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

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: DOI: Reference: S1011-1344(18)30716-4 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 Arya 1, R. Mankamna Kumari 1, Nikita Sharma 1, Nidhi Gupta 2, Ajeet Kumar 3, Sreemoyee Chatterjee2, Surendra Nimesh1,* 1 ED MA NU SC RI P T Department of Biotechnology, School of Life Sciences, Central University of Rajasthan, Ajmer 305817, India 2 Department of Biotechnology, The IIS University, Gurukul Marg, SFS, Mansarovar, Jaipur 302020 Rajasthan, India 3 Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699-5814 *Corresponding author AC CE PT 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 suc h SC RI P T as reaction time, AgNO3 concentration, extract to AgNO 3 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. NU 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 MA 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, ED 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 CE PT span of time. Further, AgNPs were assessed for their wound healing potential. AC 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 SC RI P T to many health issues including liver and kidney damage, skin problems, central nervous syste m 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. NU 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 MA properties depending upon their shape and size(9). Additionally, AgNPs can be employed as antifungal, anti-bacterial, anti-inflammatory, anti-viral, anti-angiogenesis, antiplatelet and as cancer ED 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 PT 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). CE 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 AC 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, ecofriendly 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 agai nst 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 SC RI P T 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 4aminophenol. To evaluate the wound healing potential the excision and treatment study on mice model were performed. 2. Experimental NU 2.1. Materials Silver nitrate (AgNO 3), Luria Agar, Luria broth, Kanamycin were purchased from Central Drug House, India. 4-nitrophenol, Congo red, methylene blue and sodium borohydride were MA 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 ED 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 PT 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 ddH 2O at 60ºC CE (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 AC 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 AgNO 3 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 SC RI P T 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. NU 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 AgNO 3 were taken as 1:40, 1:20, 1:10, 1:6.5 and 1:5 MA 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, ED 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. PT 2.6. Physicochemical characterization After UV-vis spectra confirmation, the synthesised AgNPs were evaluated through Dynamic light CE scattering (DLS) for the analysis of their average hydrodynamic diameter along with polydispersity index (PDI) using Zetasizer Nano ZS (Malvern Instruments UK). Fully dispersed AC 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 SC RI P T (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 NU 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 MA 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 ED 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 PT 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 CE with 2 ml water. Control 2 involved control 1 as well as 1 ml NaBH 4 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 AC 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 periodically monitored 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% CO 2 in CO 2 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 SC RI P T 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 spectrop hotometrically 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 NU with 1X-PBS. The cells were then treated with different concentration (0.1µM to 3µ M) of AgNPs, PJL extract, 1mM AgNO 3 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 MA 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 2.11 ED taken as 100 % viable that were used to compare the relative cell viability in the test wells. Animal experiment for wound healing The animal ethical committee of The IIS University approved the use of mice used in PT experiments and approved the protocols used. 2.11.1 Preparation of ointment CE 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 AC 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 SC RI P T 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 1 st, 6th, 10th and 15th days. The formula for calculation of wound healing is as follow: NU % of WH = (WA0 -WAn / WA0) x 100 Where WA0 = Wound area on day 0 MA WH= Wound healing WAn = wound area on day n (n= 1, 6, 10 and 15) Statistical analysis ED 2.12 All the experiments were done in duplicates, with three separate experiments to demonstrate reproducibility and presented as mean ±standard deviation (±SD). Statistical analysis PT was performed using Student's t-test. The differences were considered significant for p < 0.05 and p < 0.01 indicative of a very significant difference. Results and discussion CE 3 3.1 Biosynthesis of AgNPs synthesis AC 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), AgNO 3 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 SC RI P T 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 AgNO 3 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 NU 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 MA 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). ED 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. PT 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 CE 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 AC 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 SC RI P T 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 NU 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 MA appearance of dry crystalline black colonies on the plate (A(i), B(i)) where AgNPs were not supplemented. ED 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 PT the anti-biofilm activity of PJL-AgNPs. 3.6 Evaluation of catalytic activity of AgNPs CE 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 NaBH 4 along with silver nanoparticles that AC act as catalyst for the reaction. Though reduction of 4-NP into 4-AP with NaBH 4 can be possible, it is limited by the kinetic barriers due to difference in the thermodynamic potential of electron donor (NaBH 4) 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 NaBH 4 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 a fter 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 SC RI P T MB was evaluated through biosynthesised AgNPs at a stipulated time points in comparison with NaBH 4 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 NaBH 4 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 NU other hand, second reaction includes a mixture of dye and NaBH 4 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 MA 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 ED 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, PT 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. CE 3.7 In-vitro cell viability assay The cell viability result was evaluated via alamar blue assay after 24 hr of treatment. All the AC 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. T Conclusion SC RI P 4 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 NU are optimised including time, AgNO 3 concentration, extract to AgNO 3 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 MA 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 ED 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 PT showed that the synthesized PJL-AgNPs exhibiting the contraction of wound and promote wound healing very efficiently within 15 days. CE Conflict of Interest Authors declare that they have no conflict of interest. AC 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. 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Ballauff (2010) Kinetic Analysis of Catalytic Reduction of 4-Nitrophenol by Metallic Nanoparticles Immobilized in SC RI P T Spherical Polyelectrolyte Brushes. The Journal of Physical Chemistry C 114, 88148820. 37. Premkumar, T., K. Lee and K. E. Geckeler (2011) Shape-tailoring and catalytic function AC CE PT ED MA NU 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) AgNO 3 solution, (c) color change due to AgNPs synthesis. 2. UV-vis spectra data of optimisation studies (A) Reaction time, (B) AgNO 3 concentration, (C) extract to AgNO 3 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) SC RI P T 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 NU 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 MA by A-Bacillus subtilis and B- Pseudomonas aeruginosa bacteria, respectively. Whereas the addition of PJL-AgNPs block the exopolysaccharide secretion by ED 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) PT MB with NaBH 4, (D) MB with AgNPs, (E) Kinetic curve of MB degradation (F) CR with NaBH4, (G) CR with AgNPs (H) Kinetic curve of CR degradation. CE 7. In-vitro cell viability assay of synthesised PJL-AgNPs (0.1 to 3 µg) against HEK293 cells via alamar blue reagent. AC 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. AC CE PT ED MA NU SC RI P T  Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8