Plant mediated green synthesis, catalytic activity and antibacterial assay of silver nanoparticles

Article abstract of DOI:10.14233/ajchem.2019.22085

Plant mediated green synthesis of silver nanoparticles (AgNPs) was carried out with the plant extract of Vitex negundo Linn., a simple method without using harmful chemicals. Herein, a root bark extract is used as reducing and capping agent for synthesizi

Full text of DOI:10.14233/ajchem.2019.22085

Asian Journal of Chemistry; Vol. 31, No. 9 (2019), 2037-2041
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.22085
Plant Mediated Green Synthesis, Catalytic Activity and Antibacterial Assay of Silver Nanoparticles
1
2,3
1
1
1,*
M. REDDEPPA , R. CHENNA KRISHNA REDDY , P. R. SOUGANDHI , S.S. HARINI and T. SHOBHA RANI
1
2
3
Department of Chemistry, Dravidian University, Kuppam-517425, India
Department of Chemistry, Sri Venkateswara University, Tirupati-517502, India
Department of Science & Humanties, S.V. College of Engineering, Karakambadi Road, Tirupati-517507, India
*Corresponding author: E-mail: sobha.kaushu@gmail.com
Received: 18 March 2019; Accepted: 23 April 2019;
Published online: 31 July 2019;
AJC-19494
Plant mediated green synthesis of silver nanoparticles (AgNPs) was carried out with the plant extract of Vitex negundo Linn., a simple
method without using harmful chemicals. Herein, a root bark extract is used as reducing and capping agent for synthesizing AgNPs from
silver nitrate solution. The characterization ofAgNPs were obtained by UV-visible spectrophotometer, X-ray diffraction, Fourier transform
infrared spectroscopy, scanning electron microscopy and energy dispersive spectroscopy. The results were confirmed the presence of
silver nanoparticles with UV-visible absorption peak at around 433-443 nm and its crystalline nature by XRD analysis. The FTIR spectrum
confirmed that the synthesized AgNPs are stabilized and protected by various functional groups of biomolecules in extract. The SEM
morphology showed the triangular or spherical shape of AgNPs with the size in the range of 80-100 nm and EDS confirmed the presence
of elemental silver in the sample. The catalytic activity ofAgNPs was investigated in the synthesis of 5-substituted 1H-tetrazole. Furthermore,
antibacterial profile showed that biopotent AgNPs exhibited moderate to good antibacterial activity.
Keywords: Green synthesis, Vitex negundo L., Silver nanoparticles, Catalytic activity, Antimicrobial activity.
particular, silver nanoparticles are excellent antimicrobial agents
and catalysts in many chemical reactions [10].
INTRODUCTION
Nanotechnology considered that the nanoparticles acting
Because of huge applications in various fields, metallic
nanoparticles can be synthesized by physical, chemical and
biological methods. Physical and chemical approaches of
synthesis includes laser ablation, pyrolysis, decomposition,
electrochemical methods, microwave assisted techniques, sol-
gelandwetchemicalprocedures [11,12]. However, thoseexpensive
methods have toxic impact on human health, which restricts
their applications in many areas [13,14]. Hence, there is an
urgent need to promote green eco friendly processes in
chemistry and its technologies to minimize the generation of
hazardous and toxic materials to establish a sustainable process
as bridge in between atomic structures and bulk size materials
1]. In recent years, noble metal nanoparticles such as gold,
[
platinum and silver has been extensively studied due to their
unique size dependent properties, large specific surface area
and their important biological applications in the scientific
fields of bioengineering, optoelectronics, catalysis and bio-
sensing [2]. Among these noble metal nanoparticles, silver is
the most important nanomaterials for the researchers in which
5
00 tons of silver nanoparticles (AgNPs) were produced per
year and is estimated to grow in the upcoming years [3]. It
has been acknowledged that silver nanoparticles have gained
considerable attention for their unique nature and high perfor-
mance in the different scientific fields like solar energy, optical
receptors [4,5], energy storage devices, batteries, catalysis in
chemical synthesis [6], food processing industry [7], bio-labeling
and antibacterial agents [8,9]. In ancient times, silver metal is
used to treat open wound infections and chronic ulcers and in
[
15]. Recently, green methods of synthesis using renewable
materials, non-toxic chemicals, environmentally benign solvents
are used to synthesize metallic nanoparticles [16,17]. So, these
biological methods of nanoparticle synthesis are devoted as
eco friendly green method when compared to the other physical
and chemical approaches.
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2
038 Reddeppa et al.
Asian J. Chem.
Bio-molecules from several biological resources are used
this study were cleaned with HNO
3
and distilled water. Root
to synthesize metallic nanoparticles by biological methods. It
is well known that the extracts derived from various parts of
plant consists of different biomolecules will act as reducing
and capping agents in green synthesis of metallic nanoparticles
in a variety of shapes and sizes. This method of biosynthesis
is a cost effective for large scale production which consists of
utilization of environmentally benign plant extracts which
contain complex biomolecules [18]. Therefore, in this green
method of synthesis different types of non-toxic herbal chemicals
like flavones, terpenes, aldehydes, ketones, carboxylic acids
and amides are directly involved in the reduction of silver ions
into silver nanoparticles [19].
Heterocyclic compounds were bagged great importance
in the form of pharmaceuticals, agrochemicals, additives and
modifiers due to their biological properties [20]. Researchers
continuously searching for new synthetic protocols for the
synthesis of heterocyclic compounds and are succeeded with
the field of nano catalysis which involves the use of metallic
nanoparticles as catalysts under various conditions[21].Tetrazoles
are five membered heterocyclic compounds which contain four
nitrogen atoms and one carbon. It is an isosteric replacement
for carboxylic acids having a wide scope of biological and
commercial applications [22,23]. In concern with its wide appli-
cations, several methods of synthesis of tetrazoles are well
documented in the literature. Silver nanoparticles synthesized
by biological method are used as catalysts for the synthesis of
tetrazole analogs [24,25].
bark of Vitex negundo was collected in the vicinity of Punganur
town, Chittoor District, India.
Preparation of root bark extract: The healthy root bark
from Vitex negundo plant was collected for the green synthesis
of silver nanoparticles. It was washed thoroughly with running
tap water first and then with double distilled water and later
dried for a few days properly at room temperature. The dried
root bark was crushed into tiny pieces and grounded into fine
powder. Fine powder (5 g) was dissolved in 100 mL of Milli-Q
water in an Erlenmeyer flask. The above mixture is constantly
rotated with the help of magnetic stirrer with a hot plate at 80
ºC for about 0.5 h. After it was allowed to room temperature
the mixture was filtered by using Whatman filter paper No.1
and the filtrate was kept in refrigerator at 4 ºC for doing further
experiments.
Preparation of silver nanoparticles:The root bark extract
(20 mL) from Vitex negundo was added to 80 mL of 1 mM
AgNO solution in 250 mL Erlenmeyer flask followed by stirring
3
at room temperature for 0.5 h with the help of magnetic stirrer.
The change of colour of the solution from brown to dark brown
was observed and kept under incubation for 24 h after covering
the Erlenmeyer flask with silver foil. Finally, silver nanoparticles
were collected through centrifugation and drying at hot air oven.
Characterization of silver nanoparticles: The absor-
ption maxima of green synthesized nanoparticles were studied
by UV-visible spectrophotometer at wavelength range from
200-700nm. FT-IR spectra were recorded in the range of 4000-
-1
Vitex negundo commonly called as Chinese chaste tree or
five leaved chaste tree or Monk′s pepper′ belongs toVerbenaceae
family. This aromatic shrub is source for flavonoids, flavones,
glycosides, volatile oil, tri terpenes, tannins and many others
400 cm and the average particle size was measured with the
help of XRD instrument by using a powder diffractometer.
Morphological characterization of the sample was measured
using scanning and electron microscopy and energy dispersive
spectroscopy (EDS) techniques.
[
26]. Though, all parts of Vitex negundo are used in the indige-
nous system of medicine, the leaves are the most potent for
medicinal use. The roots of plant are long woody, cylindrical,
tortuous with grey brown colour possessing medicinal values
of tonic, febrifuge, diuretic, expectorant antirheumatic and
useful as a demulcent in dysentery, colic, incephalalgia, uropathy,
otalgia, wound and ulcers. The extract of root bark is extensively
used as a source of herbal medicine for the presence of medi-
cinal phytochemicals [27-29]. Considering the importance of
metal nanoparticle synthesis using different plant extracts, the
present study is aimed at synthesizing the AgNPs by Vitex
negundo root bark extract at room temperature without using
toxic chemicals. The method applied for synthesis is simple,
rapid and environmentally benign. After going through the
literature survey, as per the authors′ knowledge Vitex negundo
has not yet been utilized for the synthesis of silver nanopar-
ticles. In addition, we report the catalytic utility of silver nano-
particles for the synthesis of 5-substituted 1H-tetrazoles and
antibacterial activity against some bacterial strains.
Preparation of 5-(4-fluorophenyl)-1H-tetrazole: Briefly,
4-fluorobenzonitrile (2.0 mmol), sodium azide (2.5 mmol) and
AgNPs (20 mol %) were taken into a round-bottomed flask
and dissolved with 5 mL of glycerol. The reaction mixture was
stirred for 4 h at 95 ºC and examined the growth of reaction by
TLC. Then after completion reaction, pH of reaction mixture
was adjusted with 10 mL of HCl (0.1 N) and then extracted with
ethyl acetate (3 × 18 mL). The crude product of 5-(4-fluoro-
phenyl)-1H-tetrazole (Scheme-I) was obtained after washing
with water, drying with anhydrous Na
2
SO and concentrated
4
under reduced pressure. Finally, this crude product was purified
by column chromatography using 50 % ethyl acetate:n-hexane
as an eluent and subsequently characterized by mass, elemental
1
analysis and H NMR techniques. Colour: white solid, yield:
1
81 %, m.p.: 75-77 ºC, H NMR (400 MHz, DMSO-d
6
): δ 8.20-
8.26 (m, 2H, J = 7.6 Hz, Ar-H), 7.50-7.59 (m, 2H, J = 7.6 Hz,
Ar-H), 3.31 (br s, 1H, NH, overlap with solvent) ppm. LC-MS
+
(positive) m/z (%): 165 [M+H] (100 %). Anal. calcd. (found)
7 5 4
for C H N F: C, 51.22 (39.80); H, 3.07 (2.28); N, 34.13 (38.45).
EXPERIMENTAL
Vitex negundo root bark extract was used as reducing and
stabilizing agent to synthesize silver nanoparticles. Analytical
N
F
CN
N
N
AgNPs, Glyceral
95 oC, 4h
F
grade AgNO
3
was purchased from Sigma-Aldrich, Nutrient
+
N
H
and Muller-Hilton agar from Himedia and DMSO was procured
from SD Fine-Chem. Limited, India. The glasswares used for
NaN3
Scheme-1: Synthesis of 5-(4-fluorophenyl)-1H-tetrazole

Vol. 31, No. 9 (2019)
Plant Mediated Green Synthesis, Catalytic Activity and Antibacterial Assay of Silver Nanoparticles 2039
Antibacterial assay: The AgNPs synthesized from Vitex
negundo root bark extract were tested for their antibacterial
activity by agar well diffusion method [30] with small modifi-
cations [31]. The bacterial strains used for examination was
against Staphylococcus aureus, Bacillus subtilis, Pseudomonas
aeruginosa and Escherichia coli. Seeded agar medium was
prepared using Muller-Hilton, sterilized and allowed to cooling
4.5
4
.0
.5
3
3.0
2.5
7
to room temperature. To this 0.1 mL of diluted in oculum (10
cfu/mL) of test organism was added just before solidification
and then it was poured into sterilized plates under sterile condi-
tions. Then wells of 4 mm diameter were punctured with cork
borer maintaining aseptic environment. These wells were filled
with 10, 20, 30 and 40 µL of nanoparticle solution and solvent
DMSO is used as control. The plates were incubated at 35 ºC
for 18 h and then measured the zone of inhibition (in mm) against
test organisms. The standard drug benzyl penicillin at 20 µg/
mL concentration was used as positive control.
2
.0
1.5
1
.0
.5
0
0
3
50
450
Wavelength (nm)
Fig. 1. UV-visible spectra of green synthesized AgNPs
550
650
RESULTS AND DISCUSSION
Characterization of silver nanoparticles: Generally, in
all experiments related to plant mediated synthesis of metal
nanoparticles visual observation plays an important role to
judge its formation. The colour change from brown to dark brown
is noticed after treating the aqueous solution of plant extract
with silver nitrate. It has been identified that the yields of silver
nanoparticles depending on some parameters like concentration
of silver nitrate, concentration of plant extract, time, etc. Silver
nanoparticles were synthesized from root bark extract of Vitex
acid, amine or amide functional moieties were attributed to
alkaloids, phenolic compounds, flavanoids and fatty acids of
plant extract coated strongly on nanoparticle.
The crystalline nature of silver nanoparticles was confi-
rmed by XRD studies and the pattern showed 2θ angles at the
range of 38.41º, 44.53º, 64.72º and 77.96º corresponding to
the 111, 200, 220 and 311 (Fig. 3) confirmed the reflection
from face centered cubic stricture of silver crystal. From SEM
images (Fig. 4), it can be noticed that a well defined triangular
or spherical shapedAgNPs were formed without agglomeration.
Finally the strong signals of silver element and all silver peaks
were observed in the range between ~ 2.6-3.9 KeV on EDS
spectrum of green synthesized silver nanoparticles (Fig. 4).
Catalytic activity: Silver nanoparticles were utilized as
catalyst in (3+2) cycloaddition synthesis of 5-substituted 1H-
tetrazole. The quest for use of green methods in synthesis we
motivated to choose glycerol, a green solvent for the preparation
of 5-(4-fluorophenyl)-1H-tetrazole in good yields. Herein,
silver nanoparticles act as Lewis acid by activating nitrile group
which enhances its electro philic character which later combines
with sodium azide to produce tetrazoles [32]. The structure of
negundoand 1 mM ofAgNO were analyzed by using UV-visible
3
spectroscopy. It has been observed that a single plasmon resonance
band observed at 433-443 nm (Fig. 1) confirmed the formation
of AgNPs and no such peak was identified for UV spectra of
pure plant extract.
FT-IR analysis for pure root bark extract and silver nano-
particles were carried out to confirm the presence of biomolecules
and their dual role as reducing and capping agents. The spectrum
-
1
of pure extract showed major absorption peaks at 3314 cm
-1
-1
(
O-H str.), 2853 cm (C-H str.), 1630 cm (C=O str.), 1442
cm (O-H def.) and 1074 cm (Fig. 2a) confirms the presence
-1
-1
of plant biomolecules. The absorption peaks present at 3358,
-1
2
862, 1650, 1470 and 1095 cm (Fig. 2b) which revealed that
1
biomolecules were bound to silver nanoparticles and act as
capping agents. This results revealed that hydroxyl, carboxylic
newly synthesized tetrazole molecule was confirmed by H
NMR, mass spectral data and C,H,N analysis.
100
(A)
100
(B)
80
60
40
20
95
90
85
80
75
3500
3000
2500
Wavenumber (cm )
2000–
1500
1000
3500
3000
2500
Wavenumber (cm )
2000 – 1500
1000
1
1
Fig. 2. FT-IR spectra of (A) Pure Vitex negundo root bark extract (B) AgNPs capped by Vitex negundo root bark extract

2
040 Reddeppa et al.
Asian J. Chem.
Ag(111)
3
07.00
50.00
2
Ag(220)
Ag(200)
150.00
Ag(311)
50.17
-33.00
20
50
(°)
80
2
θ
Fig. 3. XRD patterns of synthesized silver nanoparticles
1
.00 K
.90 K
.80 K
.70 K
.60 K
.50 K
.40 K
.30 K
.20 K
.10 K
C Kα1
0
0
0
0
0
0
0
0
0
Ag M
α
α
Ag Lα
O K
1
Ag L
β
Ag L
β2
0
K
0
1.3
2.6
3.9
eZAF Smart quant results
Element Weight % Atomic % Net Int. Error % K ratio
5.2
6.5
7.8
9.1
10.4
11.7
13.0
C K
O K
Ag L
31.78
38.76
29.46
49.53
45.36
5.11
83.44
40.64
69.77
6.69
12.28
6.35
0.2260
0.0645
0.2500
Fig. 4. SEM-EDX analysis of synthesized silver nanoparticles
Antibacterial assay:The antibacterial assay of silver nano-
TABLE-1
ANTIBACTERIAL ACTIVITY OF GREEN
SYNTHESIZED SILVER NANOPARTICLES
particles synthesized using Vitex negundo root bark extract
was evaluated by agar well diffusion method. The biologically
synthesizedAgNPs have showed excellent range of activity at
the concentration of 40 µg/mL against all the tested organisms
such as Staphylococcus aureus, Bacillus subtilis, Pseudomonas
aeruginosa and Escherichia coli. The results of zone of inhibition
AgNPs
µg/mL)
S.
aureus
B.
subtilis
P.
E.
coli
(
aeruginosa
10
20
30
40
10 ± 1.1
12 ± 0.2
11± 0.6
13 ± 0.5
21 ± 1.5
10 ± 0.8
12 ± 1.2
14 ± 1.0
16 ± 1.4
23 ± 0.5
10 ± 1.2
11 ± 1.1
12 ± 0.5
14 ± 1.2
25 ± 1.0
11 ± 0.8
13 ± 0.6
14 ± 1.4
18 ± 1.6
22 ± 1.5
(
Table-1) was calculated and compared with standard drug
benzyl penicillin. Escherichia coli (18 ± 1.6 mm) and Bacillus
subtilis (16 ± 1.4 mm) showed excellent sensitivity (concentra-
tion ofAgNPs: 40 µg/mL) when compared with Staphylococcus
Std*
*
Standard drug: benzyl penicillin

Vol. 31, No. 9 (2019)
Plant Mediated Green Synthesis, Catalytic Activity and Antibacterial Assay of Silver Nanoparticles 2041
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Conclusion
The synthesis of silver nanoparticles by Vitex negundo
root bark extract is reported via green chemistry approach which
provides efficient, eco-friendly, cost effective ways for the synt-
hesis of metallic nanomaterials. The characterization results
confirmed that the reduction of silver ions to silver nanoparticles
when it was treated with plant extract. This biogenic silver
nanoparticles exhibited excellent catalytic activity for the
formation of 5-substituted 1H-tetrazole in good yields. Finally,
the antibacterial activity profile revealed their potentiality in
biomedical applications.
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ACKNOWLEDGEMENTS
(
2012);
The authors thank to Sri Jayachamarajendra College of
Engineering, Mysore, India and Mr. C.M. Narendra Reddy,
Department of Biotechnology, Dravidian University, Kuppam,
India, for providing instrumentation facilities and antibacterial
activities.
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