Catalytic, antibacterial and antibiofilm efficacy of biosynthesised silver nanoparticles using Prosopis juliflora leaf extract along with their wound healing potential

Article abstract of DOI:10.1016/j.jphotobiol.2018.11.005

The present study focuses on the catalytic, antibacterial and antibiofilm efficacy of silver nanoparticles (AgNPs) in an easy, rapid and eco-friendly pathway. Herein, we have synthesised AgNPs using an aqueous extract of P. juliflora leaf. The bioactive compounds present in the extract are responsible for the reduction of Ag⁺ to Ag⁰. The particle synthesis was first observed by visual color change and then characterized using UV–visible spectroscopy to confirm the formation of AgNPs. The synthesis conditions were then optimised using critical parameters such as reaction time, AgNO₃ concentration, extract to AgNO₃ ratio and temperature of the reaction. The hydrodynamic size of the AgNPs with Dynamic light scattering (DLS) was 55.24 nm, while, was in the range of 10–20 nm as determined through Transmission Electron Microscopy (TEM). Further, Fourier transform infrared spectroscopy (FTIR) studies were conducted to discern the functional groups or compounds responsible for the reduction of silver nitrate as well as the capping of silver nanoparticles. Later, X-ray diffraction (XRD) results showed crystalline nature of the biosynthesized AgNPs. To evaluate their antibacterial potential, AgNPs were assessed through disc-diffusion assay, which resulted in an appreciable dose-dependent activity. The antibacterial potential was investigated through disc-diffusion assay against E. coli and P. aeruginosa. The Congo red agar (CRA) plate assay successfully revealed the anti-biofilm activity against B. subtilis and P. aeruginosa. Further, the catalytic activity of synthesised AgNPs was assessed against azo dyes such a Methylene Blue (MB) and Congo Red (CR) that resulted in its effective degradation of toxic compounds in a short span of time. Further, AgNPs were assessed for their wound healing potential.

Full text of DOI:10.1016/j.jphotobiol.2018.11.005

Accepted Manuscript
Catalytic, antibacterial and antibiofilm efficacy of biosynthesised
silver nanoparticles using Prosopis juliflora leaf extract along with
their wound healing potential
Geeta Arya, R. Mankamna Kumari, Nikita Sharma, Nidhi Gupta,
Ajeet Kumar, Sreemoyee Chatterjee, Surendra Nimesh
PII:
S1011-1344(18)30716-4
DOI:
Reference:
https://doi.org/10.1016/j.jphotobiol.2018.11.005
JPB 11395
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date:
Revised date:
Accepted date:
3 July 2018
6 November 2018
9 November 2018
Please cite this article as: Geeta Arya, R. Mankamna Kumari, Nikita Sharma, Nidhi
Gupta, Ajeet Kumar, Sreemoyee Chatterjee, Surendra Nimesh , Catalytic, antibacterial
and antibiofilm efficacy of biosynthesised silver nanoparticles using Prosopis juliflora
leaf extract along with their wound healing potential. Jpb (2018), https://doi.org/10.1016/
j.jphotobiol.2018.11.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Catalytic, Antibacterial and Antibiofilm Efficacy of Biosynthesised Silver
Nanoparticles Using Prosopis Juliflora Leaf Extract Along with Their
Wound Healing Potential
Geeta Arya1, R. Mankamna Kumari1, Nikita Sharma1, Nidhi Gupta2, Ajeet Kumar3,
Sreemoyee Chatterjee2, Surendra Nimesh1,*
1Department of Biotechnology, School of Life Sciences, Central University of Rajasthan,
Ajmer 305817, India
2Department of Biotechnology, The IIS University, Gurukul Marg, SFS, Mansarovar, Jaipur
302020 Rajasthan, India
3Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam,
NY 13699-5814
*Corresponding author
Dr. Surendra Nimesh
Department of Biotechnology,
School of Life Sciences,
Central University of Rajasthan,
Bandarsindri, N.H. 8, Teh.-Kishangarh,
Dist. - Ajmer - 305817, Rajasthan, India
Tel. - +91-9468949252, Fax: +91-1463-238722
Email: surendranimesh@gmail.com, surendranimesh@curaj.ac.in

ACCEPTED MANUSCRIPT
Abstract
The present study focuses on the catalytic, antibacterial and antibiofilm efficacy of silver
nanoparticles (AgNPs) in an easy, rapid and eco-friendly pathway. Herein, we have synthesized
AgNPs using an aqueous extract of P. juliflora leaf. The bioactive compounds present in the
extract are responsible for the reduction of Ag+ to Ag0. The particle synthesis was first observed
by visual color change and then characterized using UV-visible spectroscopy to confirm the
formation of AgNPs. The synthesis conditions were then optimized using critical parameters such
as reaction time, AgNO3 concentration, extract to AgNO3 ratio and temperature of the reaction.
The hydrodynamic size of the AgNPs with Dynamic light scattering (DLS) was 55.24 nm while
was in the range of 10-20 nm as determined through Transmission Electron Microscopy (TEM).
Further, Fourier transform infrared spectroscopy (FTIR) studies were conducted to discern the
functional groups or compounds responsible for reduction as well as capping of silver nitrate.
Later, X-ray diffraction (XRD) results showed crystalline nature of the biosynthesized AgNPs. To
evaluate their antibacterial potential, AgNPs were assessed through disc diffusion assay, which
resulted in an appreciable dose dependent activity. The antibacterial potential was investigated
through disc diffusion assay against E. coli and P. aeruginosa. The CRA plate assay successfully
revealed the anti-biofilm activity against Bacillus subtilis and Pseudomonas aeruginosa. Further,
catalytic activity of synthesized AgNPs was assessed against azo dyes such a Methylene Blue
(MB) and Congo Red (CR) that resulted in its effective degradation of toxic compounds in a short
span of time. Further, AgNPs were assessed for their wound healing potential.
Keywords: Silver nanoparticles, Prosopis juliflora, antibacterial, antibiofilm, catalytic,
wound healing.

ACCEPTED MANUSCRIPT
1. Introduction
Organic dyes, one of the major effluent from textile industries, consists of mutagenic and
carcinogenic constituents that are considered as the most hazardous pollutant worldwide(1). The
presence of coloured dyes in the discharged effluent reduces the penetration of light in the water
bodies, and further disturbs the photosynthesis and development of aqua communities (2, 3).
These organic dyes cannot be degraded easily due to their greater stability and remain in
environment for long period of time. These toxic dyes pose several environmental risks and leads
to many health issues including liver and kidney damage, skin problems, central nervous system
poisoning and various blood disorders(4, 5). Although various physical and conventional methods
are being used such as absorption, precipitation and ozonation to decolorize and treat the effluent
but these methods require high cost and energy and are associated with harmful side products(6,
7). So, there is a need to develop an environmentally safe method to combat this problem.
Nanotechnology has emerged out as a promising approach in environmental remediation.
Among nanomaterials, metal nanoparticles plays a major role in this field(8). In particular, silver
nanoparticles (AgNPs) are gaining attention owing to their unique catalytic, electrical and optical
properties depending upon their shape and size(9). Additionally, AgNPs can be employed as anti-
fungal, anti-bacterial, anti-inflammatory, anti-viral, anti-angiogenesis, antiplatelet and as cancer
theranostic. Also, silver is known to have a recorded history of its medical and therapeutic benefits
more than its limitations which date back to the period before realization of microbes as the source
of infections. These particles also exhibit surface Plasmon resonance (SPR) that resulted in a
characteristic band that occurs due to collective oscillation of free electrons in resonance with
frequency of the light in visible and infrared region(10-12).
Numerous physical and chemical approaches have been established for AgNPs synthesis, but
their use is limited because of involvement of high cost and energy, stringent conditions along
with the risk pose using hazardous chemical and their bi-products. Recently, green synthesis
approach using plant extract, has gained importance as it involves the use of cost effective, eco-
friendly and non-toxic products(13). The bioactive compounds such as alkaloids, phenolics,
tannins, terpenoids, amino acids and proteins which are ubiquitously found in plants, are
responsible for the reduction of Ag ions into AgNPs and their stabilization by capping. A variety
of plant extract such as Gloriosa superb, Terminalia arjuna, Cordia dicotoma, Canarium ovatum,
Prosopis juliflora bark, Cicer arientinum leaf, coffee bean Olax scandens leaf and many more
have been used for AgNPs synthesis(14-21).
The present work focuses on the biosynthesis of AgNPs using leaf extract of Prosopis
juliflora. This plant belongs to Fabaceae family and has been used since ancient time due to its

ACCEPTED MANUSCRIPT
medicinal value against several digestive, rheumatic and skin diseases. These beneficial properties
have been attributed to the bioactive compounds such as flavonoids, alkaloids and phenolic agents
present in the plant(22). Here, in the current study we have focused on biosynthesis of AgNPs
with optimised parameter conditions. Further, the AgNPs were characterized through UV-vis
spectroscopy, FTIR, DLS and zeta analysis. Biological activity was also investigated against E.
coli and P. aeruginosa, known for multi-drug resistant properties. For anti-biofilm activity of
these AgNPs were assessed against gram-positive bacteria Bacillus subtilis and gram-negative
bacteria Pseudomonas aeruginosa. Catalytic potential of these AgNPs were confirmed from
degradation of congo red, methylene blue and conversion of 4-nitrophenol to non-toxic 4-
aminophenol. To evaluate the wound healing potential the excision and treatment study on mice
model were performed.
2. Experimental
2.1. Materials
Silver nitrate (AgNO3), Luria Agar, Luria broth, Kanamycin were purchased from Central
Drug House, India. 4-nitrophenol, Congo red, methylene blue and sodium borohydride were
purchased from SRL, India. The leaf samples of P. juliflora were collected from Central
University of Rajasthan campus, Ajmer. Double distilled water was used for the preparation of
leaf extract.
2.2. P. juliflora leaves sampling and extract preparation
Fresh healthy leaves of P. juliflora were procured from the campus of Central University of
Rajasthan Bandarsindri, Kishangarh, Ajmer in the month of January. The leaves were washed,
dried and grounded. 10g of this powder was weighed and boiled in 100 ml of ddH2O at 60ºC
(Stuart UC152, Biocote) for 20 min. The obtained mixture was then cooled and the filtrate was
obtained in reduced pressure condition (Lab. Companion VE-11, Korea) using Whatman filter
paper no. 1. The filtrate was further stored at 4°C for future use.
2.3. Biosynthesis of silver nanoparticles
The synthesis reaction was performed by dropwise addition of this extract into aqueous
AgNO3 solution under continuous stirring at 500 rpm. The reaction was then observed for a color
change. Further, conditions were optimised under various parameters that play a crucial role in the
synthesis of silver nanoparticles. The synthesised AgNPs were then separated by centrifugation at
10000 rpm (Heraeus, Fresco 17, Thermo scientific) for 10 min. The obtained pellet was washed
thrice, re-dispersed in milli-Q water.
2.4. UV-vis Spectrophotometric analysis

ACCEPTED MANUSCRIPT
The synthesis of AgNPs was confirmed after assessment by UV-visible spectrophotometer. The
scanning was done in the range of 300 to 700 nm due to unique optical properties of AgNPs. The
procured data gives the rough estimation about the size and morphology of nanoparticles that helps
in the optimisation studies of reaction parameters.
2.5. Optimisation of different parameters
Further, various crucial parameters that are essential for the synthesis of AgNPs were optimized
including time point of the reaction, ratio of extract to AgNO3, concentration of AgNO3 and finally
temperature of the reaction that controls the size, morphology, yield and agglomeration state. The
UV-visible spectrophotometer was used to generate the scanning data of synthesis reaction at
different time points (10, 20, 40, 80, 120, 160 and 200 min), after centrifugation of reaction
mixture followed by washing and sonication. The other three parameters were kept constant. The
spectrum was evaluated for primary study and the optimised time was used for further reactions.
Similarly, different concentrations of silver nitrate solution (0.1, 0.5, 1.0, 1.5 and 2 mM) were
prepared and used for the biosynthesis reaction and assessed by UV-visible spectrophotometer.
Correspondingly, varying ratio of extract to AgNO3 were taken as 1:40, 1:20, 1:10, 1:6.5 and 1:5
for the optimisation and the other parameters were kept constant during the biosynthesis, further
determined through spectroscopically. The temperature optimisation was done at 4°C, 25°C, 40°C,
60°C, and 80°C, followed by analysis through the UV-visible spectrophotometer.
Further, stability analysis was done by comparing 8 month earlier synthesised PJL-AgNPs to the
freshly prepared PJL-AgNPs through UV-vis spectroscopy.
2.6. Physicochemical characterization
After UV-vis spectra confirmation, the synthesised AgNPs were evaluated through Dynamic light
scattering (DLS) for the analysis of their average hydrodynamic diameter along with
polydispersity index (PDI) using Zetasizer Nano ZS (Malvern Instruments UK). Fully dispersed
AgNPs in milli-Q water were employed with a nominal 5mW HeNe laser run at 633 nm
wavelength followed by a scatted light at 173° angle.
For the functional group analysis, vacuum dried samples of both extract and AgNPs were assessed
by Fourier transform infrared spectroscopy (FTIR). The pellets were formed along with KBr and
scanned in the range of 4000 to 450 cm-1 against blank KBr pellet. The procured data revealed the
possible interaction of functional groups that are involved in synthesis and capping of AgNPs.
Morphological characterisation was done using transmission electron microscopy (TEM) and
X-ray diffraction (XRD) was done to determine the crystalline nature of the synthesised AgNPs.
2.7 Evaluation of antibacterial activity using disc diffusion assay

ACCEPTED MANUSCRIPT
Further, disc diffusion assay was performed against E. coli and P. aeruginosa to evaluate the
antibacterial efficacy of the synthesised AgNPs. For the preparation of primary culture, a single
colony was picked from the LB agar culture plate and inoculated in LB broth. The culture was
kept at 37º C for 16 hr at 150 rpm. From this primary culture, 1% inoculum was inoculated in
fresh broth (secondary) and incubated to grow till 0.4 optical density at 600 nm. Secondary
cultures of both were streaked on separate agar plates with a density of 105 CFU ml-1. Sterile paper
discs were placed on the streaked plates impregnated with different concentrations of AgNPs
(0.25, 0.5, 0.75 and 1µg) along with a positive and negative control (kanamycin and deionised
water, respectively). The plates were then observed for zone of inhibition after incubating at 37º C
for 24 hr.
2.8 Anti-biofilm activity of PJL-AgNPs by CRA plate method
Congo red agar (CRA) plate method have been performed to evaluate the antibiofilm activity
of synthesized PJL-AgNPs. Here in this method, a special media- brain heart infusion (BHI) broth
supplemented with 5% sucrose, 1% agar and 0.08% Congo red have been utilized against four
biofilm forming gram-positive bacteria including Bacillus subtilis and gram-negative bacteria
Pseudomonas aeruginosa. The plates were poured and after solidification the colonies of bacteria
were inoculated on separate plates with and without PJL-AgNPs and inoculated aerobically at
37ºC for 24 to 48 hour (23, 24).
2.9 Evaluation of catalytic activity of AgNPs
To evaluate the catalytic potential, the synthesized AgNPs were used against anthropogenic
pollutant 4-nitro-phenol (4-NP). For this, three sets of reactions were carried out with 4-NP
(2mM) and NaBH4 (0.03M). The three set of reactions included control 1, having 200 µl of 4-NP
with 2 ml water. Control 2 involved control 1 as well as 1 ml NaBH4 and the third set was the test
reaction having control 2 along with 30 µg/ml AgNPs(25, 26). The reaction was then observed for
stipulated time points at 0, 5, 10, 20 and 40 min. Thereafter, the reaction mixture was subjected to
centrifugation and supernatant was monitored through UV-vis spectroscopy.
For dye degradation in both cases (MB and CR), 1ml of NaBH4 (10mM) was mixed with 1.5
ml of dye (1mM) and the reaction volume (10 ml) was made up with ddH2O. This reaction was
considered as control and was monitored through UV-visible spectrophotometer at periodic time
points after centrifugation. Similarly, test reaction was performed using above composition along
with sufficient amount of AgNPs (27, 28) for their degradation. These reactions were also
periodicallymonitored in the same manner.
2.10 In-vitro cell viability assay
2.10.1 Cell Culture

ACCEPTED MANUSCRIPT
Human embryonic Kidney 293 (HEK 293) cells were maintained as a monolayer in
DMEM HG media supplemented with 1.85 gl-1 of NaHCO3 (Sodium bicarbonate) along with 10%
FBS and 1% Pen-strep. The cultured cells were maintained at 37ºC with 5% CO2 in CO2 incubator.
The culture flask having passage no 27 with 80% confluency were used in the cell viability
experiment.
2.10.2 Alamar Blue assay
The cell viability assay of biosynthesised AgNPs were performed against HEK293 cells
using alamar blue assay. This assay is a colorimetric assay where blue coloured reagent i.e.
resazurin is converted in pink coloured compound resorufin due to the metabolic activity of viable
cells. This colour shift intensity from blue to pink are quantified spectrophotometrically via ELISA
plate reader. To start the experiment, cells were trypsinised and seeded at a density of 10,000
cells/well in 96-well plate. The used media were discarded after 24 hr and cells were washed thrice
with 1X-PBS. The cells were then treated with different concentration (0.1µM to 3µM) of AgNPs,
PJL extract, 1mM AgNO3 and the untreated cells were considered as control. After incubation of
24hr, the treatment mixtures were discarded and after washing, each treated well were replaced
with 90 µl fresh media and 10 µl of alamar blue reagent (0.15mg/ml). The absorbance was
recorded at 570 and 600 nm after 4 h using micro-plate reader. The untreated control cells were
taken as 100 % viable that were used to compare the relative cell viability in the test wells.
2.11 Animal experiment for wound healing
The animal ethical committee of The IIS University approved the use of mice used in
experiments and approved the protocols used.
2.11.1 Preparation of ointment
An ointment is prepared using synthesized PJL-AgNPs along with Carbopol for topical
application on the wound area. Carbopol is used due to its hydrogel forming property when
combined with water which provide viscosity to AgNPs to use as applicator. Carbopol hydrogel
alone have been used as negative control and Povidine-Iodine used as positive control for the
experiment (29).
2.11.2 Excision and treatment of wound
For mice model handling, all the procedures were performed according to the guideline of
national institute of health Guide for care and use of amine. All the mice were kept in a
ventilated room on a 12-hour light/dark cycle at 22-25ºC and supplied with standard diet and
water. On the dorsal side of the body, the mice were shaved a day before the excision. The
shaved skin was scaled and marked for 10mm using a dermatological pencil. The marked area

ACCEPTED MANUSCRIPT
was then excised using a surgical blade under anaesthetic condition. The excised mice were
randomly divided into three group
Group-I: negative control (Carbopol hydrogel used as applicator)
Group-II: where Carbopol hydrogel having PJL-AgNPs used as ointment
Group-III: positive control (Povidine-Iodine is used)
The wound in all the group was regularly treated by their respective ointment (30, 31).
2.11.3 Percentage of wound healing
The wound area is regularly treated and reduction in wound area was measured and
photographed. The percentage of wound healing or wound are reduction was determined by the
reduction curve which was plotted by calculating the percentage of wound healing through a mean
of meter ruler in all three groups on 1st, 6th, 10th and 15th days. The formula for calculation of
wound healing is as follow:
% of WH = (WA0 -WAn / WA0) x 100
Where
WH= Wound healing
WA0 = Wound area on day 0
WAn = wound area on day n (n= 1, 6, 10 and 15)
2.12 Statistical analysis
All the experiments were done in duplicates, with three separate experiments to
demonstrate reproducibility and presented as mean ±standard deviation (±SD). Statistical analysis
was performed using Student's t-test. The differences were considered significant for pꢀ<ꢀ0.05 and
pꢀ<ꢀ0.01 indicative of a very significant difference.
3 Results and discussion
3.1 Biosynthesis of AgNPs synthesis
A change in color from light yellow to dark brown was observed within five minutes which
indicates that Ag+ get reduced into Ag0 (fig 1.) The reduced reaction mixture was then subjected to
centrifugation for 10 min at 1000 rpm and washed thrice followed by monitoring via UV-visible
spectrophotometer. The resulting reduction of Ag+ can be attributed to the action of
phytochemicals present in the extract. The synthesized AgNPs were then subjected to
centrifugation followed by washing thrice with ddH2O and resuspension in milli-Q water.
3.2 UV-visible spectrophotometric analysis and optimisation study
The confirmation of synthesis was done by UV-vis spectroscopy analysis. The procured
data indicates the characteristic peak at 420 nm that was due to the Surface Plasmon Resonance
(SPR) of the electrons in the conduction band of AgNPs.

ACCEPTED MANUSCRIPT
Further, optimisation of some important parameters as time (fig.2A), AgNO3 concentration
(fig.2B), extract ratio (fig.2C) and temperature (fig.2D) was done for the synthesis of small sized,
mono-dispersed particles. Plethora of studies has revealed that the AgNPs showing absorption
maxima around 420 nm tends to have spherical shape. A bathochromic shift in the absorption
peak indicates larger size of particles due to aggregation and broader peak for smaller size
particles. On the other hand, particles with larger size become narrow and show increase in the
absorption intensity (32, 33). Further, the study also revealed that increase in the absorption peak
height indicates an increase in concentration of AgNPs. But as the yield of AgNPs increases,
aggregation is likely to occur due to increase in collision frequency of nanoparticles (33, 34).
These studies suggested that the optimum synthesis was obtained at 25° C when 9.5 ml of 1 mM
AgNO3 was reduced with 0.5 ml of extract for 40 min. It was also observed that the synthesis
reaction was faster under microwave (fig.2E). Further, the synthesized AgNPs were observed
after 8 months that depicts the stability of AgNPs (fig.2F).
3.3 Characterization of synthesised nanoparticles
The optimized AgNPs were further characterized by DLS, TEM, XRD and FTIR to
evaluate their physicochemical properties. From the DLS studies, the hydrodynamic size of the
nanoparticles was found to be in the range of 55.24 nm along with a PDI of 0.2 (fig.3A).
XRD confirms the crystalline structure of the formed AgNPs (fig.3B). For average size
and morphological analysis, the AgNPs were evaluated through TEM where the image (fig.3C)
revealed that the size of AgNPs was in the range of 10-20 nm along with spherical morphology.
FTIR spectroscopy was conducted in order to determine the functional group of extract
that were involved in the synthesis and capping. The leaf extract band (band A) indicated peak
near 3412 and 2935 cm-1 depicting the presence of –OH (alcoholic) and phenolic compounds
with carboxylic group stretching. Peak near 1612 and 1450 cm-1 corresponds to N-H and C-H
bend, respectively. On the other hand, AgNPs showed (band B) peak of –OH bend shifted from
3412 to 3477 cm-1 and similarly, peak of phenolic compound shifted from 2935 to 2734 cm-1.
These data indicate that alcoholic and phenolic compounds are involved in the synthesis of
nanoparticles. Along with this, peak near 1574 cm-1 showed that aromatic compounds could be
responsible for the capping and stability of the AgNPs (fig.3D).
3.4 Evaluation of antibacterial activity using disc diffusion assay
To evaluate the potent antibacterial activity of synthesised AgNPs, disc diffusion assay was
conducted with different concentrations of AgNPs against E. coli and P. aeruginosa. In both the
cases, the antibacterial activity was evaluated for stipulated concentrations (0.25, 0.5, 0.75 and 1

ACCEPTED MANUSCRIPT
µg) of AgNPs on discs (C, D, E, F) in fig.4. Disc ‘A’ and ‘B’ were taken as negative and positive
control with autoclaved deionized water and antibiotic (kanamycin), respectively.
As evident from the image (fig.4A and 4B) the growth of bacteria was repressed in an
appreciable manner in both the cases. Furthermore, the zone of inhibition for both the bacterium
were quantitatively presented by bar graph (fig.4C) revealing the concomitant decrease in the
growth rate with increase in the concentration of PJL-AgNPs from 0.25 µg to 1 µg indicating that
the antibacterial potential was highly dose-dependent. The diameter of the zone of inhibition was
presented in millimeter and taken as mean ± SD of duplicates. Thus, AgNPs could play an
important role in surmounting the problem of multi-drug resistance.
3.5 Anti-biofilm activity of PJL-AgNPs by CRA plate method:
To evaluate anti-biofilm potential of PJL-AgNPs CRA plate method was performed as
described by Freeman et. al (35). In this method the appearance of dry crystalline black
colonies on plate indicate the secretion of exopolysaccharide which is responsible for the
adhesion of microorganisms to form biofilm that protects them from unfavourable
environmental factor. Figure 5 is showing the results of the experiment which indicates the
appearance of dry crystalline black colonies on the plate (A(i), B(i)) where AgNPs were not
supplemented.
On the other hand, the plates which was supplemented with PJL-AgNPs (A(ii), B(ii)) the
organism were grown but the absence of dry crystalline black colonies which indicates the
secretion of exopolysaccharide inhibited due to PJL-AgNPs treatment. These results indicate
the anti-biofilm activity of PJL-AgNPs.
3.6 Evaluation of catalytic activity of AgNPs
To evaluate the catalytic activity of AgNPs, 4-NP, MB and CR degradation studies were done.
The reduction of 4-NP was investigated with aqueous NaBH4 along with silver nanoparticles that
act as catalyst for the reaction. Though reduction of 4-NP into 4-AP with NaBH4 can be possible,
it is limited by the kinetic barriers due to difference in the thermodynamic potential of electron
donor (NaBH4) and acceptor (4-NP). This potential difference decreases the feasibility of the
reaction. So, to overcome this barrier, silver nanoparticles were used as nano-catalysts where it
facilitates the electron relay from donor to acceptor. This results in the conversion from 4-NP,
with an intermediate formation of sodium phenolate and finally to 4-AP. Here in, three sets of
reactions were performed as mentioned above and monitored through UV-visible
spectrophotometer by scanning in the range of 200-500 nm. Control 1 having 4-NP exhibited
absorbance peak at 317 nm, which red shifted to 400 nm upon addition of NaBH4 in control 2(36).
This peak was obtained due to the formation of sodium phenolate. In the third set of reaction,

ACCEPTED MANUSCRIPT
AgNPs were added (0.2, 0.4, 0.8, 1.6, 3.2 and 4µg) to the reaction mixture of control 2 and
monitored after 10 min and 2 hr followed by centrifugation. The procured data of UV-visible
spectrophotometer revealed that the reduction of 4-NP immediately started with addition of
AgNPs and a significant decrease in absorbance intensity after 10 min was observed (fig.6A). This
indicates a great reduction along with appearance of a new absorbance peak at 296 nm
(characteristic peak of 4-AP). These results showed the potent catalytic activity of synthesised
AgNPs(26, 37). Figure 6B is showing the kinetic curve graph of 4-NP. Catalytic degradation of
MB was evaluated through biosynthesised AgNPs at a stipulated time points in comparison with
NaBH4 degradation as control. Two sets of reaction were conducted as control and test,
respectively. In set 1, aqueous solution of dye was mixed with aqueous solution of NaBH4 and the
reaction was monitored periodically using UV-visible spectrophotometer by scanning in the range
of 450-750 nm. The procured data revealed very slow reduction even after 90 min (fig.6C). On the
other hand, second reaction includes a mixture of dye and NaBH4 along with AgNPs as a catalyst.
This was also observed gradually with stipulated time point and assessed by UV-visible
spectrophotometer after centrifugation. The obtained data (fig.6D) revealed that the reduction of
MB become faster with AgNPs. Further, degradation of CR through AgNPs was evaluated at
different time intervals and UV-visible spectra data were compared between control and test
reaction after centrifugation. Figure .6E is showing the kinetic curve of MB degradation.
In first control reaction, the UV-visible spectra data (fig.6F) revealed that reduction of CR is
not efficient even after 80 min wherein, second reaction data (fig.6G) having AgNPs as a catalyst,
revealed that the reduction occurred very fast that started immediately after 2 min(28, 27). Figure
.6H is showing the kinetic curve of CR degradation.
3.7 In-vitro cell viability assay
The cell viability result was evaluated via alamar blue assay after 24 hr of treatment. All the
treatment conditions were compared with untreated well that were considered as 100 % viable. The
procured data (figure 7) suggested that our synthesised PJL-AgNPs are not showing toxicity even
at 3 µg concentration where more than 94% cells are viable. On the other hand, the cells treated
with same concentration of PJL extract and 1 mM AgNO3 showed viability percent as 52 and 22,
respectively. Therefore, from the results obtained, it could be concluded that PJL-AgNPs can be
implied for biomedical applications.
3.8 Animal experiment for wound healing
The wound healing percentage was quantified from the observation taken after treatment by
calculating the size reduction of wound area after 1, 6, 10 and 15 days. The figure 8 (A) is showing
the visual observation in the size reduction of the wound that indicates the rate of wound closure in

ACCEPTED MANUSCRIPT
the mice treated with PJL-AgNPs-Carbopol was much faster than the other two groups (Carbopol
only and Povidine-Iodine). The group-I where the wound is treated with only Carbopol, the
percentage of wound healing was 25, 50 and 65 after 6, 10 and 15 days, respectively. Whereas in
group-II (treatment of PJL-AgNPs-Carbopol), the percentage of wound healing was 50, 85 and
99.9. Further, foe group-III where the wound is treated with povidine-iodine the percentage of
wound healing was 12, 56 and 90. These results confirmed that PJL-AgNPs-Carbopol promoted
the wound contraction and accelerated the healing of wounds.
4 Conclusion
Here, in our study, we are presenting a facile, quick and environmentally friendly approach for
the synthesis of spherical AgNPs from plant extract. Here, P. juliflora leaf extract have been used
which is having various secondary metabolite or phytochemicals that are responsible for reducing
as well as capping agent in the synthesis process. Various parameters that are crucial for a reaction
are optimised including time, AgNO3 concentration, extract to AgNO3 ratio and temperature of the
reaction. The synthesised particles showed SPR around 420 nm along with a hydrodynamic
diameter of 55.24 nm with 0.2 PdI. Whereas, TEM image revealed that the actual average size was
in the range of 10-20 nm with spherical morphology. Further, FTIR data revealed that phenolic
compounds are responsible for the reduction of Ag+ into Ag0. The antibacterial potential of AgNPs
resulted in positive dose-dependent manner. The CRA plate method exhibit remarkable
antibiofilm activity of synthesised AgNPs. The effective degradation of MB and CR dyes through
AgNPs on very short time revealed their strong catalytic behaviour. Further, wound healing assay
showed that the synthesized PJL-AgNPs exhibiting the contraction of wound and promote wound
healing very efficiently within 15 days.
Conflict of Interest
Authors declare that they have no conflict of interest.
Acknowledgements
Geeta Arya and Nikita Sharma acknowledge the receipt of fellowship from CSIR and UGC,
Government of India, India. Surendra Nimesh acknowledges the financial assistance from
Department of Biotechnology (grant no. 6242-P82/RGCB/PMD/DBT/SNMH/2015), Government
of India.

ACCEPTED MANUSCRIPT
Reference
1. Vidhu, V. K. and D. Philip (2014) Catalytic degradation of organic dyes using
biosynthesized silver nanoparticles. Micron 56, 54-62.
2. H Karimi and S. M. a. B. Sadeghian (2012) Silver nanoparticle loaded on activated carbon
as efficient adsorbent for removal of methyl orange. Indian Journal of Science and
Technology 5, 2346-2353.
3. Sannino, D., V. Vaiano, O. Sacco and P. Ciambelli (2013) Mathematical modelling of
photocatalytic degradation of methylene blue under visible light irradiation. Journal
of Environmental Chemical Engineering 1, 56-60.
4. Lefebvre, O. and R. Moletta (2006) Treatment of organic pollution in industrial saline
wastewater: A literature review. Water Research 40, 3671-3682.
5. Mohmood, I., C. B. Lopes, I. Lopes, I. Ahmad, A. C. Duarte and E. Pereira (2013)
Nanoscale materials and their use in water contaminants removal—a review.
Environmental Science and PollutionResearch 20, 1239-1260.
6. Patel, R. and S. Suresh (2006) Decolourization of azo dyes using magnesium–palladium
system. Journal of Hazardous Materials 137, 1729-1741.
7. Gomathi Devi, L., S. Girish Kumar, K. Mohan Reddy and C. Munikrishnappa (2009)
Photo degradation of Methyl Orange an azo dye by Advanced Fenton Process using
zero valent metallic iron: Influence of various reaction parameters and its degradation
mechanism. Journal of Hazardous Materials 164, 459-467.
8. Sana, S. S., V. R. Badineni, S. K. Arla and V. K. Naidu Boya (2015) Eco-friendly
synthesis of silver nanoparticles using leaf extract of Grewia flaviscences and study of
their antimicrobial activity. Materials Letters 145, 347-350.
9. H. Reza Ghorbani, A. Akbar Safekordi, H. Attar and S. M. R. Sorkhabadi (2011)
Biological and Non-biological Methods for Silver Nanoparticles Synthesis. Chemical
and Biochemical Engineering Quarterly 25 317–326.
10. Alexander, J. W. (2009) History of the Medical Use of Silver. Surgical infections 10,
289-292.
11. Parsons, J. G., J. R. Peralta-Videa and J. L. Gardea-Torresdey (2007) Use of plants in
biotechnology: Synthesis of metal nanoparticles by inactivated plant tissues, plant
extracts, and living plants. Developments in Environmental Science 5, 463-486.
12. Patra, C. R., S. Mukherjee and R. Kotcherlakota (2014) Biosynthesized silver
nanoparticles: a step forward for cancer theranostics? Nanomedicine 9, 1445-1448.

ACCEPTED MANUSCRIPT
13. V. V. Makarov, A. J. Love, O. V. Sinitsyna, S. S. Makarova, I. V. Yaminsky, M. E.
Taliansky and N. O. Kalinina (2014) Green nanotechnologies: synthesis of metal
nanoparticles using plants. Acta Naturae, 35-44.
14. Muthukrishnan Saradha Devia, Kaliyaperumal Ashokkumarb and S. Annapooranic
(2017) Phytofabrication and encapsulated of silver nanoparticles from Gloriosa
superba. Materials Letters 188, 197-200.
15. Ahmed, Q., N. Gupta, A. Kumar and S. Nimesh (2016) Antibacterial efficacy of silver
nanoparticles synthesized employing Terminalia arjuna bark extract. Artificial Cells,
Nanomedicine, and Biotechnology 45, 1192-1200.
16. R Mankamna Kumari, Nikita Thapa, Nidhi Gupta, A. Kumar and S. Nimesh (2016)
Antibacterial and photocatalytic degradation efficacy of silver nanoparticles
biosynthesized using Cordia dicotoma leaf extract. Advances in Natural Sciences:
Nanoscience and Nanotechnology 7, 045009.
17. Geeta Arya, Nitin Kumar, Nidhi Gupta, Ajeet Kumar and S. Nimesh (2016) Antibacterial
potential of silver nanoparticles biosynthesized using Canarium ovatum leaves
extract. IET Nanobiotechnology 11, 506-511.
18. Arya, G., R. M. Kumari, N. Gupta, A. Kumar, R. Chandra and S. Nimesh (2017) Green
synthesis of silver nanoparticles using Prosopis juliflora bark extract: reaction
optimization, antimicrobial and catalytic activities. Artificial Cells Nanomedicine and
Biotechnology, 1-9.
19. Arya, G., N. Sharma, J. Ahmed, N. Gupta, A. Kumar, R. Chandra and S. Nimesh (2017)
Degradation of anthropogenic pollutant and organic dyes by biosynthesized silver
nano-catalyst from Cicer arietinum leaves. Journal of Photochemistry and
Photobiology B 174, 90-96.
20. Wang, M., W. Zhang, X. Zheng and P. Zhu (2017) Antibacterial and catalytic activities of
biosynthesized silver nanoparticles prepared by using an aqueous extract of green
coffee bean as a reducing agent. RSC Advances 7, 12144-12149.
21. Mukherjee, S., D. Chowdhury, R. Kotcherlakota and S. Patra (2014) Potential
theranostics application of bio-synthesized silver nanoparticles (4-in-1 system).
Theranostics 4, 316.
22. Pragnesh N. Dave and J. Bhandari (2013) Prosopis julifera A review. International
Journal of Chemical Studies 1, 181-196.
23. Kalimuthu Kalishwaralal, Selvaraj BarathmaniKanth, Sureshbabu Ram Kumar Pandian,
Venkentaraman Deepak and S. Gurunathan (2010) Silver nanoparticles impede the

ACCEPTED MANUSCRIPT
biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis.
Colloids and Surfaces B: Biointerfaces 79, 340-344.
24. MA Ansari, HM Khan, AA Khan, SS Cameotra and M. Alzohairy (2015) Anti-biofi lm
effi cacy of silver nanoparticles against MRSA and MRSE isolated from wounds in a
tertiary care hospital. Indian Journal of Medical Microbiology 33, 101-109.
25. Al-Marhaby, F. A. and R. Seoudi (2016) Preparation and Characterization of Silver
Nanoparticles and Their Use in Catalytic Reduction of 4-Nitrophenol. World Journal
of Nano Science and Engineering 06, 29-37.
26. Prasad, C. H. and K. Srinivasulu (2015) Catalytic Reduction of 4-Nitrophenol Using
Biogenic Silver Nanoparticles Derived from Papaya (Carica papaya) Peel extract.
Industrial Chemistry 01.
27. Vidhu, V. K. and D. Philip (2014) Spectroscopic, microscopic and catalytic properties of
silver nanoparticles synthesized using Saraca indica flower. Spectrochim Acta A Mol
Biomol Spectrosc 117, 102-108.
28. Kolya, H., P. Maiti, A. Pandey and T. Tripathy (2015) Green synthesis of silver
nanoparticles with antimicrobial and azo dye (Congo red) degradation properties
using Amaranthus gangeticus Linn leaf extract. Journal of Analytical Science and
Technology 6, 33.
29. Moideen, M., R. Varghese, E. Kumar and C. Dhanapal (2011) Wound healing activity of
ethanolic extract of Hemidesmus indicus (Linn) R. Br leaves in rats. Res J Pharm
Biol Chem Sci 2, 643-651.
30. Gong, C. P., S. C. Li and R. Y. Wang (2018) Development of biosynthesized silver
nanoparticles based formulation for treating wounds during nursing care in hospitals.
Journal of photochemistry and photobiology. B, Biology 183, 137-141.
31. Wilkinson, L., R. White and J. Chipman (2011) Silver and nanoparticles of silver in
wound dressings: a review of efficacy and safety. Journal of wound care 20, 543-549.
32. Stamplecoskie, K. G. and J. C. Scaiano (2010) Light emitting diode irradiation can
control the morphology and optical properties of silver nanoparticles. Journal of the
American Chemical Society 132, 1825-1827.
33. Zhang, W., X. Qiao and J. Chen (2007) Synthesis of silver nanoparticles—effects of
concerned parameters in water/oil microemulsion. Materials Science and
Engineering: B 142, 1-15.
34. Sathishkumar, M., K. Sneha, S. Won, C.-W. Cho, S. Kim and Y.-S. Yun (2009)
Cinnamon zeylanicum bark extract and powder mediated green synthesis of nano-

ACCEPTED MANUSCRIPT
crystalline silver particles and its bactericidal activity. Colloids and Surfaces B:
Biointerfaces 73, 332-338.
35. Freeman, D. J., F. R. Falkiner and C. T. Keane (1989) New method for detecting slime
production by coagulase negative staphylococci. Journal of clinical pathology 42,
872-874.
36. Wunder, S., F. Polzer, Y. Lu, Y. Mei and M. Ballauff (2010) Kinetic Analysis of
Catalytic Reduction of 4-Nitrophenol by Metallic Nanoparticles Immobilized in
Spherical Polyelectrolyte Brushes. The Journal of Physical Chemistry C 114, 8814-
8820.
37. Premkumar, T., K. Lee and K. E. Geckeler (2011) Shape-tailoring and catalytic function
of anisotropic gold nanostructures. Nanoscale Research Letter 6, 547.

ACCEPTED MANUSCRIPT
Figure Legends
1. Biosynthesis reaction of AgNPs via Prosopis juliflora leaf (a) leaf extract, (b) AgNO3
solution, (c) color change due to AgNPs synthesis.
2. UV-vis spectra data of optimisation studies (A) Reaction time, (B) AgNO3
concentration, (C) extract to AgNO3 ratio, (D) Reaction temperature (E) microwave
assisted, (F) stabilty over time.
3. Characterisation of AgNPs (A) DLS that comes in the range of 55.24, (B) XRD, (C)
TEM image (D) FTIR.
4. Representative results of antibacterial assay of AgNPs by using disc diffusion assay
against (A) E. coli, (B) P. aeruginosa, (In both cases E. coli and P. aeruginosa- A is
negative control, B is positive control, and C to F is different concentration of AgNPs)
(C) Quantitative evaluation of zone of inhibition of both E. coli and P. aeruginosa that
indicate antibacterial efficacy in a dose dependent manner.
5. Evaluation of the antibiofilm activity of PJL-AgNPs by CRA plate method. The
appearance of black colonies (A(i), B(i)) indicates the exopolysaccharide production
by A-Bacillus subtilis and B- Pseudomonas aeruginosa bacteria, respectively.
Whereas the addition of PJL-AgNPs block the exopolysaccharide secretion by
bacteria and weakened their growth.
6. Represented data of catalytic degradation of dye (A) 4-NP with different
concentration at a time point of 10 min (B) Kinetic curve of 4-NP degradation (C)
MB with NaBH4, (D) MB with AgNPs, (E) Kinetic curve of MB degradation (F) CR
with NaBH4, (G) CR with AgNPs (H) Kinetic curve of CR degradation.
7. In-vitro cell viability assay of synthesised PJL-AgNPs (0.1 to 3 µg) against HEK293
cells via alamar blue reagent.
8. (A) Photographs of the wound healing activity of all the three groups on 0, 6, 10 and
15 days under the treatment of Carbopol only, PJL-AgNPs, Povdine-Iodine. (B)
Quantitative data showing the % of wound healing after treatment.

ACCEPTED MANUSCRIPT
Highlights
 Development of potent silver nanoparticles using Leaf extract of Prosopis juliflora.
 Characterized the ANB-AgNPs via UV, DLS, FTIR, TEM and XRD.
 Evaluated the antibacterial and antibiofilm potential of PJL-AgNPs.
 Evaluated the in-vitro cytotoxicity of AgNPs.
 Evaluated the Catalytic and wound healing potential of PJL-AgNPs.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8