Abstracts

The application of plant phytometabolites in comparison to other biological approaches to

nanoparticle bioreduction has piqued the interest of [Link] paper reports the
phytofabrication of silver [Link] leaf extracts of Acokanthera oppositifolia and
Leucaena leucocephala were utilized for the reduction and capping of silver ions. The structural
properties of nanoparticles were studied using scanning electron microscope (SEM),
transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive analysis
(EDAX), and Fourier transform infrared spectroscopy (FTIR). The antibacterial activity of
synthesized silver nanoparticles was tested against Bacillus cereus, Staphylococcus aureus,
Enterobacter aerogenes and Pseudomonas aeroginosa. To determine the enhancement in
antibacterial activity, the zone of inhibition of the prepared AgNPs was compared to the fresh
aqueous extracts. The finding reveals that highly crystalline spherical silver nanoparticles
produced with an average size of 13.38 nm and 11.93 nm for Acokanthera oppositifolia and
Leucaena leucocephala, [Link] inhibition zone was reduced from 1.13 x 105 to 3.04 x
102 and 2.16 x 102 CFU/mL after reduction and capping of silver ion with phytomolecules
ofAcokanthera oppositifolia and Leucaena leucocephala, respectively. The results conclude that
polyurethane membranes doped with silver nanoparticles are very effective in wastewater
treatment and hold great promise for wastewater remediation [Link] –
coated nanoparticles synthesis was found to be a simple, time-saving, cost-effective, and eco-
friendly ideal strategy with superior properties.

Keywords: Nanoparticles, Phytomolecule, capping, diffractogram, antibacterial

 1. Introduction

Nanotechnology NP intro SNP fabrication application antimicrobial

Over the past few decades, nanotechnology has grown into a powerful force at the atomic level.
Nanoparticles are the result of manipulating matter at atomic scales from 1 to 100 nm and exhibit
improved, novelproperties compared to their bulk form (2). Surface to volume ratio and stronger
confinement are the two important factors that play a major role in the change of behavior in
nanomaterials. These two principal factors can change or enhance properties such as chemical
reactivity, optical activity, strength, and electrical characteristicscompared to the same material
in bulk (3).These improved properties make nanoparticles accessible for use in various fields.

Various physical, chemical, and biological approaches can be used to fabricate nanoscale
particles,among them biological has gained popularity. The problems of physicochemical
methods, including the use of toxic chemicals, high temperatures and pressures, and the
formation of dangerous by-
[Link],biologicalapproachesusingmicrobes,fungi,
enzymes,[Link] plant-derived phytochemicals for nanoparticles
synthesis has intrigued researchers due to superiority over other biological [Link]
microbes, plant extractsdoes not require complex protocols to maintain sterile microbial
cultures and does not exhibit genetic mutations. The plants are widespread,readilyavailable,
and [Link] plants apreferred source over
[Link] studies have documented the use of environmentally friendly green
approaches to reducing and capping agents in nanoparticle synthesis.
synthesis of nanoparticles is due to the presence
of a number of metabolites and reductive biomolecules in the
plant extract, which are responsible for the reduction of metal [Link] the process of silver
nanoparticle
synthesis, the silver atoms enter inside the plant cells through specific
channels and the coalesces or nucleation of silver atoms inside
the channels lead to the formation of silver nanoparticles [70].Furthermore, plant leaf extract contains
carbohydrate and proteins
which serves as a reducing agent for the reduction of silver
ions to silver nanoparticles [86]. Also the proteins with functional
amino groups might act as reducing agent for silver ions [87].

Gade and colleagues [91] studied the phytofabrication of

silver nanoparticles using Opuntiaficus-indicaand proposed the
possible mechanism for the fabrication of silver nanoparticles.
Based on the study of the bioactive compounds secreted by this
plant, it was found that the plant secretes mainly terpenoides and
flavonoids, of which quercetin and kaempherol are the major components.
Out of which quercetin possesses reducing capacity [90]
which reduced the silver ions possibly by formation of an intermediate
complex. Therefore, it was suggested that quercetin plays a
major role in the reduction of silver ions to form silver nanoparticles.
Fig. (4) showed the schematic representation of reduction of
silver ions with quercetin leading to formation of silver nanoparticles.
 Different phytochemicals such asflavonoids, ketones, terpenoids, amides, carboxylicacids, and

aldehydes,present in plants are responsible for the reduction, capping, and stabilization of

nanoparticles (Raiet al., 2013). The mechanism of nanoparticle synthesis with plants is complex,

and many hypotheses have been proposed, the actual mechanism is not fully understood

yet(Ghotekaret al., 2018).A hypothetical reaction process for the production of AgNPs from

Leucaena leucocephala is proposed in Figure 1.

Metallic nanoparticles such as gold, silver, copper, and zinc are of most interest in the present
scenario because of their unique physicochemical properties and superior magnetic, electrical,
medical, and optical capabilities.

Silver nanoparticles is the most conductible, sensitive, stable, light-absorbing, and has the
highest surface plasmon resonance (SPR).Among other nanoparticles,nanosilver is well known
for its antibacterial and antioxidant potential. Due to their diverse applications, these are the most
commonly used nanoparticles and are exploited in various fields, especially biomedical
applications.16-21

In recent years, the use of various chemicals and antibiotics has increased rapidly, and antibiotic
resistance has become a problem due to the inappropriate use of antibiotics.(Mboya et al.,
2018).Addressing this challenge requires a multidisciplinary strategy, including the development
of novel antimicrobial agents that have the potential to affect a broad spectrum of microbes.
(Gunasekaran Suriyakala2021)

Nanotechnology beings a technological mammoth at the atomic scale, it had gained more
importance in the past few decades. Although nanoparticles are considered a modern scientific
invention by Richard Feynman in 1959, they have a very long history since they were used in the
9th century in the Mesopotamian culture for glittering effect on the pot's surface (1). The fact is
that Nanoscale particles are the basic foundation of life on earth, as genetic material essential
biomolecules for the formation and function of all living cells exist in nano forms. This sentence
contains false information. Atoms (or organic cells) should not be linked to nanoparticles. The sizes of atoms
are in the range of 0.1 to 0.5 nm. Nanoparticles usually contain a few hundred atoms therefore their
dimensions are around two orders of magnitude larger than atoms.
Nanotechnology, according to the National Nanotechnology Initiative (NNI), is the study and
engineering of matter at atomic dimensions ranging from 1 to 100 nm that has novel properties
that are applicable in various fields(2).

Surface to volume ratio and stronger confinement are the two important factors that play a major
role in the change of behavior in nanomaterials. These two principal factors can change or
enhance properties such as chemical reactivity, optical activity, strength, and electrical
characteristics as compared to their in bulk form (3). Due to these properties, nanoparticles are
excessively exploited in different sectors(4).Based on the material used for the fabrication,
nanoparticles can be categorized into the following groups' carbon, metal, ceramic, polymeric,
and semiconductor. (I do not agree with such narrow categorization. Authors cite paper by Santos et al.
(2015) where such categorization is much broader and more accurate, but do not exhaust all views.)

Phytofabrication

Nanoscale material can be fabricated using various physical, chemical, and biological
approaches. However, phytofabrication has gained prominence as a simple method this not only
makes the approach environmentally friendly but also more cost-effective.(refrence)

The use of different chemicals and antibiotics have been increased drastically in past few years
leads to the problem of bacterial resistance in the treatment of infectious diseases.(In addition, such
information given in introduction falsely indicate that authors are study of treatment of viruses instead of
bacteria. Furthermore, if authors claim that the COVID-19 pandemic led to problem of bacterial resistance,
they should support it with proper reference. )Therefore,there is a need for new antimicrobial
substances that could affect a wide range of microbes that are either bacteriostatic or bactericidal.
Metal-based nanosilver particles have tremendous potential to combat such kinds of problems for
the benevolent of mankind(5,6). In the Previous findings, metal-based nanoparticles can prevent
or overcome biofilm formation, i.e., Gold (7) Silver (8) Magnesium (9) NO,(10), [Link] authors
mean by writing "In the Previous findings, metal-based nanoparticles can prevent or overcome biofilm
formation, i.e., Gold (Yu Q 2016) Silver (Markowska 2013) Magnesium (Lellouche 2012) NO, (Hetrick 2009),
etc."? Why this is relevant in the current study?
Finding the different literature concludes that, silver nanoparticles can be used in diverse areas
safely i.e., in agriculture (11) biomedical (12) cosmetic (13) food packaging (14)textile (15)
water purification (16).

Based on finding concluded through different studies, here a comparative study was designed for
the phytofabrication and characterization of Acokanthera oppositifolia and Leucaena
leucocephala nanoparticles. It is not clear which study exactly prompted towards such research, as
references listed in the previous paragraph are related to wide area of research that involves nanoparticles.
Further, the difference in antibacterial activity of pure leaves extracts and their prepared
nanoparticles also has been investigated.
1.1 Envisaged Reaction Mechanism of phytofabricated approach

There are three major steps in bio-reduction of Ag+ to Ag0; reduction and nucleation,
aggregation, third capping, and stabilization which are represented in (Figure1A). Plant's
secondary metabolites play a crucial role like reducing and capping agents (17). In a previously
reported study, Acokanthera oppositifolia seeds contained palmitate;lup-20(29)-en-3β-O-(3′-β-
hydroxy) triterpene; lupeol, cardiac glycoside; acovenoside A and a sterol; β-sitosterol (18).
Leucaena leucocephala leaf extracts possess Mimosine, Luteolin-7-o-glucoside, caffeic acid,
chrysoeriol, isorhamnetin 3-O-galactoside, kaempferol-3-O-rubinoside, Quercetin-3-O-
rhamnoside biomolecules(19, 20, 21, 22). These phytomolecules are assumed to play a key role
in reduction and provide stability during nanoparticle fabrication. An envisaged reaction
mechanism for the generation of AgNPs represents in (Figure 1B) however, the exact mechanism
is still unknown and opens the door for a new research area (23).

2. Materials and Methods

2.1 Chemicals

Silver nitrate (AgNO3) salt was purchased from Sigma-Aldrich. Microbial strains of Gram-
positive bacteria (Bacillus cereus; MTCC430, Staphylococcus aureus; MTCC 1144) and Gram-
negative bacteria (Pseudomonas aeruginosa; MTCC 1864, Enterobacter aerogenes MTCC2822)
imported from IMTECH Chandigarh were tested for antibacterial potential.

2.2 Preparation of Plant Extracts

Fresh leaves of Acokanthera oppositifoliaand Leucaena leucocephala were collected,

taxonomically identified, rinse with tap and distilled water, and air-dried. The dried leaves were
coarsely crushed into powder using a grinder. 2g powder dissolved in 100 ml of distilled water
and heated at 60°C for one hour. After cooling obtained solution was filtered and stored at 4–8
°C. (24).

2.3 Silver nanoparticles phytofabrication

5 ml (25mM) AgNO3 mixed with 1 ml of plant extracts (1:5) ratio,for silver nanoparticles
fabrication and color change, was observed at different time intervals. One sentence about
fabrication process is insufficient (and should not stand as separate section). Authors should expand the
description of fabrication of nanoparticles or refer to adequate references.

2.4 Structural Characterization

To investigate information about the formation, average size, morphology shape, aggregation
state, the functional groups involved, and crystalline nature of synthesized nanoparticles a set of
characterization techniques were applied (25, 26).
The optical properties which confirm the fabrication of nanoparticles were detected as a
consequence of the wavelength range of 200 to 800 by using a double beam UV-VIS
spectrophotometer (Labtronics Model LT-2201) after optimization. "The optical properties which
confirm the fabrication of nanoparticles were detected as a consequence of the wavelength range of 200 to
800 by using a double beam UV-VIS spectrometer (Labtronics Model LT-2201) after optimization." This
sentence makes no sense. Firstly, optical properties are not the proof of fabrication of nanoparticles. Instead,
the specific observations of optical response can confirm fabrication of nanoparticles. Secondly, the numbers
are missing units. Thirdly, the wavelength range cannot be a consequence of detection of nanoparticles as
authors have chosen such wavelength range. Finally, authors mention optimization but there is no
explanation what that means. For Sem analysis, Zeiss EVO 18 with acceleration voltage 0.2 to 30
kV was [Link] Sem analysis, Zeiss EVO 18 with acceleration voltage…" the "Sem" refers to the acronym
of the measurement technique (Scanning Electron Microscopy), therefore the whole acronym should be
capitalized.

For this purpose synthesized aqueous nanoparticles diluted with DW and ultrasonicate, a drop of
sample loaded on aluminum stub allowed to dry in stub holder. The morphology and average
size of AgNPs were analyzed by transmission electron microscopy (JEOL JEM-1400 maximum
accelerating voltage of 120 kV). For transmission electron analysis, ultrasonic dispersion of the
synthesized nanoparticles in ethanol and a droplet of this dispersedsample was put onto a carbon-
coated copper grid, and dried at room temperature. Energy-dispersive X-ray microanalysis was
carried out by using RONTEC’s EDX system (Model QuanTax 200) for getting the elemental
details. The structural characterization and crystalline nature of powder nanoparticles obtained
after centrifugation was analyzed by X-ray profile using X’Pert PRO (PANalytical Netherlands)
in the region of 2θ from 30ᵒ to 80ᵒ. The functional groups associated with the production of silver
nanoparticles were determined using FTIR [Link] of dried powder was carried out by
NicoletTM - IS-50 FTIR (Thermo Scientific) through the KBr pellet technique, in the
transmission mode range 4000–500 cm-1 through the attenuated total reflectance (ATR)
method.

2.5 Comparative Antibacterial potential of phytofabricated AgNPs against their pure

extract

2.5.1 Agar well diffusion assay

Antibacterial activity was determined by utilizing an agar well diffusion assay (27). For this
purpose, 20 ml of molten NAM was poured into sterile petri dishes and allowed to solidify.
Tested microorganisms were swabbed uniformly over the surface of solidified nutrient agar and
wells were punched. Prepared well filled with 50 µL and 100 µL of AgNPs and pure plant
extracts of both the [Link] antibiotic ampicillin (10mg) has been used as a [Link]
plates were incubated for 24 hours at 37 degrees [Link] Petri plates were photographed and
the diameter of the inhibition zone was measured through this RIZD% was calculated. The
acronym RIZD% is not explained. In the sentence it refers to inhibition zone diameter (IZD) rather than to
relative one. Any acronym used in the text or in the equation should be explained. Each
experiment was
performed in triplicate to calculatethe mean ±standard deviation by using origin software. The
antibacterial activity was calculated using the formula given below (28, 29):
 A ( Npsinhibition zone ) – B ( PE inhibition zone )
Percent RIZD = ×100 equation: The notation
C ( Std . inhibition zone ) – B ( PEinhibition zone )
should be explained and consistent with the one used in the text. In addition, instead of using full words
authors should use short notation that make equation compact.

2.5.2 Micro-broth dilution method

The minimal inhibitory concentration (MIC) was determined by the micro-broth dilution method.
For this, 0.1ml of the cultural broth was inoculated with nanoparticles and pure extracts at
concentrations ranging from 1.0 to 50 mg/ml and incubated at 37°C with mixing at 150 rpm in
the [Link] 24 hours, microbial growth was identified by Uv-Visible spectrometric analysis
at 600 [Link] medium which was free of nanoparticles used as a positive [Link] lowest
concentration of nanoparticles that was able to completely inhibit bacterial growth was
considered as MIC value (30).
It is not clear from the description how the technique works. Authors should explain what was determined
with spectrometer. Authors should also specify what concentrations exactly they used in this test. It seems
bizarre that they do not determine minimum bactericidal concentration (MBC) as well.

3 Results and Discussion

3.1 AgNPs' UV spectra and visible inspection for colour variation

Biosynthesis of AgNPs has been confirmed by color variation,startinglight yellowish to orange

to finally brownat the primary level and further examined by UV-visible spectroscopydepicted at
different time intervals (Figure 2 a, b).After 24 hours further no color change was observed
suggesting that the silver salt of the solution was completely reduced.A similar color shift was
seen in earlier investigations (31). The color change is due to the strong surface plasmon
resonance (SPR) of nanoparticles in an aqueous solution, which is determined by factors like
morphological structure, dimension, etc. (32).authors link change in color to the strength of SPR. Such
statement is wrong. In the case of nanoparticles localized-surface plasmon resonance (LSPR) is more
important than SPR. In addition, the specific size of spherical nanoparticles determines the spectral position
of LSPR and the color of solution is related to the absorption wavelength. Therefore, the shift in the spectral
position in the range observed in Fig. 2 cannot be explained by such statement.
The UV-Visible spectrum of plant leaf extracts Acokanthera oppositifolia and Leucaena
leucocephalashows a sharp band at 293nm, and 261nm respectively but AgNO 3 in aqueous form
gives maximum absorbance at 287. ." The discussion refers to data from supplementary materials,
therefore there should be proper citation to these. The same compound AgNO3 plotted in two different
figures in supplementary materials is different (see the deep in the spectrum at about 238 nm in the upper
figure and at about 250 nm in the lower figure) what makes all presented results unreliable. Whereas,Uv
spectra at 200-600 nm of nanoparticles give a sharp absorbance band at 431nm and 436 nm for
Acokanthera oppositifoliaand Leucaena leucocephala [Link] reference to specific plots
in the specific figures. In addition, authors claim that they observe sharp peaks, while described peaks show
broadening or some modification in comparison to the other spectra presented in the same figure.
After 24 hours, the maximum absorption peak intensity is observed, which indicates complete
phytofabrication of silver nanoparticles has taken place in the solution. The optical density in all
cases is roughly the same therefore I don't understand what the maximum absorption peak refers to.

3.2 Structural Characterization

3.2.1 SEM analysis

The shape of the synthesized silver nanoparticles was analyzed by SEM micrograph. Figures 3
and 4 (i) demonstrate that biosynthesized silver nanoparticles are spherical in shape.
The SEM images show area of about 25 μm x 20 μm, while the diameter of nanoparticles is below 30 nm,
therefore, contrary to authors' claim, it is not possible to observe the shape and size of particles in these
images.
3.2.2 TEM analysis

The TEM micrographs of biosynthesized AgNPs are displayed in Figures 3 and 4 (ii), which
show well-dispersed and spherical-shaped nanoparticles. However, some nanoparticles were
observed to be aggregated, but their borders were seen to be lighter than their cores. Our results
supported a previous finding (Das, B. in 2017). Some light-colored material found on the surface
of nanoparticles indicates phytomolecule capping of the silver nanoparticles, which are
responsible for fabrication, these factsare similar to the finding of the previous study by other
researchers (33, 34).Particle size distribution of Acokanthera oppositifolia AgNPs, was in the
ranges of 4 to 27.5 nm with the average size of 11.71nm ±0.0273,
11.34nm±0.04713.88nm±0.019 at 80,000X 40,000X and 30,000X magnification respectively, as
compared to Leucaena leucocephala AgNPs, was in the ranges from 4.2 to 23.5 nm with the
average size of 9.43nm ±0.028, 14.15nm±0.015,12.68nm±0.018 at 80,000X 50,000X and
30,000X respectively shown by histogram please see supplementary(S1).The result of TEM is
good agreement crystalline size obtained by XRD. The incorporation of diverse phytomolecules
for reduction as well as capping may result in different size distributions of nanoparticles (29).
Our findings are comparable to earlier reported 25–50 nm AgNPs produced from Leucaena
leucocephala seed and leaf extract (19, 35).The notation of average particles sizes is wrong. Firstly,
there is lack of information to what parameter these numbers refer to. Authors should inform what they
mean by size of the nanoparticle (assumed is the diameter of nearly spherical particle). Secondly, the units
should be given for the whole number. Thirdly, the notation of the value and standard error should agree
(the same significant figures) and the rounding rules should be applied.

3.2.5 FTIR scanning

Infrared light scanning, or FTIR, was used to decipher the involvement of phytochemical
constituents in the capping and stability of nanoparticles during fabrication. Apart from a few
peaksnearby shifting and the emergence of some bands as well, the FTIR spectra of leaf extracts
are nearly the same as nanoparticles in both plants, as shown in figures 3 and 4 (iii).This
resemblance justifies that some phytochemical constituents of plant extracts hold on to the
surface of silver, which is responsible for the reduction as well as stabilization of nanoparticles
during the fabrication.
Figure 3 (iii) represents a strong and wide peak at 3410cm -1 characterized by OH stretch of
phenolic hydroxyl group presence, 2906 cm-1 assigned to C-H stretching of alkane, peak at 2829
cm-1 indicates C-H stretching of aldehyde, band at 2111 cm -1assigned to C≡C stretching of
Alkyne monosubstituted, 1638 cm-1peakdesignated toalkene C=C stretching, little and sharpen
signal at 1408 cm-1characteristics of O-H bending of alcohol, 1255 cm -1 position represent (C-N)
Stretching of amine, 1042 cm-1 indicate CO-O-CO stretching in anhydride, 782 for C=C
bending alkane disubstituted (Cis), a signal at 615 cm -1designated for C-Br halo compound
stretching(36, 37, 38). After the reduction and stabilization of silver, the following peaks indicate
nearby shifting: 615 to 611, 782 to 794,1042to 1130, 1255 to1252, 1408 to1390, 1638 to
1640,2829 to 2842, 2906 to 2933, 3410 to3423 cm -1 indicating that hydroxyl, amide, bromine
halo groups may be participating in nanoparticle synthesis. The absence of a 2111 cm -1signal in
nanoparticles indicates that alkyne oxidation coupled with silver reduction and increment in
signal intensity at 1042 cm-1 CO-O-CO stretching in anhydridewas also observed.

Figure 4 (iii) depicts, that theN-H stretching of aliphatic primary amine can be observed by the
strong and broadening peak at 3395 cm -1, C-H stretching of alkanes at 2906 cm -1, and little and
sharp band at 1592 cm-1designated for N-H stretching of amide, S=O stretching of sulfonyl
chloride represent by a small and sharp peak at 1408 cm -1, C-N stretching of amine at 1225 cm -1,
C-O stretching of primary alcohol at 1057, C-Br stretching at 645-507. Whereas after
nanoparticle synthesis following peaks indicate nearby shifting from 1225 to 1213, 1408 to 1380,
1592 to1553, 2829 to 2832, 2906 to 2894, 3395 to 3418 to cm -1 which indicates that alcohol,
bromine halo compound, amide, sulfonyl chloride responsible for silver reduction and
stabilization.

The absence of 1225 cm-1and 1057 cm-1 bands in nanoparticle spectra suggests that nitrogen-
containing compounds such as mimosine amino acid and alcohol were involved in nanoparticle
synthesis. There was also an increase in intensity at 2832 cm -1and 2894 cm-1, which represent the
involvement of aldehyde and alkane, respectively.A new peak at 1553 cm -1 indicates N-O
stretching of the nitro compound, and a 1614 cm -1α,β-unsaturated ketone was also detected. The
difference in intensity, location and the number of peaks indicate the silver ion reduction.

3.2.3 EDAX analysis

The study of the EDX indicates a substantial signal from the silver zone, supporting the silver
nanoparticle fabrication represented in Figures 3 and 4 (iv). Besides this, the signal for Cu and Al
is also present which has come from the TEM grid and holder respectively. Other elements such
as chlorine, potassium, carbon, oxygen, calcium, and iron as recorded possibly due to elements
from enzymes or proteins (39). The signal for nitrogen might be due to a partial silver nitrate
reduction in the reaction mixture or may be present in metabolites of plants.
3.2.4 XRD Profile
The crystalline nature of nanoparticles is depicted in Table 1, by X-ray diffraction profile.
Different brags reflection peak was obtained for the phytofabricated nanoparticles of
Acokanthera oppositifolia and Leucaena leucocephala which represent in Figures 3 and 4 (v).
All these peaks are representative of the fcc structure as compared to JCPDS International center
for data diffraction File No.: 04-0783 (40, 41). The calculated d spacing was 2.35, 2.04, 1.44,
1.23Å, for Acokanthera oppositifolia nanoparticles, whereas, 2.35, 2.03, 1.44, 1.24, for
Leucaena [Link] numbers are wrong in the case of plane (311), for which the
value of diffraction angle is clearly wrong with the results on Fig. 3v and 4v. Such diffraction angle doesn't
As we can see
correspond to presentedinterplanar spacing. Authors should double check their results.
from the figure, the highest peak intensity for (111), among all which depicted maximum amount
of nanoparticles growth in this plane and peak sharpening represent particles are in the nano
range. Various unassigned peaks may be due to the presence of a capping and stabilizing agent
of plant metabolites adsorbed over the surface of nanoparticles. The mean crystallite size was
calculated using the Debye-Scherrer’s equation: D=Kλ/βCosθ,

λ x-ray wavelength (1.54 Å), k Scherer’s constant (0.9), D crystal diameter, and β XRD peak
FWHM. The average crystalline size reported for Acokanthera oppositifolia and Leucaena
leucocephalae phytofabricated nanoparticles were 13.38 nm and 11.93 nm, respectively
(Table2). The outcomes of this analysis also confirmed conformity for SEM and TEM findings.
The values given in the Table 2 do not agree with the numbers than can be obtained based on the provided
data (diffraction angle, FWHM, X-ray Wavelength and Scherer's constant). Authors should double check these
calculations. In addition, presented numbers for the sizes of fabricated nanoparticles are given as average
numbers based on the calculations for each observed diffraction peak. These numbers are wrong as they
were obtained from the incorrect values (particularly for the last considered peak). In addition, authors
present numbers without estimation of an error, while in the case of NPs sizes estimated from TEM they gave
standard error values. I do not see the reason why the error is not estimated also in the case of XRD
analysis.
3.3 Antimicrobial activity

3.3.1 Zone of inhibition

The antibacterial property of the phytofabricated AgNPs using two different concentrations (50
and 100 µl) of Acokanthera oppositifolia and Leucaena leucocephala, against four pathogenic
bacteria: Bacillus cereus, Staphylococcus aureus (Gram-positive), Enterobacter aerogenes, and
Pseudomonas aeroginosa (Gram-negative) was employed using the agar welldiffusion
[Link] 5 (a;h) petri plates showing inhibition [Link] inhibition zones of nanoparticles
and plant extracts are compared to the ampicillin reference.

It is observed in Table 3, the antimicrobial efficacy of nanoparticles prepared from leaf extracts
of Acokanthera oppositifolia is found to be more significant at 13±0.88, 14±0.66, 15±0.88,
16±0.66 mm against B. cereus, S. aureus, E. aerogenes, P. aeroginosa bacteria respectively at
100 µl as compare to nanoparticles ofLeucaena leucocephalaat the same [Link] the
other hand, when bacterial strainsare treated with pure plant extracts inhibition zone is less as
compared to nanoparticles. In contrast, P. aeroginosa resistsAcokanthera oppositifolia pure plant
extracts at both the concentration, and B. cereus E. aerogenes resist Leucaena leucocephala pure
plant extracts whereas phytofabricated nanoparticles of these plant extract were found to be
effective against these selected microorganisms. Because we detected a larger inhibition zone in
the case of bacterial strains treated with nanoparticles, the antibacterial activity of
phytofabricated silver nanoparticles is significantly higher than that of pure plant extract. The
nanoparticles exhibited antibacterial efficacy in a dose-dependent manner against all tested
microbial cultures because a bigger inhibition zone was observed when using a 100 µl
concentration than 50 µl. A similar result was found earlier, in Leucaena leucocephala AgNPs
where dose-dependent activity was observed and more effective against gram-negative bacteria
(19). The result in Table 4 indicates that the relative inhibition zone diameter obtained for the
gram-negative bacterial strain was more as compared to gram-positive when treated with
nanoparticles of these plants. Enhancement in antibacterial activity against gram-
negative bacteria may be the result of liberated silver ions by nanoparticles which destroy the
cell wall or the result of secondary metabolites adsorbed on the nanoparticle surface, as well as
because the cell wall composition is different structurally in gram-positive and gram-negative
bacteria (42, 43).
The statement "It is observed in Table 3, the antimicrobial efficacy of nanoparticles prepared from leaf
extracts of Acokanthera oppositifolia is found to be more significant at 13±0.88, 14±0.66, 15±0.88, 16±0.66
mm against B. cereus, S. aureus, E. aerogenes, P. aeroginosa bacteria respectively at 100 µl as compare to
nanoparticles of Leucaena leucocephala at the same concentration." is true only in two cases (B. cereus and
P. aeroginosa) and in the case of other two the values are comparable.

The claim from the following statement "On the other hand, when bacterial strains are treated with pure
plant extracts inhibition zone is less as compared to nanoparticles." should be quantified.

"In contrast, P. aeroginosa resists Acokanthera oppositifolia pure plant extracts at both the concentration,
and B. cereus E. aerogenes resist Leucaena leucocephala pure plant extracts whereas phytofabricated
nanoparticles of these plant extract were found to be effective against these selected microorganisms." It is
not clear against what authors oppose this statement, as it agrees with the previous information. However,
in the same sentence, authors claim the same behavior of nanoparticles from both plants in the case of all
bacteria, while in the table 3 there is lack of information in several cases, therefore it is not possible to make
such strong claim.

- Section 3.3.1, lines 40-43: Sentence repeats information from the discussion that precedes it.
- Section 3.3.1, lines: 47-50: "The result in Table 4 indicates that the relative inhibition zone diameter
obtained for the gram-negative bacterial strain was more as compared to gram-positive when treated with
nanoparticles of these plants.". This is again false statement. For example, the RIZD% of APN100 in the case
of gram-negative bacterial strain (E. aerogenes) is smaller than the same concentration of the same plant in
the case of gram-positive bacterial strain (S. aureus). The same behavior is observed in the case of LNP100
for the same bacterial strains. Therefore, the explanation of this false statement, that is presented in the
next sentence (lines 50-53) is not valid too.
- Authors do not show values of zone of Inhibition (Table 3) in several cases. However, these results are
needed to calculate RIZD% values that are discussed in the section 3.3.1 and shown in table 4. Surprisingly,
only in one case in the table 4 the value is missing (the case with no change in zone of inhibition). Authors
should explain and provide values that were used to calculate all other cases that are impossible to estimate
with missing numbers in table 3.

3.3.2 MIC values

After 24 h of incubation, no growth of B. cereus, S. aureus, E. aerogenes, P. aeroginosawas
observed in the test tubes supplemented with 1.5, 1.5, 1.5, 1.0 mg/ml nanoparticles solution of
Acokanthera oppositifolia which comparable to MIC value 2.0,1.5, 1.5, 25.0 mg/ml of aqueous
extracts of this plant as shown in (figure 6a).The optical density of nanoparticles at 600 nmwas
found to be 0.40, 0.46, 0.64,and 0.74 against tested pathogenic microorganisms (Figure6 b).
Similarly MIC of Leucaena leucocephala aqueous extracts was found to be 10.0, 2.0, 2.5, 2.0
mg/ml as compared to their phytofabricated nanoparticles the inhibitory concentration was 2.0,
1.5, 1.5,1.5 mg/ml with optical density 0.40, 0.44, 0.63,0.73 at 600 nm (Figure6 c).

At last, we conclude that antibacterial activity depends on the three-factor Concentration of

nanoparticles and type of plant-specific phytochemicals and metabolites in plants, and the type of
bacteria due to structural [Link] are supposed to be various mechanisms that make the
nanoparticles more susceptible to working as an antibacterial agent. These particles cause
damage to bacterial cells by permeating and interfering with DNA, proteins, and other cellular
constituents as a consequence of alteration in physical and chemical properties as well as
impaired electron transport, permeability, respiration, and osmoregulation (44, 45, 46).Silver
nanoparticles liberate silver ions, which act as an antimicrobial according to their dosage and
size (47).
- Section 3.3.2, line 60: The MIC value of APE for S. aureus do not match the one in the table 4 and Fig. 6a.
- Section 3.3.2, lines 4 and 8: The values of optical density at wavelength 600 nm, given in the text, do not
correspond to the ones presented in Figs 6b,c.
- Authors do not draw any reasonable conclusion from MIC test; therefore, it seems to be useless. Instead,
they formulate strong claim about "damage to bacterial cell by permeating and interfering with DNA,
proteins and other cellular constituents…" what was not presented in this work.

4. Conclusions

In this work, we effectively produced spherical-shaped silver nanoparticles through aqueous leaf
extracts of Acokanthera oppositifolia and Leucaena leucocephala of 13.38 and 11.93 nm,
[Link] nanoparticles possess good antibacterial activity as compared to their pure
aqueous extracts against gram-positive as well as gram-negative but are more effective against
gram-negative bacteria. This may be the consequence of their, size or large surface-to-volume
ratio which makesthem accessible to readily absorb by cell surface and interact with their
intracellular molecules with greater efficiency. Based on the results of the present study, we can
conclude that the phytofabricationprocessis not only a simple, time-saving, cost-effectiveeven
environment-friendly and ideal strategy in comparison to other biological chemical and physical
methods of nanoparticle synthesis. However, there are still several unsolved questions about how
nanoparticles cross the cell membrane and what is the exact antibacterial mechanism? Therefore,
this branch is still in its early stages and requires more research, which opens the door to various
unanswered problems in this field and paves the way for a broad future [Link] studies for
other prospective applications of these silver nanoparticles are in progress.
Section 4, lines: 21-22: Although authors obtained sizes of nanoparticles with two different techniques, they
do not discuss agreement or disagreement of these values. Why? Instead, they present averaged values
from XRD analysis without information how these values were obtained and without standard error.
- The conclusions that authors draw in the section 4 do not agree with the results.

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