Water Science and Engineering xxxx, xxx(xxx): xxx

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Green synthesis of silver nanoparticles using aqueous leaf extract of

Saussurea obvallata for efficient catalytic reduction of nitrophenol,
antioxidant, and antibacterial activity
P.S.R. Vidya Sagar a,b, Dharmasoth Ramadevi c, Keloth Basavaiah a,*, Sathish Mohan Botsa d,**
a
Department of Chemistry, Andhra University, Visakhapatnam 530003, India
b
Department of Chemistry, Adikavi Nannaya University, Rajahmahendravaram 533296, India
c
Samuel George Institute of Pharmaceutical Sciences, Markapur 523316, India
d
Bio Enviro Chemical Solutions, Visakhapatnam 530017, India
Received 12 December 2022; accepted 6 September 2023
Available online ▪ ▪ ▪

Abstract

Of several noble metal nanoparticles, silver nanoparticles (AgNPs) have attracted special attention due to their distinct properties, such as
favorable electrical conductivity, chemical stability, and catalytic and antibacterial activities. Green synthesis of AgNPs using plant extracts
containing phytochemical agents has attracted considerable interest. This environmentally friendly approach is more biocompatible and cost-
efficient and has the capability of supporting large-scale synthesis. This study developed an eco-friendly method for the preparation of
AgNPs using the aqueous leaf extract of Saussurea obvallata as reducing and capping agents. Ultraviolet visible spectroscopy (UVeVis),
Fourier transform infrared spectroscopy (FTIR), Raman, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron
microscopy (SEM), and transmission electron microscopy (TEM) analyses were conducted to characterize the synthesized AgNPs. The
morphology of AgNPs was found to be spherical with an average crystallite size of 12 nm and a maximum absorbance at 410 nm. 10 mg of
AgNPs had potential to reduce 4-nitrophenol to 4-aminophenol in 16 min and exhibited strong biological activities against the Gram-negative
bacteria Escherichia coli (12 mm) and Gram-positive bacteria Enterococcus faecalis (13 mm). The antioxidant activity of the synthesized
AgNPs was investigated against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and exhibited up to 61.21% ± 0.02% at an AgNPs
concentration of 500 mg/mL.
© 2023 Hohai University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
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Keywords: Saussurea obvallata; Reduction of 4-nitrophenol; Capping agent; Antimicrobial activity; Antioxidant assay

1. Introduction broad array of scientific sectors. Creation, exploitation, and

synthesis are the terms associated with nanotechnology, which
The utilization of nanotechnology for constructing nano- generally considers the materials that measure less than 1 nm.
scale products in research and development divisions is Nanotechnology supports diverse unique industries, such as
growing (Albrecht et al., 2006). Nanotechnology can be used electronics, pesticides, medicine, and parasitology, and thus
to produce a broad range of products applicable to an equally provides a platform for collaboration (Bhattacharyya et al.,
2010). Nanobiotechnology provides an example, wherein
research and development combine multiple scientific sectors,
* Corresponding author. including nanotechnology, biotechnology, material science,
** Corresponding author.
physics, and chemistry (Rai et al., 2008; Huang et al., 2007).
E-mail addresses: [email protected] (Keloth Basavaiah), bsathish401@
gmail.com (Sathish Mohan Botsa). Biologically synthesized nanoparticles with antimicrobial,
Peer review under responsibility of Hohai University. antioxidant, and anticancer properties are possible through the

https://doi.org/10.1016/j.wse.2023.09.004
1674-2370/© 2023 Hohai University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Vidya Sagar, P.S.R et al., Green synthesis of silver nanoparticles using aqueous leaf extract of Saussurea obvallata for efficient
catalytic reduction of nitrophenol, antioxidant, and antibacterial activity, Water Science and Engineering, https://doi.org/10.1016/j.wse.2023.09.004
2 P.S.R. Vidya Sagar et al. / Water Science and Engineering xxxx, xxx(xxx): xxx

collaboration of different natural science sectors. These (SEM), and transmission electron microscopy (TEM) analyses
nanotechnologies may provide novel resources for the evalu- were conducted to characterize the synthesized AgNPs. The
ation and development of new, safe, and effective drug for- synthesized AgNPs were evaluated for catalytic reduction of
mulations (Dipankar and Murugan, 2012). Of the several 4-nitrophenol to 4-aminophenol, the antioxidant activity
noble metal nanoparticles, silver nanoparticles (AgNPs) have against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and
attracted special attention due to their distinct properties the antibacterial activity against the Gram-positive bacteria
including favorable electrical conductivity, chemical stability, Enterococcus faecalis and Gram-negative bacteria Escherichia
and catalytic and antibacterial activity (Bharathi et al., 2018). coli.
Silver at the nanoscale also has properties different from bulk
silver. Synthesis of AgNPs is an emerging area and has been 2. Experimental methods
much sought after (Song and Kim, 2009). Green synthesis of
AgNPs has been accomplished using plants, microorganisms, 2.1. Phytochemicals in aqueous leaf extract of S.
and other biopolymers. Wet chemical synthesis can be obvallata
robustly scaled for the large-scale synthesis of AgNPs in
tunable shapes and sizes through the optimization of synthesis S. obvallata is an endemic herb in the Himalayan region and
conditions. However, wet chemical methods use toxic chem- distributed between 3 000 and 4 800 m. It is commonly known
icals that are hazardous to the environment and usually result as Brahma Kamal and is the state flower of Uttarakhand in
in the adsorption of toxic chemicals onto the surface of syn- India due to its traditional, medicinal, ornamental, and religious
thesized AgNPs, making them unsuitable for biomedical ap- purposes (Pant and Semwal, 2013). It has been used to treat
plications. In contrast, physical methods are expensive and various diseases or disorders, such as paralysis, cerebral
cumbersome for the large-scale production of nanoparticles. ischemia, wounds, cardiac disorders, and mental disorders, and
Therefore, the development of environmentally conscious, some people use it as an antiseptic in healing cuts. The phy-
energy-efficient, facile, and rapid green synthesis methods that tochemicals in the aqueous leaf extract of S. obvallata, such as
avoid toxic and hazardous chemicals has attracted significant carbohydrate, triterpenic acid, flavonoid, phenol, and saponin,
interest (Mohapatra et al., 2015). In addition, due to their are responsible for the formation of AgNPs (see Table 1).
potent antimicrobial activity, AgNPs have also been used in The major components in the aqueous leaf extract of S.
clothing (Tharani et al., 2020), food (Nandana et al., 2022), obvallata (Fig. A.1 in Appendix A) are linoleic acid (22.5.%),
sunscreens, and cosmetics (Martinez-Gutierrez et al., 2010; dehydrocostus lactone (21.98%), palmitic acid (11.84%),
Kokura et al., 2010). pregnanolone (11.43%), doconexent (9.00%), litsomentol
The use of plants for nanoparticle synthesis offers a wide (2.25%), stearic acid (2.38%), and bupleuronol (1.23%)
range of benefits over other biological synthesis methods (Prabhakar and Sakshi, 2019). Phenols, flavonoids, and tri-
because it does not require the maintenance of cell cultures terpenes in the plant extract contain active oxygen that has the
and incorporates support for the large-scale synthesis of potential to donate electrons for reduction of silver precursors
nanoparticles (Shankar et al., 2004). Extracellular nanoparticle into AgNPs. Carboxylic acids in the plant extract have the
synthesis, which utilizes extracts from individual leaves rather eCOO
group that acts as a chelating group for capping the
than from entire plants, may be more inexpensive due to its synthesized AgNPs. Thus, the phytochemical constituents in
easier downstream processing. Shankar et al. (2003a, 2003b), the aqueous leaf extract of S. obvallata can act as reducing and
Rai et al. (2006, 2007), and Chandran et al. (2006) are the capping agents for the formation of AgNPs.
pioneers to synthesize nanoparticles using plant extracts.
Green synthesis of AgNPs using plant extracts containing 2.2. Chemicals and reagents
phytochemical agents has attracted considerable interest. This
environmentally friendly approach is more biocompatible and All analytical-grade chemicals (i.e., AgNO3, H3PO4,
cost-efficient and has the capability of supporting large-scale NaOH, and ethanol) were purchased from Merck and used
synthesis (Arunachalam et al., 2013). The synthesis of without further purification. MilliQ water was utilized for all
AgNPs in different green phytochemical conditions and with experiments.
numerous microorganisms has been investigated.
Saussurea is an important genus of the family Asteraceae
Table 1
due to a variety of reasons, and most species of this genus have
List of phytochemical compounds in aqueous leaf extract of S. obvallata.
been well investigated (Mittal et al., 2015; Pandey et al., 2007;
Phytoconstituent Performed test Test result
Chick et al., 2015; Fan et al., 2015; Chen et al., 2016).
However, few studies have been conducted on Saussurea Carbohydrate Molisch's test Present
Alkaloid Dragendorff's test Present
obvallata. This study reported an eco-friendly method for
Saponin Foam test Present
preparation of AgNPs using the aqueous leaf extract of S. Phenol Ferric chloride test Present
obvallata as reducing and capping agents. Ultraviolet visible Tannins Alkaline reagent Absent
spectroscopy (UVeVis), Fourier transform infrared spectros- Flavonoid Lead acetate test Present
copy (FTIR), Raman, X-ray diffraction (XRD), X-ray photo- Triterpenoid LiebermanneBurchard test Present
Carboxylic acid Test with Na2CO3 Present
electron spectroscopy (XPS), scanning electron microscopy

Please cite this article as: Vidya Sagar, P.S.R et al., Green synthesis of silver nanoparticles using aqueous leaf extract of Saussurea obvallata for efficient
catalytic reduction of nitrophenol, antioxidant, and antibacterial activity, Water Science and Engineering, https://doi.org/10.1016/j.wse.2023.09.004
 P.S.R. Vidya Sagar et al. / Water Science and Engineering xxxx, xxx(xxx): xxx 3

2.3. Preparation of aqueous leaf extract of S. obvallata media) was dissolved in the water, and it was distributed in a
100-mL conical flask and sterilized in an autoclave at 121 C in
S. obvallata leaves were washed with water and shaded 15-L base pairs for 15 min after the media-poured sterilized
dried for 20 d. Dried leaves were grounded into powder, stored Petri plates were autoclaved. Chloramphenicol was taken as a
in an airtight container, and kept in a desiccator for further use. positive control for antibacterial activity. The antibacterial
To prepare the 1% aqueous leaf extract, 1 g of the leaf powder activity of the compounds was evaluated with the agar well
was taken in a 250-mL Erlen Meyer flask, and 100 mL of Milli diffusion method. Inoculums were spread over the surface of
Q water was added and heated at 70 Ce80 C for 20 min. agar plates with a sterile glass spreader. Four wells were made
Afterwards, the extract was cooled and filtered with What- at an equal distance using a sterile cork borer. To examine the
mann Paper No. 1. The filtrate was stored at 4 C in a refrig- antibacterial activity of the compounds, the extracts at final
erator for the preparation of AgNPs. concentrations of 40, 60, and 80 mg/mL were poured on each
well, and then the plates were incubated for a period of 24 h at
2.4. Preparation of AgNPs with aqueous leaf extract of 37 C in an incubator after the incubation diameter of a clear
S. obvallata inhibitory zone forming around the well was measured.

AgNPs were prepared with the aqueous leaf extract of S. 2.8. Antioxidant activity test
obvallata (Fig. A.2 in Appendix A). In a typical procedure,
10 mL of the 1-g/L aqueous extract of the S. obvallata leaf DPPH was used to assay the antioxidants. In brief, 3 mL of
powder was added to 100 mL of the 5-mmol/L AgNO3 aqueous the test extract was added to 1 mL of the 0.1-mmol/L solution
solution in a 250-mL Erlenmeyer flask. Then the reaction of DPPH in methanol. After 30 min of incubation at 37 C, the
mixture was stirred in a water bath for 60 min at 90 C. The pH maximum absorbance was measured at 517 nm against the
value of the reaction mixture was adjusted to 10 with 0.100 mol/L controls with a spectrophotometer. Ascorbic acid was used as
of NaOH and 0.033 mol/L of H3PO4. The color of the reaction the reference material. The percentage of inhibition (I ) indi-
mixture turned to reddish brown, indicating the formation of cating the radical scavenging activity was calculated by
AgNPs. The formed mixture solution was centrifuged, thor- comparing the absorbance values of the test samples (At) with
oughly washed with water and followed by ethanol, and finally those of the control (Ac):
dried in an oven overnight. The same procedure was adopted for
At
the synthesis of AgNPs at different AgNO3 concentrations, plant I¼  100% ð1Þ
extract concentrations, pH values, and temperatures. Ac

2.5. Characterization of prepared AgNPs 3. Results and discussion

Analytical instruments were used to determine the elec- 3.1. Characterization of synthesized AgNPs
tronic, optical, and morphological properties of the prepared
AgNPs. XRD analysis was carried out with Bruker AXS D8 Fig. 1 shows the UVeVis absorption spectra of the syn-
with the Cu -Ka wavelength at a scan rate of 0.02 . FTIR thesized AgNPs and the aqueous leaf extract of S. obvallata.
analysis was conducted with IR Prestige 21 (Shimadzu), with The characteristic absorption peak at 430 nm indicated the
a range of 4 000e500 cm
1. XPS analysis was conducted with formation of AgNPs, primarily due to the surface plasmon
PHI 5000 Versaprobe III, and a UVeVis spectrometer (Shi- resonance (SPR) absorption in AgNPs (Mulvaney, 1996). In
madzu 2 600R) was used to study the optical properties of the order to find out the best conditions for the synthesis of
prepared samples in a wavelength of 200e800 nm. AgNPs, optimization was carried out with different experi-
mental parameters, such as the concentration of AgNO3, the
2.6. 4-nitrophenol reduction experiment

The reduction of 4-nitrophenol (4-NP) to 4-aminophenol

(4-AP) with the prepared AgNPs in the presence of NaBH4
was selected as a model reaction. The whole experiment was
carried out in a quartz cuvette. 2 mL of the 0.000 7-mol/L 4-
NP aqueous solution, 2.5 mL of 0.09-mol/L freshly prepared
NaBH4, and the prepared AgNPs were taken to examine the
reduction of 4-NP to 4-AP, and a UVeVis spectrometer was
used for monitoring.

2.7. Antimicrobial activity assay

Bacterial cultures that grew overnight at 37 C were used to Fig. 1. UVeVis spectra of aqueous leaf extract of S. obvallata and
test the antibacterial activity. The nutrient agar medium (high synthesized AgNPs.

Please cite this article as: Vidya Sagar, P.S.R et al., Green synthesis of silver nanoparticles using aqueous leaf extract of Saussurea obvallata for efficient
catalytic reduction of nitrophenol, antioxidant, and antibacterial activity, Water Science and Engineering, https://doi.org/10.1016/j.wse.2023.09.004
4 P.S.R. Vidya Sagar et al. / Water Science and Engineering xxxx, xxx(xxx): xxx

concentration of the aqueous leaf extract of S. obvallata, clearly shows that the alkaline environment improved the
temperature, and pH. reduction and stabilization of the potential of the aqueous leaf
Fig. 2(a) shows the UVeVis spectra of AgNPs prepared at extract of S. obvallata towards the formation of AgNPs. The
different AgNO3 concentrations. The intensity of the SPR number of formed AgNPs increased with alkaline pH due to
absorption peak increased from 424 to 437 nm as the con- the enhanced reactivity of the aqueous leaf extract of S.
centration of AgNO3 was increased from 1 to 3 mmol/L, and obvallata. Thus, the absorption peaks of formed AgNPs un-
the peak was red-shifted (424e437 nm). Fig. 2(b) shows the derwent a blue shift with smaller particle sizes. However, at a
UVeVis spectra of AgNPs prepared with different concen- pH value greater than 10, agglomeration of AgNPs was
trations of the aqueous leaf extract (1%, 2%, and 3%). As the observed.
concentration of the aqueous leaf extract was increased, the The FTIR spectra of the aqueous leaf extract of S. obvallata
intensity of the SPR peak of AgNPs also increased. The in- and AgNPs were recorded to identify the interaction of the
crease in the intensity of the SPR absorption peak could be functional groups of the aqueous leaf extract of S. obvallata
ascribed to the presence of a greater number of biomolecules responsible for the reduction of AgNO3 and the capping of
in the aqueous leaf extract of S. obvallata, which was subsequently formed AgNPs. As shown in Fig. A.3 in
responsible for the reduction of Agþ ions to AgNPs. The SPR Appendix A, both the FTIR spectra of the aqueous leaf
peak was blue-shifted (426e410 nm), indicating the formation extract of S. obvallata and AgNPs were similar, with some
of AgNPs in smaller sizes (Smitha et al., 2008). marginal shifts in the peak position, clearly indicating the
The effect of temperature on the formation of AgNPs is presence of the residual plant extract on AgNPs. The FTIR
shown in Fig. 2(c). The intensity of the SPR peak increased spectrum of the aqueous leaf extract of S. obvallata exhibited
with temperature. The reaction temperature increase led to a characteristic stretching frequencies at 3 300, 2 011, and
quick reduction of Agþ ions, thereby causing successive ho- 1 635 cm
1. The characteristic bands at 3 300 and 1 635 cm
1
mogeneous nucleation of AgNPs in smaller particle sizes. As were ascribed to the presence of an OeH group of phenolic
the temperature was increased from 30 C to 90 C, the in- compound or carboxylic acid and an acid group of C]O. In
tensity of the SPR peak also increased with a blue shift the case of AgNPs synthesized with the aqueous leaf extract of
(427e410 nm). A further increase in the temperature above S. obvallata, there was a shift in the absorbance peak of the
90 C caused the intensity of the SPR peak to decrease. Thus, OeH group of phenolic compound or carboxylic acid from
the optimum temperature for the synthesis of AgNPs was 3 300 to 3 600 cm
1. This implied that the reduction of silver
90 C. The UVeVis spectra of AgNPs obtained with different ions with hydroxyl groups and the capping of the synthesized
pH values are shown in Fig. 2(d). The intensity of the SPR AgNPs with the carboxyl group of the extract were evident.
peak increased with an increase in pH from 2 to 10. The rate of Furthermore, one additional peak at 580 cm
1 was observed in
the formation of AgNPs was high at a pH value of 10. Fig. 2(d) the case of AgNPs. This was ascribed to the symmetric and

Fig. 2. UVeVis spectra of AgNPs prepared with different AgNO3 concentrations, plant leaf extract concentrations, temperatures, and pH values.

Please cite this article as: Vidya Sagar, P.S.R et al., Green synthesis of silver nanoparticles using aqueous leaf extract of Saussurea obvallata for efficient
catalytic reduction of nitrophenol, antioxidant, and antibacterial activity, Water Science and Engineering, https://doi.org/10.1016/j.wse.2023.09.004
 P.S.R. Vidya Sagar et al. / Water Science and Engineering xxxx, xxx(xxx): xxx 5

asymmetric vibration of AgeO, where oxygen came from the The surface morphology of the synthesized AgNPs was
hydroxyl group of the plant extract. The phytochemical anal- investigated via the field emission scanning electron micro-
ysis of the aqueous leaf extract of S. obvallata based on the scopy (FEeSEM) and TEM. The representative FEeSEM
FTIR spectrum strongly suggested the presence of carboxylic images of AgNPs synthesized with the aqueous leaf extract
acid, flavonoids, and polyphenols, apart from other phyto- of S. obvallata are shown in Fig. A.4(c) and (d) in Appendix A.
chemicals, which was mainly responsible for the preparation The FEeSEM results clearly showed the spherical shape of
of AgNPs by reducing AgNO3 (Kumar et al., 2016). AgNPs. The scanning electron microscopyeenergy dispersive
The crystalline nature of AgNPs was confirmed by powder X-ray spectroscopy (SEMeEDX) results (Fig. A.4(b) in
XRD. The XRD pattern of AgNPs synthesized with the Appendix A) revealed the presence of elemental silver (Ag)
aqueous leaf extract of S. obvallata is shown in Fig. A.4(a) in and confirmed the successful formation of AgNPs.
Appendix A. Bragg reflection peaks were observed at 2q (q is The TEM images of AgNPs synthesized with the aqueous
the diffraction angle) values of 38.49 , 44.62 , 64.85 , and leaf extract of S. obvallata are shown in Fig. 3(a). The TEM
77.68 , corresponding to (111), (200), (220), and (311) lattice results revealed that AgNPs were in a spherical morphology
planes of the face centered cubic structure of AgNPs, with an average particle size of 17.0 nm. The selected area
respectively. The intense reflection of the (111) plane in electron diffraction (SAED) pattern (Fig. 3(b)) showed a ring-
contrast to other planes might specify the growth direction of like diffraction pattern, indicating the polycrystalline nature of
the nanocrystals. Moreover, with the DebyeeScherrer formula AgNPs (Kumar et al., 2016). The particle size distribution
(Eq. (1)), the crystallite size of the synthesized AgNPs was (Fig. A.5 in Appendix A) showed that the average particle size
calculated to be 16 nm, which was fitted to the Joint Com- was 17.7 nm.
mittee Powder Diffraction Standards (JCPDS) Card No. 04- Fig. 4(a) shows the XPS spectrum of the surface of AgNPs
0783 (Sheny et al., 2011). capping with the leaf extract of S. obvallata. As shown in

Fig. 3. TEM images of AgNPs synthesized with aqueous leaf extract of S. obvallata and SAED pattern.

Fig. 4. XPS spectra of AgNPs, Ag 3d, O 1s, and C 1s.

Please cite this article as: Vidya Sagar, P.S.R et al., Green synthesis of silver nanoparticles using aqueous leaf extract of Saussurea obvallata for efficient
catalytic reduction of nitrophenol, antioxidant, and antibacterial activity, Water Science and Engineering, https://doi.org/10.1016/j.wse.2023.09.004
6 P.S.R. Vidya Sagar et al. / Water Science and Engineering xxxx, xxx(xxx): xxx

Fig. 4(b) through (d), the elements of Ag, O, and C were The extract consisted of several organic constituents like
detected. The atomic concentration of O was slightly greater carboxylic and hydroxyl groups. In the Raman spectrum, the
than that of S. obvallata, but the atomic concentration of Ag bands at 1 363 and 1 606 cm
1 were obtained due to the
was low. This indicated that AgNPs were well protected by S. carboxylic symmetric and anti-symmetric C]O stretching
obvallata. Two peaks of Ag appeared at 365.6 and 371.6 eV, vibrations of the carboxylic group, respectively (Chen et al.,
corresponding to Ag 3d5/2 and 3d3/2 binding energies, 2009; Huang et al., 2008).
respectively. In comparison to Ag0 (368.3 and 374.3 eV), the AgNPs were synthesized with the aqueous leaf extract of
peaks were shifted to lower binding energies, indicating that S. obvallata as capping and reducing agents. Fig. 6 shows
the chemical environment around Ag atoms was changed. the plausible mechanisms for the formation of AgNPs using
Surface-enhanced Raman spectroscopy (SERS) is a helpful the leaf extract of S. obvallata as a reducing agent based
analytical tool for sensitive chemical analysis and interfacial on UVeVis and FTIR spectroscopic analyses. The car-
studies. Molecules adsorbed on rough silver surfaces provide boxylic group of doconexent present in the aqueous leaf
stronger SERS signals (viz., silver nanoparticles in various extract of S. obvallata acts as a reducing agent as well as a
shapes). Since the last decade, researchers have focused on capping agent. The eOH group of doconexent reduces Agþ
manipulating the size and form of nanomaterials, which af- ions to AgNPs, and the eCOO
group acts as a capping
fects their physical, chemical, and optical properties. Fig. 5 for AgNPs.
shows the Raman spectrum of AgNPs. It consisted of vibra-
tional modes at 146, 401, 518, 639, 1 363, and 1 606 cm
1. 3.2. Reduction of 4-nitrophenol to 4-aminophenol with
AgNPs

The catalytic activity of AgNPs synthesized with the

aqueous leaf extract of S. obvallata was investigated in terms
of the reduction of 4-NP to 4-AP in the presence of excess
NaBH4. This reduction reaction occurred via the formation of
intermediate 4-nitrophenolate ions (Fig. 7(a)). Usually, the
NaBH4 concentration was greater than the 4-NP concentra-
tion during the catalytic reduction reaction. Thus, this reac-
tion was the pseudo-first-order reaction with respect to 4-NP.
The reduction of 4-NP to 4-AP using the aqueous leaf extract
of S. obvallata as a control without AgNPs in the presence of
NaBH4 was initially investigated for 24 h, and 4-NP was not
Fig. 5. Raman spectrum of AgNPs synthesized with aqueous leaf reduced to 4-AP in the presence of excess NaBH4 (Fig. 7(b)).
extract of S. obvallata. Afterwards, the synthesized AgNPs were used as a catalyst

Fig. 6. Plausible reduction mechanisms for formation of AgNPs with aqueous leaf extract of S. obvallata as reducing agent and capping of plant
extract through carboxyl group.

Please cite this article as: Vidya Sagar, P.S.R et al., Green synthesis of silver nanoparticles using aqueous leaf extract of Saussurea obvallata for efficient
catalytic reduction of nitrophenol, antioxidant, and antibacterial activity, Water Science and Engineering, https://doi.org/10.1016/j.wse.2023.09.004
 P.S.R. Vidya Sagar et al. / Water Science and Engineering xxxx, xxx(xxx): xxx 7

Fig. 7. Formation of 4-AP from 4-NP using AgNPs in presence of NaBH4, reduction of 4-NP using plant extract in presence of NaBH4 without
AgNPs, reduction of 4-NP using AgNPs in presence of NaBH4, and kinetics of reduction.

for the reduction of 4-NP to 4-AP in the presence of NaBH4. concentration of AgNPs was ascribed to an increase in the
When 10 mg of AgNPs was introduced to the reaction number of active reaction sites for the reduction of 4-NP
mixture, the intensity of the absorption peak at 400 nm (Palem et al., 2016).
decreased, a new peak at 300 nm was observed, and its in-
tensity increased with time (Fig. 7(c)), indicating the effec- 3.3. Antioxidant activity
tive reduction of 4-NP to 4-AP. After the reaction was
completed, AgNPs were collected from the reaction system The antioxidant activity of AgNPs was investigated with
through centrifugation and successive washing with deion- the DPPH assay method. AgNPs were used as a radical
ized water and dried for use in the next cycle. As shown in scavenger, and DPPH was used as a radical source. The color
Fig. 7(d), the kinetics of the reduction as a function of of the DPPH solution was changed from deep violet to pale
catalyst dose was obtained as a linear plot from the pseudo- yellow in the presence of AgNPs. The absorbance of DPPH at
first-order kinetic model (ln (At/A0) versus time (t), where 517 nm gradually decreased with an increase in the concen-
At and A0 are the absorbances at time t and the initial time, tration of AgNPs, which further confirmed the free radical
respectively). The rate constant (k) of the reduction of 4-NP scavenging activity of AgNPs.
using different doses of AgNPs was determined (Fig. 7(b) As shown in Table 3, the antioxidant efficiency of AgNPs
and Table 2). An increase in the rate constant with the was dose-dependent, and the free radical scavenging activity

Table 2 Table 3
Kinetic parameters obtained from reduction of 4-NP to 4-AP using AgNPs Antioxidant efficacy of different concentrations of AgNPs synthesized with
synthesized with aqueous leaf extract of S. obvallata. aqueous leaf extract of S. obvallata against DPPH.
Sample Amount k (min
1) R2 Time for Sample Concentration Percentage of
number of completion number of AgNPs (mg/mL) DPPH scavenging effect (%)
AgNPs of reduction 1 100 59.45 ± 0.01
(mg) of 4-NP (min) 2 200 59.56 ± 0.02
1 5 0.147 7 0.983 4 24 3 300 60.33 ± 0.04
2 10 0.159 6 0.971 8 14 4 400 60.44 ± 0.03
5 500 61.21 ± 0.02
Note: R2 is the coefficient of determination.

Please cite this article as: Vidya Sagar, P.S.R et al., Green synthesis of silver nanoparticles using aqueous leaf extract of Saussurea obvallata for efficient
catalytic reduction of nitrophenol, antioxidant, and antibacterial activity, Water Science and Engineering, https://doi.org/10.1016/j.wse.2023.09.004
8 P.S.R. Vidya Sagar et al. / Water Science and Engineering xxxx, xxx(xxx): xxx

of AgNPs exhibited up to 61.21% ± 0.02% at a 500-mg/mL 4. Conclusions

concentration of AgNPs (Abdel-Aziz et al., 2014).
This study was a first attempt to develop an eco-friendly
3.4. Antimicrobial activity and convenient green method for the synthesis of AgNPs
with the leaf extract of S. obvallata. The size of AgNPs and
The antibacterial activity of the synthesized AgNPs was the formation rate can be monitored by varying the concen-
investigated with 3% dimethylsulfoxide (DMSO) as a nega- tration of the plant leaf extract, the AgNO3 concentration, and
tive control. The negative control and 5% leaf extract did not pH. The leaf extract of S. obvallata was suitable for the green
show antimicrobial activity (Fig. 8). The antimicrobial ac- synthesis of AgNPs within 20 min. As a result, spherical and
tivity of AgNPs was found to be dose-dependent. As the highly crystalline AgNPs were obtained. The UVeVis spectral
concentration of AgNPs was increased, the antimicrobial analysis indicated the formation of AgNPs. The XRD and
activity of AgNPs also increased due to an increase in the SEAD analyses confirmed the formation of highly spherical
interaction between AgNPs and sulfur containing the proteins structured AgNPs. In addition, the formation and stabilization
of bacteria, resulting in cell death (Feng et al., 2000). Hence, of AgNPs with the aqueous leaf extract of S. obvallata were
the concentrations of AgNPs represented in Table 4 were identified by FTIR and Raman analyses. The catalytic activity
compared. AgNPs at a concentration of 1 mg/mL were found of the synthesized AgNPs was investigated through the
to have antimicrobial activity against the Gram-negative reduction of 4-NP to 4-AP with NaBH4 as a reducing agent.
bacteria E. coli (12 mm) and Gram-positive bacteria (E. Thus, the synthesized AgNPs showed significant catalytic
faecalis) (13 mm) (Fig. 8) (Ibrahim, 2015). The bacterial activity. The DPPH method was used to quantify the antiox-
interaction of AgNPs can generally be described with three idant activity of the synthesized AgNPs, and the percentage of
possible reasons. First, there could be electrostatic attraction inhibition increased with the concentration of AgNPs. This
between positively charged membrane proteins present on the confirmed the antioxidant activity of AgNPs. The synthesized
surface of bacteria and negatively charged AgNPs (stabilized AgNPs possessed potent antibacterial activity against E. fae-
carboxylate) during the interaction. Second, there could be calis and E. coli bacterial strains.
physicochemical changes in the bacterial cell wall, which
may cause the extrusion of intracellular materials and cell Declaration of competing interest
death. Third, AgNPs tend to penetrate bacterial membranes.
As a result, AgNPs may prevent DNA replication and The authors declare no conflicts of interest.
respiration of bacteria cells and may cause bacterial cell
death (Agnihotri et al., 2014).
Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.wse.2023.09.004.

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Please cite this article as: Vidya Sagar, P.S.R et al., Green synthesis of silver nanoparticles using aqueous leaf extract of Saussurea obvallata for efficient
catalytic reduction of nitrophenol, antioxidant, and antibacterial activity, Water Science and Engineering, https://doi.org/10.1016/j.wse.2023.09.004