Environment Conservation Journal 25 (1):16-21, 2024

Journal homepage:https://www.environcj.in/

Environment Conservation Journal

ISSN 0972-3099 (Print) 2278-5124 (Online)

Antimicrobial activity of silver nanoparticles synthesized from

Wrightia tinctoria fruit extracts
Arpana Ashokrao Durge
Department of Biochemistry, Guru Nanak College of Science, Ballarpur (Maharashtra) India
Utpal Dongre
Department of Biochemistry and Biotechnology, Dr Ambedkar College, Deekshabhoomi, Nagpur (Maharashtra) India
Utkarsh Ravindra Moon
Department of Microbiology and Biotechnology, Mahatma Gandhi College of Science, Gadchandur (Maharashtra) India

ARTICLE INFO ABSTRACT

Received : 28 July 2023 Nanoparticles are synthesized by using various chemical methods in higher
Revised : 13 September 2023 yields but they are not very environmentally friendly and have hazardous
Accepted : 20 October 2023 effects on living cells. This can be attributed to the overuse of hazardous
chemicals involved in the process. The green approach of nanoparticle
Available online: 10 January 2024 synthesis is widely gaining attention worldwide as it is considered as nontoxic,
harmless and ecofriendly. Apart from this they also have multiple applications
Key Words: in various fields of science and technology. Nanoparticles synthesized by using
Antimicrobial activity various phytochemicals are also effective against a variety of microbial
Green synthesis populations. The objective of this study is to synthesize silver nanoparticles
Nanoparticles (AgNPs) from the fruit extracts of Wrightia tinctoria and evaluating its
Silver nanoparticles antimicrobial capacities against gram-positive and negative bacterial strains.
Silver nitrate Silver nanoparticles were synthesized using different solvent extracts of
Wrightia tinctoria Wrightia tinctoria pods. The formation of silver nanoparticles was noted by
detecting the change in color of the solution. The presence of nanoparticles was
detected by performing UV visible spectroscopy and monitoring the spectrum
from 400 nm to 800 nm. A small peak at 425 nm suggested the presence of silver
nanoparticles. In a later part of the study, the inhibitory effect of green
synthesized silver nanoparticles on the growth of E. coli and S. aureus was
monitored. The appearance of a zone of inhibition further confirmed the
antimicrobial effect of the synthesized nanoparticles.

Introduction
NPs are fine and thin particles ranging between 1 and biomolecules, such as amino acids and proteins,
100 nm in diameter (Mohanraj and Chen, 2006). sugars, certain enzymes and other traces of metals.
This process involves the collection of atoms bonded These metabolites are strongly involved in the
together to form a structure with a radius less than bioreduction process. Plant materials also contain
100 nm (Kumari, 2018). Metal nanoparticles ranging many phytochemicals, such as phenols, saponins,
in size possess different shapes. Metal nanoparticles terpenes, alkaloids, and alcohol, which tend to
of Au, Ag, Zn, Pt, Pd, Se, Ti, Cu, etc., have been reduce these metal salts (Adeyemi et al., 2019). Due
synthesized successfully (Khandel et al., 2018). To to the large surface area, the activity of the
make metal nanoparticles eco-friendly and in the nanoparticles is enhanced. The green synthesis of
quest for ‘going green’, various bioinspired methods nanoparticles can be an eco-friendly alternative for
for their synthesis have been employed. the production of nanoparticles. Natural plants have
Biocompatibility and environmentally benign been verified to have dual effects, acting both as
properties make green synthesized nanoparticles the capping agents and reducing agents, thus improving
preferred choice. Plant extracts carry a variety of the pharmacological effectiveness and constancy of

Corresponding author E-mail: [email protected]

Doi:https://doi.org/10.36953/ECJ.26582638
This work is licensed under Attribution-Non Commercial 4.0 International (CC BY-NC 4.0)
© ASEA
 Antimicrobial activity of green synthesized silver nanoparticles

NPs (Javed et al., 2020). Plant-based nanoparticles In this study, the focus was on the synthesis of silver
are gaining attention worldwide due to their multiple nitrate nanoparticles by using Wrightia tinctoria
applications in various fields of science and fruit extracts and their effect on the growth of gram-
technology. They are synthesized from different negative and gram-positive bacteria such as E. coli
plant extracts and are nonhazardous to the and S. aureus, respectively.
environment. In India, we have vast knowledge of
ayurveda since ancient times, which includes Materials and Methods
information on hundreds of plants with medicinal The following methods and materials were utilized
properties (Pandey et al., 2013). This knowledge is to synthesize silver nanoparticles and to evaluate
still being used by locals and medical practitioners their antimicrobial potential against gram-positive
to cure many health ailments. In the last few decades, and gram-negative bacteria.
the synthesis of plant-based nanoparticles (NPs) has
attracted increased amounts of attention due to the a. Selection of medicinal plants
ease of preparation. The ability of phytoconstituents Wrightia tinctoria, which belongs to the family
to act as reducing agents has made their use Apocynaceae, was selected for this study due to its
attractive (Mittal et al., 2013. Ankamwar, 2010). medicinal properties. This plant possesses a wide
Research conducted worldwide indicates that plant- range of phytochemicals that have antimicrobial
mediated nanoparticle synthesis methods progress properties, and the use of green synthesis of AgNPs
via quick extra or intracellular processes (Husen, is still unexplored. The plant was collected from
2017). Most phytoconstituents are found in various Gadchandur town located in Chandrapur district in
parts of plants, such as leaves and flowers. They are Maharashtra, India (19°41'26"N 79°10'31"E). The
also abundant in roots, fruits, and stems. Therefore, pods of the plants were collected in October.
the use of different plant parts has been reported for
the production of plant-mediated metal-based b. Preparation of plant extract:
nanoparticles (Husen, 2017). NPs synthesized from The plants were fresh and washed with tap water and
several phytochemicals are powerful against a twice with distilled water. Later, the plants were
variety of microbial populations. The green shed and dried for 30-35 days at a temperature of
synthesis of silver nanoparticles (SNPs) has been approximately 37-40°C. These were then cut into
proven to have numerous applications in scientific pieces after weighing and boiled with a mixture of
fields and shows great potential for antimicrobial methanol and distilled water to obtain a dark-colored
activity (for example, AgNPs) (Rai et al., 2009) [10]. extract. Methanol (80 ml) and distilled water (20 ml)
Silver nanoparticles (AgNPs) are among the most were mixed together and added to 7.24 g pieces of
studied metal nanoparticles. Its unique features and Wrightia tinctoria pods in a 200 ml beaker. Then, the
physical and chemical properties have led to its mixture was heated in a water bath by gradually
diverse application in medicinal fields (Zhang et al., increasing the temperature from 50°C to 130°C. The
2016). Moreover, nanoscale silver particles exhibit a mixture was heated at 50°C for the first hour, after
high surface-area-to-volume ratio (for which the size which the temperature was increased to 100°C, after
remains below 100 nm) and are of great interest due which the sample was heated for the next half an
to their strong antimicrobial effects on both gram- hour. Finally, the temperature was increased to
positive and gram-negative bacteria (Morones- 130°C, and the mixture was heated continuously
Ramirez et al., 2013. Meroni et al., 2020) viruses until the temperature was reduced to half of the
and other eukaryotic microorganisms (Gong, 2007) initial temperature.A similar procedure was repeated
compared to other metals in their nanosized forms. by replacing methanol with distilled water as a
Wrightia tinctoria is the plant used for this study and solvent.
belongs to the family Apocynaceae. The plant is
found in hilly regions in India, Australia and c.Biosynthesis/green synthesis of silver nano
Southeast Asia (Wrightia tinctoria - Wikipedia). It is particles using plant extracts:
a flowering plant, and its fruit show vast medicinal Methanol extracts from the fruits of the plants were
properties. obtained, mixed with silver nitrate solution and
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 Durge et al.

incubated overnight. Centrifugation was performed by measuring the zone of inhibition against E. coli
using a microcentrifuge at 5000 × g for 5 min. and S. aureus.
The extract prepared by this method was then Results and Discussion
filtered through filter paper to remove coarse The formation of silver nanoparticles was noted by
particles, and a brown-colored filtrate of detecting the change in color from light brown to
approximately 24.5 ml was collected in a 50 ml dark brown with silver shade (Fig. 1). The presence
beaker. Silver nitrate reagent was prepared by of AgNPs was tested by using UV‒visible
mixing 1.698 g of silver nitrate crystals in 100 ml of spectroscopy at 400 to 800 nm. A small peak at 425
distilled water (100 mM). The Wrightia tinctoria nm was observed, indicating the presence of AgNPs
extract (24.5 ml) was properly mixed with 100 ml of (Figure 2). The UV‒visible spectrum of the
silver nitrate solution and stirred for a few minutes, biosynthesis of silver nanoparticles using Wrightia
after which the color change from clear brown to tinctoria showed a peak at 425 nm corresponding to
turbid dark brown was observed. The mixture was the surface plasmon resonance of AgNPs for the
kept in the dark at room temperature for 24 h, the tested fruit extracts of Wrightia tinctoria. Peak
sample became turbid, and a precipitate was observations at 425 nm were also observed when the
collected at the bottom of the beaker. The mixture extracts of T. harzianum and T. viride were used for
was then centrifuged by using a microcentrifuge at the green synthesis of AgNPs (Elamawi et al., 2018).
5000 × g for 5 minutes. The sample was added to 15 UV‒visible absorption spectroscopy has been
ml microcentrifuge tubes. The sample was placed proven to be a sensitive method for the
into a microcentrifuge tube and centrifuged at 5000 determination of silver nanoparticles because of
× g for 5 minutes. The pellet obtained was collected surface plasmon excitation at this wavelength
by adding acetone to round bottom centrifuge tubes. (Zaheer et al., 2010). Phyllanthus acidus plant
The pellet was collected on a watch glass and kept extracts are also known to reduce silver nitrate and
overnight for evaporation. The crystals of the act as capping agents for the synthesis of AgNPs.
nanoparticles obtained weighed 1.5 g. The formation of nanoparticles was confirmed by
UV‒vis spectrophotometry at 425 nm. This is
d.UV‒visible spectroscopic analysis and anti justified by the formation of a surface plasmon
microbial activity of green-synthesized silver resonance band at 425 nm (Soumya et al., 2018).
nanoparticles The synthesis of AgNPs by sodium borohydride and
The resulting extracts were analyzed with a UV‒ Trigonella foenum-graecum leaf extract as reducing
visible spectrophotometer (Shimadzu-1780, Japan) and stabilizing agents has been confirmed. The
by scanning the sample between 440 and 800 nm. formation of AgNPs at 425 nm after overnight
The AgNPs were resuspended in distilled water, incubation with silver nitrate and leaf extracts has
after which their antimicrobial capacities were also been shown (Kishore et al., 2023). AgNPs have
studied. The broths of E. coli and S. aureus were also been used in the identification of mercury ions
prepared and incubated overnight at 37°C. Later, the in solution. The peak corresponding to the AgNPs
confluent cultures of E. coli and S. aureus were used in this process was also 425 nm in length and
spread on two agar plates carefully, and the wells was attributed to the AgNPs formed by green
were punctured by using sterilized gel puncture. The synthesis (Pomal et al., 2021). To further confirm the
silver nanoparticle sample was poured into the wells formation of silver nanoparticles by using this
created on both Petri plates. Three equidistant wells method, the sample was subjected to
were used for puncture. The silver nitrate solution characterization. This approach provides clear
was added to the first well. In the second and third information about the size and shape of the
wells, the silver nanoparticle test sample and the nanoparticles.The zone inhibition method was used
plant extract were added, respectively. These Petri to study the antimicrobial activity of the strains
plates were again kept overnight in an incubator at (Banerjee et al. 2022). A good zone of inhibition of
37°C. After this period, the Petri dishes were drawn 2.6 ± 0.2 cm (Table 1 and Fig. 3) was observed for
and observed for the appearance of zones of E. coli, and a zone of inhibition of 2.2 ± 0.1 cm
inhibition. The efficacy of the AgNPs was calculated (Table 2 and Fig. 3) was observed for S. aureus.
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 Antimicrobial activity of green synthesized silver nanoparticles

These findings suggest that the silver nanoparticles

that formed antimicrobial activity against these two
bacterial strains. AgNPs are known to have good
inhibitory effects on gram-positive and gram-
negative bacteria (Cavassin et al., 2015). These
antibacterial activities of the AgNPs can be
attributed to the physicochemical properties of the

Figure 1: Silver nanoparticles synthesized from

Wrightia tinctoria pod extracts

Table 1: Antimicrobial activity of silver nanoparticles

formed using methanol as a solvent

Microorganism Zone of inhibition (in cm) obtained with

Silver W T extract Silver
Nitrate (methanol) nanoparticles
(5 mg SNP in
0.5 ml D. W.)
E. coli (Gram 1.2 ± 1.6 ± 0.1 2.0 ± 0.1
Negative) 0.1
S. aureus (gram- 1.0 ± 1.2 ± 0.1 2.6± 0.2
positive) 0.1

Table 2: Antimicrobial activity of silver nanoparticles

formed using distilled water as a solvent Figure 3: Antimicrobial activity of methanol (A) and
distilled water (B) extracts
Microorganism Zone of inhibition (in cm) obtained with
Silver WT Silver nanoparticles
Nitrate extract The shape, size, chemical and charge of AgNPs It
(5 mg SNP in 0.5 ml
(distilled D. W.) can affect the interaction of particles with cell
water)
E. coli (Gram 1.3 ± 0.1 0.8 ± 0.1
membranes and even pass through the intracellular
2.2 ± 0.1
Negative) membrane of bacteria. The continuous release of
S. aureus (gram- 1.1± 0.1 0.8 ± 0.1 1.8 ± 0.1 silver ions from AgNPs can cause the death of
positive)
microbes (Bapat et al., 2007). Silver ions can bind to
the sulfur and phosphorous present in DNA. It can
also cling to the cell wall and plasma membrane of
microorganisms. Adherent ions are known to affect
the permeability of the cell membrane and may
generate reactive oxygen species, which can cause
oxidative stress, leading to disruption of the cell
(Khorrami et al., 2018). Modulation of the signal
transduction pathway leading to cell death is also
known. AgNPs cause dephosphorylation of
phosphotyrosines, which leads to cell signal
transduction and cell death. Aerobic conditions are
known to release Ag+ from the surface of particles;
Figure 2: UV‒visible spectrum showing the spectra of Ag+ interacts with the cell membrane and cell wall
the extract at 400-800 nm components of bacteria and plays a crucial role in

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 Durge et al.

cell death (Bapat et al., 2007). The antibacterial Thus, Wrightia tinctoria-assisted AgNPs will be
activities also depend on the sizes of the further coated with different agents, such as chitosan
nanoparticles formed (Lu et al., 2013). Among the and antibiotics, to enhance their overall antibacterial
sizes of nanoparticles formed (5 nm, 15 nm, and 55 capacities.
nm), the 5 nm AgNPs had the greatest inhibitory
effect on E. coli. This difference may be attributed Conclusion
to the oxidation of AgNPs in aqueous media when The objective of this study was to synthesize silver
exposed to air. This reduces the antimicrobial nanoparticles (AgNPs) from fruit extracts of
activity potential of large AgNPs. In contrast, the Wrightia tinctoria and evaluate their antimicrobial
larger nanoparticles showed greater antibacterial capacities. The AgNPs were synthesized from
activity, which was explained by the greater surface methanolic and distilled water extracts, and the
area available for the larger nanoparticles (Agnihotri formation of AgNPs was confirmed by observing the
et al., 2014). The positively charged AgNPs interact change in color to dark brown with silver shade. The
with the negatively charged bacterial cells to inhibit UV‒visible spectrum showed a peak at 425 nm,
their growth. The antibacterial activities of AgNPs which indicated the formation of AgNPs. The
are also affected by the process of synthesis. The antimicrobial potential of the AgNPs was further
capping agent used for the synthesis may influence tested against pathogenic strains of E. coli and S.
the effectiveness of the AgNPs as antimicrobial aureus. The AgNPs synthesized from the extracts of
agents. The organic extracts used were described by Wrightia tinctoria showed a good zone of inhibition
Murei et al., 2020, who confirmed the enhanced and hence promising antimicrobial potential against
activities of the synthesized AgNPs. When E. coli (2.2 ± 0.1 cm, gram-negative) and S. aureus
conjugated with AgNPs and antibiotics such as (2.6 ± 0.2 cm, gram-positive).
ampicillin, the extracts of Pyrenacantha grandiflora
showed a very low minimum inhibitory Conflict of interest
concentration compared to that of AgNPs alone. The authors declare that they have no conflicts of
This was due to the synergistic effects of the interest.
antibiotic-coated AgNPs.

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