 – 0
A. niger 10 13 16 19 22 – – 29 0
C. albicans 8 11 14 16 19 – – 28 0
GNPs S. aureus 7 11 15 18 20 28 27 – 0
B. cereus 5 10 12 15 17 27 26 – 0
E. coli 5 6 8 11 14 26 25 – 0
A. niger 8 9 12 14 17 – – 27 0
C. albicans 5 5 8 11 14 – – 28 0
AgNO3 S. aureus 8 11 13 16 18 26 26 – 0
B. cereus 7 9 12 14 16 25 24 – 0
E. coli 5 7 10 12 14 23 25 – 0
A. niger 5 7 9 12 14 – – 26 0
C. albicans 0 3 6 10 11 – – 25 0
HAuCl4·3H2O S. aureus 5 7 8 11 13 25 24 – 0
B. cereus 5 6 9 10 12 24 23 – 0
E. coli 3 5 7 8 10 25 24 – 0
A. niger 5 5 8 10 12 – – 25 0
C. albicans 0 3 6 8 10 – – 23 0

showed that the synthesized SNPs at different concentrations
were very active against the Gram-positive bacteria (B. sub-
tilis, S. aureus) and Gram-negative bacteria (K. Pneumonia,
E. Coli, and Pseudomonas aeruginosa), P. aeruginosa being
most susceptible to the antimicrobial effect of the silver nan-
oparticles (Ezealisiji et al. 2017).
Conclusion
Using the high potential of natural resources, especially
plants, leads us to produce stable NPs. Phytosynthesized
NPs are far smaller and more widely used than NPs made
of chemical and physical processes. The present study is
a report on the phytosynthesis of GNPs and SNPs using
aqueous extract of saffron petals. Saffron plant petal is
used as a reducing and stabilizing agent in the synthesis
of NPs. Compounds existing in the extract include phenol,
flavonoids, anthocyanin, and carotenoids, which reduce
metal ions and ultimately stabilize, synthesized NPs. As a
result, the amount of these compounds, and the antioxidant
and reducing properties of the plant in the synthesis of
NPs are desirable, important, and necessary. The results
of the reducing capacity and the amount of phenol, flavo-
noids, anthocyanin, and carotenoids in the saffron petal
have shown that they have a good potential for reducing
the free radicals and metal ions. As a result, the petal of
the studied plant is an ideal candidate for the reduction of
gold and silver metal ions, converting them into GNPs and
SNPs and stabilizing them. One of the limitations in most
NPs’ synthesis methods is the high distribution of NPs in a
wide range of sizes in nano-metric dimensions. This great
constraint can be eliminated by various methods such as
pH changes, amount of plant extracts, different concentra-
tions of salt solution, and duration, to reduce the range of
nanoparticle sizes and convenient condition is achieved.
In this study, the effects of the mentioned factors were
investigated and optimized. In general, according to the
results of this study and other reports on the production
of NPs using biological materials such as microorganisms,
plant biomass, and even plant extracts, it is possible to
use biological and green methods as a complement for
physical and chemical methods of producing these NPs. In
addition, considering the safety and environmental aspects
of NPs production, these methods are also considered as
environmentally friendly. In addition, the presence of anti-
microbial activity of the extracts and GNPs and SNPs syn-
thesized from the aqueous extract leads to their use being
recommended for different areas to prevent infection and
prevalence of pathogens as a replacement for conventional
antibiotics. Therefore, plants are high potential for the syn-
thesis of metal NPs, which can be used as an antibiotic to
inhibit many pathogenic bacteria and fungi.

13
Applied Nanoscience
Acknowledgements  We thank the University of Sistan and Bal-
uchestan Deputy of Research for financial support this research.
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