Grewia tenax -Mediated Silver Nanoparticles as Efficient Antibacterial and Antifungal Agents

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Introduction
Efective treatment strategies have been developed for eradicating common microbial infections.Despite the concerning approaches, the ever-increasing incidences of microbial infections commonly caused by parasites, fungi, viruses, and bacteria are alarming.Several common bacterial infections are caused by bacteria such as Escherichia coli, Streptococcus pneumonia, Klebsiella pneumonia, and Staphylococcus aureus [1].Bacterial infections further cause several chronic infections and are the reason for the high number of mortalities, globally [2].Terefore, the discovery of antibiotics is directed as a remarkable achievement in the medical feld, from surgery to organ transplant and for various microbial infections and diseases.But bacteria have developed immunity against antibiotics which led to the occurrence of multidrugresistant bacteria [3].For decades, antimicrobial resistance (AMR) has been growing as a threat to the treatment efcacy of antiparasitic, antifungal, antiviral, and antibacterial drugs.Hence, antibiotic resistance, especially antibacterial resistance (ABR), has turned out as a dilemma in the medical feld and public health, for treating patients.
To overcome this issue of antibiotic resistance, researchers are exploiting nanotechnology to develop nanoparticles as "nano-antibiotics" for targeted drug delivery without any drug resistance problem [4].Nanoparticles (NPs) with a size range of 1-100 nm can penetrate the bacterial cell wall, thus, in turn, impeding the bacterial biochemical pathways by destroying the cell organelles followed by bacterial cell death [1].NPs generate reactive oxygen species (ROS) which damage the bacterial cell membrane [5], as well as target several cellular pathways simultaneously, making it impossible for the bacteria to develop resistance against them [6].Due to the direct interaction and binding with the bacterial cell wall, there is no requirement for cell penetration and NPs can be used as alternatives to antibiotics, and their mode of action is irrelevant to the majority of the antibiotic resistance mechanisms [2].
Unique properties of nanoparticles, such as size, large surface area, exceptional mechanical strength, high stability, and lower melting points, make NPs the best candidate as drug carriers for several clinical applications [7].Tese properties drove the interest of researchers from conventional synthesis to a biological synthesis approach, i.e., using biological systems for synthesis of nanoparticles, the process being termed as "green synthesis" [1].
Te green synthesis of metallic NPs (Ag, Zn, Au, etc.) is gaining global acceptance by the scientifc community [1,8] due to its cost-efectiveness, ease of synthesis, biodegradability, nontoxicity, and environmental benignity [9][10][11].Several types of NPs have attracted interest for their properties in the feld of biomedical applications.Carbon-based NPs gained interest because of their interesting biomedical applications owing to the biocompatibility.Carbon NPs are extensively studied in several forms, from bulk to nanoscale [12], along with their methods of preparation and modelling [13].Moreover, among a variety of nanomaterials, currently available in biomedical and biosensing research, silicon nanoparticles [14] are biocompatible and biodegradable and exhibit peculiar physicochemical properties, making Si-based nanostructures one of the most promising candidates as theragnostic material.Furthermore, among the metallic NPs, it is necessary to mention gold NPs (AuNPs) prepared by diferent methods [15] that attract the interest of researchers due to their enormous diagnostic importance of several diseases including diagnosis of cancer or imaging capabilities related to their optical properties, such as exhibiting a strong surface plasmon resonance [16].
Silver nanoparticles (AgNPs) have gained much popularity and are known to be a material of the future [17].Recently, nanotechnologists in the feld of biomedical science are more concerned with the biogenic/green synthesis of AgNPs to conquer the major drawbacks associated with conventional approaches.Plants have gained attention as a production assembly for the green synthesis of silver nanoparticles, whether from their fruit, seed, leaf, stem, bark, fower, or root extracts [18,19].Plant-mediated synthesis provides an ecofriendly, cost-efective, rapid, operative, and nontoxic/nonpathogenic approach with only a single-step technique for the biosynthesis of silver nanoparticles [20,21].Biomolecules or biocomponents of the plant(s) (phytochemicals) serve as reducing and stabilizing agents for silver ions.
Hence, to solve the problems associated with the nonbiological synthesis approach of AgNPs and to explore a new mode of antimicrobial therapeutic agent, this study has been designed.Te research work aims to develop a biological/ green approach to synthesize silver nanoparticles using Grewia tenax plant leaf extract and testing these GTAgNPs for their antimicrobial activity with a special focus on antibacterial activity against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains and antifungal activity against Aspergillus niger and Candida albicans.Tis work gives us more insights into the applications and mechanism of biological therapeutics of green synthesized silver nanoparticles from Grewia tenax, a plant known for its medical ailment properties.Tis study might prove useful in future selection of biogenic AgNPs for their antimicrobial applications to use as alternatives to antibiotics for the beneft of patients as well as the environment.

Materials.
Fresh and disease-free leaves of Grewia tenax were collected from neighbourhood nurseries in Mansarovar, Jaipur, India, and identifed from the University of Rajasthan, Jaipur, India.All the chemicals were of analytical grade and obtained from HiMedia Laboratories Pvt. Ltd., India, and Sigma Aldrich, St Louis, Missouri, USA.Te bacterial and fungal cultures were obtained from the Central Governmentapproved MTCC facility of IMTECH, Chandigarh, India.

Preparation of Grewia tenax Leaf
Extract.Fresh leaves of the plant were thoroughly washed and dried for two to three days under shade before they were ground to powder form.Te powdered leaves were used to prepare 10% (w/v) extract by heating at 60 °C for 20 min with continuous stirring.Ten, the mixture was cooled and fltered through Whatman flter paper No. 1 with reduced pressure conditions.Tis extract was utilized for NP synthesis and stored at −20 °C for future use.

Biosynthesis and Optimization of Parameters for Silver
Nanoparticles.Optimization of physical conditions was done following the rule of one variable at a time (OVAT).Te extract used was prepared by dissolving 10 g of leaves powder in 100 ml water.Under the process, for the green synthesis of GTAgNPs, diferent volumes of extracts (50 μl of extract in 950 μl of AgNO 3 solution; 100 μl of extract in 900 μl of AgNO 3 solution; 150 μl of extract in 850 μl of AgNO 3 solution; and 200 μl of extract in 800 μl of AgNO 3 solution so that the ratio used was 1 : 5, 1 : 7.5, 1 :10, and 1 : 20) were added in a dropwise manner to the varying concentrations of AgNO 3 (1.0 mM, 1.5 mM, 2.0 mM, and 2.5 mM) solution under constant stirring at 600 rpm for varied time intervals and temperatures.Color variation was an essential indication for the synthesis of NPs.Te synthesized nanoparticles were centrifuged and the pellet was washed three times with deionized water.Ten, it was resuspended in deionized water, and formation of GTAgNPs was confrmed by UV-visible spectroscopy.

Optimization of Process Parameters.
Te essential parameters which are important in the process of biosynthesis of GTAgNPs including the time of reaction, temperature, the concentration of AgNO 3 , and the ratio of extract to AgNO 3 were addressed during the green synthesis of NPs.By varying one parameter and keeping other factors constant, the biosynthesis process was carried out for 10 minutes and UV-visible spectrum was recorded to determine the synthesis.After optimizing the time required for synthesis, other three factors such as concentrations of AgNO 3 (1.0,1.5, 2, and 2.5 mM), temperature (4 °C, 25 °C, 40 °C, and 60 °C), and variable extract to AgNO 3 ratio (1 : 20, 1 : 10, 1 : 6, and 1 : 5) were analysed similarly and each time UV-visible spectrophotometric analysis was performed to conclude the best obtained yield.

Design Expert Systematic Optimization of GTAgNPs by
Using Box-Behnken Design (BBD).Using Design Expert, Box-Behnken design (BBD) ver.12.0 software (Stat-Ease Inc., Minneapolis, USA), the most signifcant parameters, i.e., volume of leaf extract, concentrations of AgNO 3 , and reaction temperature, were chosen as self-regulating factors for optimization.Te trial runs were performed, and based on experimental trials, polydispersity index (PDI) and size of particles were calculated by the dynamic light scattering (DLS) technique.BBD results were reviewed through mathematical modelling, and numerical desirability function along with graphical optimization essential for the biosynthesis of GTAgNPs was obtained.Various important parameters that were taken into consideration for BBD study are summarized in Table 1.

Physicochemical Characterization of GTAgNPs.
Te dispersed AgNPs were analysed using UV-visible spectrometry within 300-700 nm wavelength range.Te method is useful to estimate the size, concentration, and aggregation level of the particles.Determination of the hydrodynamic diameter and polydispersity index of GTAgNPs was done using dynamic light scattering (DLS).Te Brownian motion of the NPs causes the scattering of the projected laser light at varying intensities.Te particles were dissolved in deionized water and sonicated for 15 min, before being screened with a 5 MW He-Ne laser run at 633 nm wavelength followed by scattered light at 173 °angle.
To identify the functional groups responsible for the reduction and stabilization of GTAgNPs, Fourier transform infrared (FTIR) spectroscopy of both plant extract and NPs was carried out.For this, KBr was utilized for tablet construction and scanned in an array of 4000 to 450 cm −1 .Te spectrum revealed possible interaction between Ag and the bioactive molecules present in the extract.
Te microstructural study for evaluation of defnite shape-size and surface morphology of GTAgNPs was performed by scanning electron microscopy (SEM), and the chemical components of these particles were studied by using EDX.In SEM, during the interaction of the electrons and the sample, a wide variety of signals arise which reveal a lot of information about the surface morphology of the particles.During this process, NP solution was frst converted into a dry powder and mounted on a sample holder which is followed by coating with a conductive metal, such as gold, using a sputter coater.A focused fne beam of electrons is used to scan the sample.Te surface characteristics of the sample were obtained from the secondary electrons emitted from the sample surface.

Antimicrobial Potential of GTAgNPs.
Te biosynthesized GTAgNPs were explored for their antimicrobial potential against Gram (+) and Gram (−) bacteria as well as against fungal strains.Tese particles were also evaluated for their potency in inhibiting the formation of bioflm.
(1) Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of GTAgNPs.2, 3, 5-Triphenyl tetrazolium chloride (TTC) assay was carried out to evaluate the MIC of the synthesized GTAgNPs.TTC was used as a chromogenic marker which got reduced to TPF (1, 3, 5-triphenyl formazan) in the presence of bacteria.100 μl of 24 h bacterial culture was poured into a 96-well plate with increasing concentrations of GTAgNPs from 0.05 mg/ml to 1 mg/ml.10 μl of freshly prepared TTC was added to each well.Te plate was incubated for 24 h at 37 °C, and the absorbance was read at 480 nm.In the presence of live bacterial cells, TTC got reduced to red formazan, which is directly proportional to the percentage of viable cells.
MBC was evaluated using broth dilution of MIC tests where no observable growth was seen followed by plating the cultures on an agar plate and incubating for 24 h at 37 °C.Te concentration at which no growth of bacterial colonies was observed was taken as the MBC.
(2) Estimation of Antibacterial Efcacy of GTAgNPs by Disc Difusion Method.Te antibacterial activities of GTAgNPs were evaluated against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) by disc difusion assay taking into consideration the zone of inhibition.Bacteria were grown as primary culture in Luria broth for 24 h at 37 °C.Te secondary inoculum was taken as 1% of the primary inoculum and incubated till an O.D. of 0.4 at 600 nm was achieved.Secondary cultures were streaked on agar plates with a density of 10 5 CFU ml −1 , and sterilized discs were placed on plates and loaded with varying concentrations of GTAgNPs (0.25, 0.5, 0.75, and 1.0 μg).Te plates were then incubated overnight at 37 °C, and the zone of inhibition was measured [22].

Antibioflm Activity of GTAgNPs
(1) Congo Red Assay.Te antibioflm activity of GTAgNPs was studied against four strains of bacteria (B.cereus, S. aureus, E. coli, and P. aeruginosa) using BHI agar media

Mechanism of GTAgNPs' Antibacterial Activity by SEM.
To understand the mode of action of AgNPs against bacteria, SEM was done to observe the changes that occur on the cell surface for nanoparticles-treated and untreated bacterial culture.For the study, B. subtilis was grown in the presence and absence of GTAgNPs, and SEM was performed.Slides were prepared for the cultures, heat fxed, and stained with 2.5% glutaraldehyde followed by washing with PBS for three times.A dehydration series of ethanol (30%, 50%, 70%, 80%, 95%, and 100%) was done before air-drying [23].Tese prepared slides were coated with gold and analysed under SEM.

Antifungal Assay
(1) MIC and MFC Determination of GTAgNPs Using Broth Dilution Method.Te biosynthesized GTAgNPs were tested against Aspergillus niger and Candida albicans for their antifungal profciency.Te strains of both were maintained on PDA and YMA, respectively, at 28 °C for 24 h.Te MIC value was well defned based on visual inspection of fungal growth at diferent concentrations of nanoparticle suspensions.In this, two sets of tubes with 5 ml PDB were taken to which diferent concentrations of GTAgNPs (5, 7.5, 10, and 12 μg/ml) were added.PDB alone was taken as negative control, and amphotericin B at 20 μg/ml was taken as the positive control.Tese tubes were inoculated with 20 μl of diluted fungal spore suspension and incubated for 3-4 days at 28 °C.Based on visualization, MIC values were taken which were further confrmed by taking O.D. at 600 nm.
(2) Disc Difusion Assay.Te antifungal efciency of GTAgNPs was studied against A. niger and C. albicans by performing a disc difusion assay.Te colonies of A. niger and C. albicans were obtained on potato dextrose agar and yeast malt agar plates, respectively.Ten, spore suspension broth was prepared in Tween 20 (0.02%), and standard (0.5) McFarland's solution was used for regulating turbidity.Prepared plates of PDA and YMA were used for harvested spores.Sterile discs impregnated with diferent concentrations of GTAgNPs (2.5, 5, 7.5, 10, and 12 μg/ml) were placed on seeded PDA and YMA plates along with negative (distilled water) and positive controls (amphotericin B 20 μg/ ml).Incubation of plates was done at 28 °C for 48 h and inhibition zones were measured.
(3) Colony Growth Assay.For colony growth inhibition assay, diferent concentrations of GTAgNPs (5, 7.5, 10, and 12 μg) were incorporated in PDA and YMA plates along with both negative (only PDA and YMA) and positive (PDA and YMA with amphotericin B 20 μg) controlled plates.Afterward, the cultures of A. niger and C. albicans were introduced at the centre of each PDA and YMA plate (6 mm wells) individually.Incubation was done for 3-5 days at 28 °C to monitor the retardation of growth, and the growth inhibition rate was calculated using the formula mentioned below: where C is the diameter of the negative control colony and T is the diameter of the treatment colony.

Mechanistic Study of GTAgNPs' Antifungal Activity.
For studying the mechanism of action of GTAgNPs on Aspergillus niger, SEM was conducted.For this, 100 μl of treated and untreated liquid fungal cultures was centrifuged at 6,000 rpm for 10 minutes.Te supernatant was discarded and the pellet was resuspended in sodium-phosphate bufer (100 mM, pH 7.0) by gentle inversion.A drop from this suspension was placed on the slide and air-dried.Dehydration series of ethanol (30%, 50%, 70%, 80%, 95%, and 100%) for the preparation of air-dried slides was carried out for 10 minutes in each step.Afterward, the gold coating was done on the slides to enhance the quality of the image.

Results and Discussion
Te plant of Grewia tenax was submitted for identifcation to the Department of Botany, University of Rajasthan, Jaipur, India, and the reference number obtained was RUBL 21279.Leaves of the plant were then collected, dried, powdered, and sieved to obtain a fne powder that was used to prepare AgNPs using silver nitrate.Tese biosynthesized NPs were investigated for their antimicrobial activity against bacteria and fungi.
4 Nanomaterials and Nanotechnology

Biosynthesis of Silver Nanoparticles Using Grewia tenax Leaf Extract and Optimization of Reaction Parameters.
Biological preparation of AgNPs involved using plant extracts which is a more environmentally sustainable approach.For this, an extracellular strategy was followed where we used 2.5 mM AgNO 3 and 10% extract prepared from the dried powder of leaves of Grewia tenax in a ratio of 1 : 10 in a dropwise manner.Several phytochemicals such as favonoids, terpenoids, polyphenols, and alkaloids are present in the leaf extract of Grewia tenax that might play a dual role in the synthesis of AgNPs.Tese phytochemicals may act as reducing as well as stabilizing agents in the biogenic preparation of AgNPs.Te reaction mixture was stirred continuously for 5 minutes at room temperature, and once the color change was visualized (Figure 1(a), top panel), it was read by UV-visible spectroscopy in the range of 300-700 nm.
Te surface plasmon resonance (SPR) spectrum of AgNPs synthesized using plant extract showed a distinct peak at around 420 nm, confrming the presence of biogenic AgNPs as shown in Figure 1(b), lower panel.As explained by Brause et al., the obtained optical absorption spectrum of AgNPs is majorly dominated by SPR and the peak corresponded with particle size [24].With the increase in the size of the particles, a shift to longer wavelengths can be observed in the SPR spectra of metallic nanoparticles.Also, it can be concluded that fabricated AgNPs were isotropic and spherical as a single SPR peak was observed.

Box-Behnken Design-(BBD-) Based Evaluation of Experimental Conditions.
To have monodispersed and optimum synthesis of NPs, physicochemical conditions such as concentration of AgNO 3 , amount of extract, and the ratio of two along with temperature and time need to be standardized.For this, multivariate in silico tools are available to optimize the experimental conditions.We have employed one such tool, namely, Box-Behnken design (BBD), for 17 experiments which was established to zero down the best conditions for biogenic synthesis.Te seventeen experimental trials established in BBD Design Expert version 12.0 that gave particle size and polydispersity index (PDI) are summarized in Table 2. Tese trial runs were then validated by wet experiments, and particles were analysed by dynamic light scattering (DLS) for the average size and zeta sizer for charge potential.
Te data thus obtained in BBD were put under a quadratic polynomial model to generate diagnostic plots and evaluate them.As shown in Figures 2(a)-2(c), the model diagnostic plots obtained for response particle size gave a linear predicted vs. actual graph where it was observed that the predicted values were in close proximity with the actual ones.Perturbation plots were obtained to study the overall efect of all the selected factors, and the result showed that response is sensitive to factor B, i.e., extract volume to be used as a curvature in response to this factor is observed.Te other two factors, factor A, i.e., AgNO 3 concentration, and factor C, temperature, also showed slight curvature.Te interaction plots obtained were also showing bending patterns, indicating that all three factors play an important role in the biosynthesis of AgNPs and any fuctuations in the three factors would eventually afect the size (Figures 2(a

)-2(c)) and PDI (Figures 2(d)-2(f )).
Tree-dimensional (3D) response surface plots obtained are in agreement with two-dimensional (2D) contours.Tese plots evaluate the cumulative impact variations in all the variables on the size distribution of metallic nanoparticles.In our study, we observed that the predicted size range of the particles was 95-105 nm with changes in any of the three variables, indicating that there might be variations in the size of AgNPs with increasing concentrations of reactants and temperature (Figures 3(a)-3(f)).Te actual size as analysed by DLS was found to be ∼109 nm and the zeta potential was 0.321, very close to the one obtained by BBD, i.e., 105 nm at 25 °C and PDI of 0.0547, as shown in Table 2.

Optimization with respect to Concentration of AgNO 3 ,
Volume of Extract, and Temperature.Concentrations of AgNO 3 , the percentage of extract, and the ratio of the two afect the size of synthesized particles.Also, temperature plays an important role in the synthesis of monodispersed, spherical AgNPs.We have, therefore, used varying concentrations of AgNO 3 and extract amounts to obtain the best suitable conditions for Grewia tenax-mediated synthesis of AgNPs.As summarized in Figures 4(a)-4(c), we identifed that optimized conditions for GTAgNPs synthesis were 2.5 mM AgNO 3 and 1 : 10 v/v ratio for AgNO 3 to 10% extract, at 37 °C in 10 minutes of continuous stirring.Te temperature range from 25 °to 60 °C was found to be suitable for synthesis as a single, sharp peak was obtained when the SPR area was obtained by a UV-visible spectrophotometer.Te narrow, sharp peak indicated the formation of monodispersed nanoparticles.

Size and Shape Analysis of Biosynthesized GTAgNPs.
Size distribution of the formulated AgNPs along with the surface charge was analysed by DLS that revealed the hydrodynamic diameter to be around 109 nm with a zeta potential of 0.321 (Figures 5(a) and 5(b)), agreeing to have less polydispersity for the biosynthesized AgNPs.
To observe the morphology along with the size of GTAgNPs, scanning electron microscopy was conducted which showed that the particles formed were spherical with a smooth texture, and the average size was determined to be around 44 nm (Figure 6(a)).EDX analysis as summarized in Figure 6(b) showed that silver atoms were predominantly present with four Bragg's difraction values.Yazdi et al. also reported the presence of four Bragg's difraction values for silver nanoparticles biosynthesized using Rheum turkestanicum shoot's extract [25].

FTIR Analysis of Biogenic GTAgNPs. Fourier transform infrared (FTIR) spectroscopy tool helps understand the involvement of functional groups present in biomolecules
Nanomaterials and Nanotechnology found in plant extract that helps in reducing AgNO 3 to AgNPs as well as in capping to provide stability to the synthesized AgNPs.Te spectra were recorded both for the extract and the synthesized GTAgNPs following the range of 4000−400 cm −1 frequency.In the GT extract (Figure 7), the peak at 3427 cm −1 corresponding to OH stretching of polyphenols was observed which then moved to shorter frequency wavelengths in the obtained spectrum of AgNPs, clearly indicating the reducing role of hydroxyl groups of polyphenols present in the extract.Te FTIR spectrum for GTAgNPs revealed major peaks at 2982−2980 cm −1 which stands for CH stretching, along with peaks at 1603 cm −1 (N-H stretching) and 1253 cm −1 (C-N stretching) [26].Te presence of relative peaks (Figure 7) confrmed the role of carboxylic acids and primary and secondary amine compounds in the capping of AgNPs.

Estimation of Antimicrobial Efcacy of Nanoparticles
Using Various Methods

Antimicrobial Activity of Nanoparticles by Disc Diffusion Study.
AgNPs are known for their biological applications such as antimicrobial activities.Te literature recommends several studies that support the antibacterial activity of plant-based synthesized AgNPs.Te green synthesis approach provides a better augmentation to the existing antimicrobial properties of the plant in terms of nanoparticles as the synergistic impact can be obtained in terms of the latter.NPs synthesized using plant extract help in reducing the concentration of the required doses for the activity against bacteria and fungi.In this study, we have chosen Grewia tenax, a locally grown berry that is used in rural and suburban regions of Rajasthan, India, for various      stomach ailments.Te antibacterial activity of the particles was assayed by observing the zone of inhibition, obtained by placing paper discs impregnated with AgNPs, water, and ampicillin as tests, negative control, and positive control, respectively.Te zone of inhibition thus obtained for these is summarized in Table 3. Te activity of synthesized AgNPs was observed to be more potent in comparison to the positive control.In the case of Gram-positive bacteria, namely, B. subtilis and S. aureus, the antibacterial activity of AgNPs was 3-5 times more than the antibiotic ampicillin.Te results demonstrated that GTAgNPs were able to penetrate the thick cell membrane of bacterial cells more potently to impart their antibacterial efect.

MIC and MBC Determination of Nanoparticles Using
TTC Assay.Initially, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of GTAgNPs were determined to calculate the minimal dose that is efective in reducing the growth of bacteria and responsible for the death of more than 99 percent of bacterial cells, respectively.Te MIC was determined to be 0.5 μg for both Gram-negative and Gram-positive bacteria.MBC was observed to be 2.0 μg for Gram-negative bacteria, namely, E. coli, and 10 μg concentration was observed for S. aureus, i.e., Gram-positive bacteria.MBC is more for Gram-positive bacteria due to hindered accessibility of AgNPs through thick cell membranes.

Estimation of Antibioflm Efcacy of Nanoparticles
Using Congo Red and Crystal Violet Assays.Most bacteria impart their antibiotic resistance due to the formation of bioflm that is made of exopolysaccharides being secreted by the bacteria when present in a group.Bioflm has a defned structure and acts as a medium for genetic transfer and communication between cells [27].Bioflm is an important health issue as medical devices harbouring bioflms are responsible for an array of microbial infections in patients and add burden on an already crippled healthcare environment [28].
Green synthesized AgNPs can be coated on medical devices and to an extent prevent postoperative infection as NPs are quite efcient in inhibiting the formation of bioflms as observed by our earlier as well as current studies [29][30][31].In the present study, GTAgNPs were evaluated for their efcacy against bioflm formation in all four bacterial strains considered.Congo red agar assay (Figure 8(a)) clearly marked the inhibitory potential of AgNPs as shown by the absence of black colonies in the treated culture which are indications of secretion of exopolysaccharide for the formation of bioflms.Quantitative analysis by crystal violet assay also showed remarkable inhibition of bioflm formation in a dose-dependent manner as summarized in Table 4.

Evaluation of Antifungal Efcacy of GTAgNPs.
Post COVID-19 pandemic, Aspergillus niger and Candida albicans have been observed to cause severe infections in case of health compromised patients, while no such symptoms have been observed in the case of healthy people [25,26].C. albicans is clinically important as it is responsible for catheter-related blood infections [32].At times, antifungal treatment cannot be provided in time in such patients.AgNPs' efcacy can be studied in such scenarios as green synthesized nanoparticles can provide a solution to such comorbidities, but more detailed investigations are required.In our study, we evaluated the benefts of GTAgNPs against the two fungi.In vitroassays confrmed that even at a low concentration of 5 µg, AgNPs were able to signifcantly inhibit the growth of both the fungal strains as shown in Figure 9, by colony growth assays and broth dilution method.In comparison to amphotericin B, a known antifungal, GTAgNPs have exhibited almost 2-fold fungal growth inhibition.Fungi are more resistant to treatment due to their complex cell structure and advanced detoxifcation system [33], but our biosynthesized AgNPs have shown potent antifungal activity and this potential can be harnessed medically with further deep research investigations.

Analysis of the Mechanism of Antimicrobial Activity
Exerted by GTAgNPs by SEM.Te biosynthesized GTAgNPs were found to possess potent antibacterial and antifungal efcacy.As per a study conducted by Rama et al., biosynthesized AgNPs were observed to possess remarkable antioxidant potential along with antimicrobial efcacy [19].Te mechanism of the antimicrobial study was then studied by scanning electron microscopy (SEM) where AgNPstreated and nontreated bacterial and fungal cultures were observed for change in cell surface morphology.On observation, it was revealed that untreated bacterial as well as fungal cells possess intact, smooth cell membranes, whereas pores were formed on the surface of nanoparticles-treated cells (Figures 10(a) and 10(b)).In certain instances, it was observed that the microbial cells were ruptured due to the binding of AgNPs on the cell surface.
Burdus ¸el and coworkers explained that AgNPs work in a Trojan-horse fashion as their initial attachment to the cell surface causes permeability and respiration impairment, Nanomaterials and Nanotechnology followed by cell-barrier penetration and the release of intracellular metallic silver ions.AgNPs exert this activity via a Trojan-horse mechanism since their initial binding to the cell surface results in permeability alteration and respiration impairment, followed by cell-barrier penetration and alteration and respiration impairment, followed by cell-barrier penetration and intracellular metallic silver ion release [30].However, various mechanisms of action have been proposed    for biogenic AgNPs targeting bacteria and fungi in works published by various groups.Tree to four mechanisms of antibacterial and antifungal activity by the biosynthesized AgNPs have been reported in the literature which can be further categorized in diferent stages as follows: (1) reactive oxygen species (ROS) generation, (2) protein denaturation, (3) binding with cell wall/cell membrane, (4) formation of irregularly shaped pits in the cell membrane that leads to changes in the permeability of the membrane, (5) entry in the cell wall, (6) interruption in the respiratory chain and diferent pathways, (7) release of lipopolysaccharides and various membrane proteins, and (8) ultimately cell death [2,34,35].Te possible mechanism of action has been illustrated in Figure 11.Terefore, it can be deciphered that nanoparticles help in making the cell membrane unstable, leading to the formation of pores through which AgNPs might get accessibility to the cell and thus impart their inhibitory impact on the microorganism.Tough the exact mechanism of antimicrobial activity is not yet deciphered, the mortality of microbes might be due to any of the suggested mechanisms of action of AgNPs on the cell [31,36,37].

Conclusion
Te green approach for the synthesis of AgNPs is associated with an arena of applications in the felds of biomedical, pharmaceutical, and even agricultural activities.Te strategy is cost-efective and eco-friendly in the synergism of benefcial aspects of plants with silver.In this study, we have explored the potential of a locally grown berry, Grewia tenax, known for its medicinal properties, especially against stomach ailments with the antimicrobial activities of silver.Te synthesized AgNPs from the leaf extract of the Grewia plant were of 109 nm in size as observed in DLS analysis, and it was at par with the optimized conditions of the BBD tool.Te biosynthesized AgNPs showed remarkable antimicrobial activity against bacterial strains such as B. subtilis, E. coli, P. aeruginosa, and S. aureus and also against fungi A. niger and C. albicans along with potent bacterial bioflm inhibition activity.Tese particles were very active as they brought morphological changes in the cell structure of Gram-positive bacteria and Aspergillus niger and Candida albicans.Tese GTAgNPs have high antibacterial efciency with antibioflm profciency and showed high antifungal efcacy which could render them a protective tool in the feld of biomedicine specifcally with catheters and other medical instruments that might be responsible for infections after medical interventions.Te synthesized AgNPs can defnitely be used for many bioapplications, but further investigations are required for their safe applications which then can be more useful for the environment and human well-being.

Figure 1 :
Figure 1: (a) Synthesis of GTAgNPs (C-E) shown by a transition in the color of silver nitrate solution (B) from colorless to deep brown color after the addition of extract (A).(b) UV-Vis spectra of GTAgNPs in the range of 300-700 nm, along with that of extract as reference.

Figure 2 :Figure 3 Figure 3 :Figure 4 :
Figure 2: Model diagnostic plots of actual vs. predicted interaction and perturbation of response for silver nanoparticles in terms of size and PDI.(a) Representation of the actual particle size vs. the predicted particle size by BBD.(b) BBD evaluation of the efect on the particle size with variation in volume of the extract and AgNO 3 concentration.(c) Efect of temperature on the size of synthesized AgNPs as evaluated by BBD.(d) Evaluation of PDI linearity by BBD for actual vs. prediction.(e) Variation in volume of the extract vs. AgNO 3 concentration deviates PDI as observed.(f ) BBD analysis showed deviation in PDI with variation in temperature.

Figure 5 :Figure 6 :
Figure 5: (a) Te graphs depict the hydrodynamic diameter of GTAgNPs determined by dynamic light scattering which is ∼109 nm with (b) zeta potential of synthesized GTAgNPs.

Figure 7 :
Figure 7: FTIR spectra of Grewia tenax leaf extract (in red) and biosynthesized GTAgNPs using the extract under optimized conditions (in blue).

Figure 8 :
Figure 8: (a) Congo red agar assay showing antibioflm efcacy of GTAgNP.Te occurrence of black colonies marked bioflm production, whereas no such formations were seen in GTAgNP-treated bacterial strains.(b) Graphical representation of percentage bioflm inhibition in crystal violet assay.

Table 4 :
Quantitative estimation of bioflm inhibition (percentage) by crystal violet assay against Gram-positive and Gram-negative bacterial strains.

Figure 10 :
Figure 10: SEM images of (a) B. subtilis culture alone, (b) B. subtilis culture treated with GTAgNPs (c) A. niger culture alone, and (d) A. niger culture treated with GTAgNPs.

Figure 11 :
Figure 11: Schematic illustration of antimicrobial mechanism of silver nanoparticles.AgNPs cluster to cause cell membrane damage, making cells porous and causing cytosol leakage.Nanoparticles can generate formation of ROS that have radicalizing efect on microbial cells causing oxidative stress.AgNPs can bind to nucleic acids and can impair the synthesis of the nucleic acid and proteins.

Table 1 :
Independent variables are taken for optimization parameters via BBD.5%) and Congo red (0.08%).24 h grown cultures of these four strains were streaked on Congo red agar plates with and without GTAgNPs and kept at 37 °C for 24 h.Te positive results in terms of formation of bioflm by microorganism were indicated by the occurrence of black colonies having a dry crystalline consistency.Te microtiter plate assay is performed for the quantitative evaluation of the antibioflm efciency of the biosynthesized GTAgNPs.It is an indirect method for the estimation of bacteria in situ.For 24 h, grown bacterial cultures (B.cereus, S. aureus, E. coli, and P. aeruginosa) were taken and adjusted to 0.5 McFarland's standard.Te individual wells of the microtiter plate were flled with 180 μl of BHI broth and 10 μl of 24 h grown bacterial cultures.10 μl of GTAgNPs, varying in concentration (0.25, 0.5, 0.75, and 1.0 μg), was added in each (2) Microtiter Plate Assay (Crystal Violet Assay).well (except control wells), and cultures were then incubated overnight at 37 °C.After incubation, the plated cultures were washed thrice with 200 μl of phosphate bufer saline (PBS, pH-7.2) to remove any nonadherent bacteria.To fx the sessile organisms in wells, each well was treated with 200 μl of 2% sodium acetate.Te wells were air-dried and then 200 μl of 0.1% (v/v) crystal violet (CV) dye was added to each well and then washed multiple times.Te dye incorporated by the adherent cells was solubilized with 200 μl of 95% (v/ v) ethanol.Te concentration of CV was determined by measuring the optical density of the solution at 595 nm using a microtiter plate reader.

Table 2 :
Experimental trial runs given by BBD.

Table 3 :
Te zone of inhibition (mm) showing the antibacterial potential of GTAgNPs in a dose-dependent manner against Gram-positive and Gram-negative bacterial strains.