Synthesis of Biogenic Silver Nanoparticles (bAgNPs) Using Leaf Extract of Mirabilis jalapa and Evaluation of Anti-vibriocidal, Anti-oxidant properties and Cytotoxicity

Acute hepatopancreatic necrosis disease (AHPND) and luminescent vibriosis are two major bacterial diseases of penaeid shrimp which are caused by gram-negative pathogenic bacteria Vibrio parahaemolyticus (Vp) and Vibrio harveyi (Vh) respectively. These diseases cause massive mortality and huge economic loss worldwide in shrimp aquaculture. Extensive and inappropriate usage of antibiotics against these pathogens resulted in antibiotic-resistant strains. Drug repurposing appears to be an appropriate solution to eliminate antibiotic resistance in pathogens. In the present study, biogenic silver nanoparticles (bAgNPs) are synthesized by reducing AgNO3 using the aqueous extract of Mirabilis jalapa (MJ) leaves. The anti-oxidant, cytotoxic, and anti-vibriocidal activity of bAgNPs against Vp and Vh are evaluated. The formation of bAgNPs was confirmed by the appearance of a dark brown coloured solution and with a maximum absorption peak at 434 nm. The characterization of bAgNPs using FESEM and EDX, TEM, XRD, FTIR, and DLS has confirmed that the nanoparticles are crystalline and spherical in shape with an approximate diameter of 50 nm and have capping agents. The diameters of microbial growth inhibition zones for Vp and Vh are 26 mm and 23 mm respectively. Further, the MIC values for Vp and Vh are 31.25 µg/mL and 93.75 µg/mL respectively. The DPPH and FRAP assays showed substantial anti-oxidant activity with IC50 values of 67.39 µg/mL and 5.509 µg/mL respectively. MTT assay to check the cytotoxicity effect of bAgNPs on Vero cells resulted in very less toxicity at the maximum concentration tested with an IC50 value of 293.5 µg/mL. Therefore, the bAgNPs synthesized from leaves of MJ showed effective anti-vibriocidal and anti-oxidant properties with negligible cytotoxic effects.


Introduction
Shrimp farming is accounted for the multibillion-dollar industry worldwide that continues to show significant development [1]. Penaeid shrimp culture is dominated by Litopenaeus vannamei (Pacific white leg shrimp), Penaeus monodon (tiger shrimp), Macrobrachium rosenbergii (giant freshwater prawn), etc. because of their fast growth rate, adaptation to wide salinity ranges, and high yield [2,3]. The high profitable value of shrimp has created excellent employment opportunities which plays a significant role in supporting the economic livelihoods of people working in the aquaculture sector [5]. Penaeid shrimps are more susceptible to diseases due to high stocking density, excess feed, and inappropriate maintenance of physicochemical parameters in culture ponds [4,5].
Vibriosis is a major bacterial disease that affects penaeid shrimp caused by an opportunistic gram-negative Vibrio bacteria of the Vibrionaceae family [6]. There are several Vibrio species that cause shrimp vibriosis; among them, Vibrio parahaemolyticus (Vp) and Vibrio harveyi (Vh) are the two major pathogens which are highly prevalent among others and cause acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS) and luminescent vibriosis, respectively, resulting in massive mortality and a billion-dollar economic loss in the shrimp aquaculture sector [4,6]. The shrimp affected with vibriosis are identified with symptoms such as stomach lethargy, discolouration of the hepatopancreas, hemocytic infiltration, etc. [5]. Vp exhibits virulence to the shrimp by producing a binary toxins Photorhabdus insect-related A (Pir A) and Pir B which are present on an extrachromosomal plasmid pVA1, whereas the virulence of Vh is caused by extracellular products (ECPs) which are lethal to the shrimp [7].
There are several traditional treatment methods are available to control shrimp vibriosis. Antibiotics such as azithromycin, oxytetracycline, oxolinic acid, florfenicol, etc. are used to inhibit both Vp and Vh but inappropriate and extensive use of antibiotics leads to the development of antibiotic resistance to almost all the antibiotics and also leaving an adverse impact on the environment [8]. An appropriate solution to eliminate antibiotic resistance in pathogens is to replace the antibiotics with biogenic, eco-friendly methods to achieve sustainable aquaculture practices [9].
One of the eco-friendly technologies to reduce the adverse environmental impact is nanotechnology. Nanotechnology is a novel science which deals with nanomaterial which has a larger surface area to volume ratio and it is one of the powerful emerging science [10]. Metal nanoparticles such as silver, gold, titanium, magnetic nanoparticles, etc. have shown anti-microbial, anti-fungal, anti-viral, anti-tumour, and anti-parasitic activities. Since 1000 B.C., silver has been used as an antiseptic agent and also has antibacterial and antitumor properties [11]. Silver nanoparticles (AgNPs) are the nanometre-sized silver particles with few to 100-nmdiameter sizes produced by physical, chemical, and biological methods [12]. Gamma irradiation, ultrasonic irradiation, and evaporation-condensation are physical methods to synthesize AgNPs. In chemical methods, sodium borohydride, polyol, etc. are used as reducing agents for the synthesis of AgNPs [13].
Recently, the biosynthesis of AgNPs using biomaterials/ phytochemicals extracted from plant materials and microorganisms as reducing agents and their broad spectrum biological activities have been extensively studied. The AgNPs are formed by the oxidation of Ag + to Ag 0 by various phytochemicals or other biomolecules such as ketones, aldehydes, carboxylic acids, tannins, flavonoids, terpenoids, amides, polyphenols, ascorbic acid, etc. [14]. The phytochemicals present in leaf extracts have a remarkable potential to reduce metal ions in a much shorter time compared to microorganisms which require longer incubation times and difficulty in maintaining cultures by avoiding contamination [15]. Secondary metabolites/phytochemicals of plants are used as reducing agents to synthesize biogenic AgNPs or bAgNPs, which possess less toxicity than physically and chemically synthesized AgNPs [16]. In recent decades, bAgNPs have gained great attention from researchers and opened new insights in science and technology which reported a more efficient anti-bacterial activity than chemical or physically synthesized AgNPs [10,17]. A thorough study of the literature has been carried out to find appropriate methods using bAgNPs against shrimp vibriosis, but unexpectedly, very few studies were done on the effect of bAgNPs on shrimp vibriosis. There is a need to synthesize bAgNPs using different plant species or other biological sources in order to meet the demand of aquaculture farmers who are depending exclusively for livelihood on shrimp aquaculture [18].
Mirabilis jalapa (MJ) L. (Nyctaginaceae) (MJ) is a perennial herb and traditionally used medicinal plant for the treatment of various human ailments and is a rich source of phytochemicals/secondary metabolites. It is popularly known as the four 'O' clock plant and exhibits incomplete dominance for flower colour [19]. Traditionally, MJ is used for the treatment of gastrointestinal disorders, recovery of external wounds, amenorrhea and dysmenorrhea in women, and also for the treatment of jaundice and hepatitis [20]. The roots of this plant have hypoglycaemic and hypolipidemic activities in animals [21]. Studies revealed that MJ contains a purgative alkaloid, i.e. trigonelline, oxymethyl anthraquinones, arabinose, galactose, and ß-sitosterol [22]. Phytochemical investigation and spectroscopy analysis of MJ extracts had shown numerous bioactive compounds such as proteins, flavonoids, alkaloids, tri-terpenes, and steroids. However, several studies also found the presence of beta amyrins, arabinose, dopamine, daucosterol, and campesterol in the MJ extracts [19], although many natural materials have been reported for the biogenic synthesis of AgNPs. However, no study has reported the anti-vibriocidal activity of bAgNP synthesized from MJ leaf extract. Therefore, this study can investigate the potential anti-vibriocidal activity of bAgNPs.
The present study proposed to synthesize bAgNPs using MJ aqueous leaf extract as the reducing agent and assessment of anti-vibriocidal activity on Vp and Vh. The antioxidant activities (DPPH, FRAP) and Vero cell cytotoxicity were also evaluated.

Preparation of Leaf Aqueous Extract
In the present study, plant MJ leaves were selected for the synthesis of bAgNPs and leaves were collected from the premises of Rajahmundry, East Godavari, Andhra Pradesh, India. Aqueous leaf extract was prepared using a decoction method according to Alvarez-Cirerol et al. [23]. Leaves were subjected to shade drying for 2-3 days and ground into fine powder. Five grammes of leaf powder was dissolved in 100 mL of distilled water and boiled for 60 min at 50 °C and the resultant solution was filtered using Whatman number 1 filter paper and final aqueous leaf extract was stored in a screwed bottle at 4 °C temperature until further analysis [23].

Biogenic Synthesis of AgNPs
The bAgNPs synthesis was done according to the protocol [25]. Aqueous leaf extract of MJ was added to the 10 mM of silver nitrate (AgNO 3 ) solution in a 1:9 ratio prepared with distilled water. The AgNO 3 solution and aqueous leaf extract were added and kept in a constant magnetic stirrer up to 4 h. The synthesis of bAgNPs was confirmed by changing of the colour from yellowish green to dark brown and the pH of the reaction mixture during the synthesis of bAgNPs was 9.2. The solution was incubated for 24 h in a dark chamber, and after incubation, the solution was centrifuged at 12000 rpm for 10 min and the pellet was washed with distilled water 2-3 times and dried into powder using a hot air oven.

UV-Vis spectroscopy
This technique was used to confirm the formation of bAg-NPs from the precursor 10 mM AgNO 3 solution reduced by leaf aqueous extract. The surface plasmon resonance (SPR) of bAgNPs was measured in the range of 200-800nm wavelength, using a Shimadzu-UV 1800 double-beam spectrometer at a step rate of 480 nm per minute at 25 °C beam aperture width 0.5 nm) during different time intervals such as 0 h, 2 h, and 4 h. Ultra-pure water was used as the blank reference for bAgNPs [24].

Fourier-Transform Infrared Spectroscopy
The FTIR analysis was performed to analyse and identify the functional groups in MJ leaf aqueous extracts and functional groups responsible for the reduction and stabilization of bAgNPs. The bAgNPs were dried in a desiccator conditioned at 45 °C for 24 h to remove water content and ground thoroughly with potassium bromide (approximately 5% bAg-NPs) and pressed to create a thin disc. The MJ leaf aqueous extract was prepared using a decoction method and subjected to drying in a rotary evaporator. Approximately 5 mg of the plant's dried powder was encapsulated with 100 mg of kBr in order to prepare the translucent sample discs. The spectrum of MJ leaf aqueous extract and bAgNPs were taken using an FTIR spectrophotometer (Perkin Elmer) and FTIR spectrometer (Bruker) respectively in the wavelength range of 4000-400 cm −1 [15].

X-ray Diffraction
The XRD method was used to analyse the crystalline phase (structure and grain size) of bAgNPs and it was conducted by XPERT-PRO using monochromatic Cu ka radiation (k = 1.5406 A˚) operated at 40 kV and 30 mA from 10 to 90° in 2θ angles. The powder sample of bAgNPs was held on a cavity slide and gently pressed to create a smooth surface. The diffractometer was run on the data scan software with a scan rate of 1.2° per minute. The obtained data was compared with the ICDD card number 04-0783. The obtained data was compared with the International Centre for Diffraction Studies (ICDS) library to account for the crystalline structure [23].

Field Emission Scanning Electron Microscope and Energy Dispersive X-ray Spectroscopy
The morphology and shape of the bAgNPs were examined using Field Emission (FEI) Quanta 200 FEG MKII SEM. The resolution of FESEM is 1.5 nm and with high output thermal field emission (> 100-nA beam current). The FESEM has a backscatter (BSE) detector with high sensitivity (18 mm) for atomic number contrast. The EDX was used to determine the presence of various elements and measure their relative proportions of each element in the bAgNPs sample [14].

High-Resolution Transmission Electron Microscope
TEM analysis was also used to determine the morphology, size, and shape of the bAgNPs. TEM measurements were done by HITACHI H-800, operating at 200 kV. The TEM grid was prepared by placing a drop of the bio-reduced diluted solution on a carbon-coated copper grid and later drying it under a lamp [15].

Dynamic Light Scattering
The DLS was used to analyse particle size distribution and zeta potential of the bAgNPs by using the Zetasizer Nano ZS90. The scattering angle of particle size distribution and zeta potential are 90° and 15° respectively and the laser beam wavelength was 635 nm. The bAgNPs were diluted in Milli-Q pore water with a concentration equal to 0.0005% (w/v) prior to the measurement. During dilution, the concentrations of pH and salts were precisely regulated and the solution was sonicated for 10 min to achieve even distribution of bAgNPs in the dispersion [25].

Anti-vibriocidal Efficiency of bAgNPs
The bAgNPs synthesized from the aqueous leaf extract of MJ was tested for anti-vibriocidal activity on Vibrio parahaemolyticus (MTCC 451) and Vibrio harveyi (ATCC 334). The anti-vibriocidal activities of the bAgNPs were determined by the well diffusion assay and micro broth dilution method using a marine nutrient agar (MNA) medium. In this method, 10 5 CFU/mL of pathogenic organisms Vp and Vh were taken from pure cultures and swabbed on marine nutrient agar (MNB) plates using a sterile cotton swab. Approximately six wells with a depth of 2.5 mm were made on agar media using sterile gel puncture. The 6 wells were earmarked and WL1 was taken as negative control, and in WL2, 20 µl of antibiotic (Azithromycin 20 mg/mL) was added. WL2 to WL6 were added with 25 µl, 50 µl, 75 µl, and 100 µl of bAgNPs (20 mg/ mL) respectively and incubated at 37 °C for 24 h. The experiment was carried out in triplicate under aseptic conditions. The mean ± SD values of the zone of inhibition were calculated [24]. Micro dilution broth was performed to determine the minimum inhibitory concentration (MIC) of bAgNPs according to the [26]. This assay was carried out in a 96-well micro titre plate using the standard broth dilution method. The bacterial inoculums of two pathogens were adjusted to the concentration of 10 5 CFU/mL. Column 1 is taken as negative control and filled with 100 µl of Maine nutrient broth in micro titre plate. 100 µl of bAgNP stock solution (20 mg/ mL) was added to columns 1 and 2, and columns 11 and 12 served as negative control. Except for negative control, 50 µl (10 5 CFU/mL) of each pathogen were added to the all other columns and plates were incubated at 37 °C for 24 h. After incubation, the wells were observed for bacterial growth. The formation of turbidity indicates the growth of bacteria. The experiment was run in triplicates and The mean ± SD values of MIC were calculated [26].

2,2-Diphenyl-1-picrylhydrazyl assay
The free radical scavenging activity of bAgNPs was determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The bAgNPs prepared at different concentrations-20, 40, 60, 80, and 100 μg/mL-were agitated thoroughly and added to 2 mL of 3 × 10 −5 M DPPH in methanol was added to all test tubes. The solutions were incubated at 37 °C in dark chamber for about 2 h and the absorbance was recorded at 517 nm wavelength using UV-Vis spectroscopy. The DPPH scavenging activity of each concentration was calculated by given below formula. Ascorbic acid was used as standard.
where A 0 = the absorbance of control.

Ferric Reduction Anti-oxidant Power Assay
Ferric reduction anti-oxidant power (FRAP) assay was done according to the [27]. This method depends upon the ability of bAgNPs to reduce Fe +3 to Fe +2 in the presence of 2, 4, 6-tripyridyl-s-triazine (TPTZ) and the formation of the intense blue complex (Fe +2 -TPTZ) in an acid environment. The bAgNPs prepared at different concentrations-20, 40, 60, 80, and 100 μg/mL-were agitated thoroughly and added to 2 mL of FRAP reagent. The solutions were incubated at 37 °C in a dark chamber for about 2 h and the absorbance was recorded at a 593-nm wavelength using UV-Vis spectroscopy. The ferric reduction capacity of each concentration was calculated by the given formula below. Ascorbic acid was used as standard.
where A 0 = the absorbance of control and A 1 = the absorbance of standard.

Cytotoxicity Assay
The cytotoxicity of bAgNPs on Vero cell lines was carried out at Apex Biotechnology Training and Research Institute, Chennai, using 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. MTT is cleaved by mitochondrial Succinate dehydrogenase and reductase of viable cells, yielding a measurable purple product formazan, which is directly proportional to the viable cell number and inversely proportional to the degree of cytotoxicity. Dulbecco's Modified Eagle Medium (DMEM) was discarded from the Vero cell subculture and trypsinized separately. To these disaggregated cells in the flask, 25 mL of DMEM with 10% FCS (foetal calf serum) was added. The cells were homogenized in the suspended medium. One millilitre of homogenized suspension was added to each well in a 24-well culture plate along with the addition of 0, 12.5, 25, 50, 100, and 200 µg/mL concentration bAgNPs in each column respectively and were incubated at 37 °C in a humidified CO 2 incubator and allowed to reach 80% confluence. Cells without being treated with bAgNPs were considered control.
After incubation, the treated cells were washed in phosphate-buffered saline of pH 7.4 and the cultured plate was loaded with 20 µl of MTT reagent and incubated at room temperature for about 3 h. After incubation, the content was removed from the wells and 100 µl of sodium dodecyl sulphate (SDS) in dimethyl sulfoxide (DMSO) was added to all wells to dissolve formazan crystals and absorbance was read at 540 nm with a Lark LIPR-96 micro titre ELISA reader. The cytotoxicity was calculated as follows: where A = the mean optical density of the control well and B = the optical density of the treatment well.

UV-Vis spectroscopy
The synthesis of bAgNPs was confirmed by observing the change in the colour of the solution from yellowish green to dark brown. This colour transition is due to the excitation of surface plasmon vibrations of bAgNPs and was primarily confirmed by UV-Vis spectroscopy at different time intervals (0, 2, and 4 h) and the absorbance peak was observed at 434 and 451 wavelengths, after 2 h and 4 h of synthesis, respectively, which indicates the formation of bAgNPs (Fig. 1). The increase in SPR after 4 h is due to increase in the size of bAgNPs by the surface accumulation of phytochemicals [29]. The AgNPs synthesized from Prosopis farcta fruit extract showed an absorbance peak around a 475-nm wavelength [30]. Another study showed that AgNPs synthesized using Brassica oleracea leaf extract gave an absorbance peak at a 415-nm wavelength [31]. The smaller the wavelength peak, the lesser the size of AgNPs and the more effective their biological activity [30].

FTIR
The phytoconstituents present in the plant extract have dual roles which act as both reducing and capping agents. The presence of functional groups in the synthesized bAgNPs was confirmed through FTIR analysis in the spectral range of 400-4000 cm −1 and depicted in Fig. 2. The observed peak broadening and noise were probably macromolecules present in the plant extract which may be responsible for the reduction of AgNPs. The FTIR bands and the corresponding functional group in the IR range are depicted in Tables 1  and 2 for MJ aqueous leaf extract and bAgNPs respectively. The functional groups obtained in the FTIR spectrum shows the presence of various phytochemicals such as phenols, flavonoids, terpenoids, saponins, etc. which suggests that these phytomolecules are acting as reducing and stabilizing agents. This finding was also supported by the previous reports of Khan et al. [32] and Nadeem et al. [27].

X-ray Diffraction
The XRD is a primary technique to determine the crystalline nature of bAgNPs [17] by predicting the spectrum of 2θ value ranges from 10 to 90°. The bAgNPs showed strong peaks at 2θ° values 36.76°, 48.20°, 68.27°, 77.25° corresponding to the intense peaks at 111, 200, 220, and 311 which strongly reflected Bragg's reflection with facecentred cubic (fcc) and confirming the crystalline nature of the bAgNP sample using ICDD (crystallographic sheet 04-0783) shown in Fig. 3. The peaks which are unassigned in XRD pattern are due to the crystallization of bioorganic phase that occurs on the surface of bAgNPs [32]. The intense peaks 111, 220 correspond to the ultra-small AgNPs and face-centred cubic (fcc) [33]. The XRD of bAgNPs synthesized using sheep's blood (Ovis aries) showed intense peaks 111, 200, 220, and 311 which is similar to the present study [34]. The AgNPs synthesized from Prosopis chilensis leaf extract XRD analysis showed four intense peaks 111, 200, 220, and 311 which strongly support Bragg's reflection and fcc [35].

FESEM
FESEM analysis was performed to determine the morphology of the bAgNPs. Figure 4a shows the SEM micrograph formed by the secondary electrons (SE) of AgNPs. The FESEM analysis showed well-dispersed bAgNPs at 0.5 μm and most nanoparticles are aggregated and spherical in shape. The AgNPs synthesized from methanolic extracts of Ipomoea carnea showed spherical-shaped and uniformly dispersed nanoparticles in FE SEM analysis [36] and bAg-NPs synthesized from Pseudoduganella eburnean showed rod-shaped and uniformly dispersed nanoparticles [37]. In contrast, FESEM analysis of the present study reported the bAgNPs with spherical and non-homogenous shapes and aggregated in form (Fig. 5). The chemical composition and relative abundance of bAgNPs were analysed using EDX    (Fig. 4b).
The other elements were bound to the bAgNP surface and serve as capping agents. The peak area obtained around 3 keV is the characteristic absorption peak of Ag [25].

HR-TEM
Selected area electron diffraction (SAED) of bAgNPs was determined by TEM analysis and it was found that diffraction rings are corresponding to the crystalline planes (Fig. 5a). The TEM images of the bAgNPs revealed variable shapes and most are spherical in shape with a particle size of 50 nm and above (Fig. 4b) which was further confirmed by the Zeta size analysis. The TEM analysis of bAgNPs synthesized from Prosopis farcta fruit extract showed 60-nm-sized bAgNPs with a corresponding UV-Vis absorbance wavelength around 475 nm, higher than the UV absorbance and size of bAgNPs of the present study [30]. bAgNPs synthesized from leaf extract of Erythrina suberosa TEM analysis showed the size of AgNPs between 15 and 34 nm with a corresponding UV-Vis absorbance peak at ~ 428 nm [38]. This clearly shows that, the smaller the absorbance of UV-Vis spectroscopy, the lesser the size of the AgNPs. In the present study, TEM analysis of bAgNPs showed the particle size was around 50 nm and a UV-Vis absorbance peak at 434 nm (Fig. 5b).

DLS
The particle size and particle dispersion index (PdI) were analysed using dynamic light scattering (DLS) which showed the size of bAgNPs to be > 50 nm (Fig. 6a) and particle dispersion index (PdI) was 0.202. The Z average is found to be 269 diameters in nanometres (d.nm). The zeta potential value of bAgNPs was found to be -24.9 mV which indicates a higher degree of stability (Fig. 6b). The negative ZP value of bAgNPs may be due to the presence of capping agents. By monitoring the dynamic fluctuation from the light scattering intensity and velocity movement of the particles in suspension, the zeta size determines the average size of AgNPs in aqueous suspension [39]. In this study, the computed PDI value of the bAgNPs was found to be in the range of 0-1, indicating that the bAgNPs were in a monodisperse phase with minimal particle aggregation [39].

Anti-vibriocidal Activity
Over the years, antibiotic-resistant strains of Vibrio species have emerged in the environment due to the extensive use of chemotherapeutic agents in aquaculture. This resulted in the evolution of new pathogenic strains and more disease outbreaks which are difficult to treat with chemotherapeutic agents [7]. Vp and Vh are more prevalent pathogens among the Vibrio species which cause vibriosis in shrimp [40,41]. In recent decades, AgNPs gained a lot of attention in different research and commercial sectors due to

ConcentraƟon of AnƟbioƟc/bAgNPs in µl
Vibrio parahaemolyƟcus Vibrio harveyi their targeted specific action and anti-microbial activity. The application of AgNPs in commercial aquaculture sectors has been increased to combat various disease-causing pathogens [42,43]. In this study, a well diffusion assay was performed to determine the anti-vibriocidal activity of bAgNPs and the zone of inhibitions are 20.075 mm (25 µl (Fig. 8).
In contrast to the present study, colloidal AgNPs synthesized by a photo-assisted reduction method obtained two different-sized bAgNPs 16.62 nm and 22.22 nm that showed 42.1 mm and 43.12 mm of the zone of inhibition against Vh [44]. The green AgNPs synthesized by extracellular products of Bacillus subtilis produced a size range of 10-25 nm and showed a zone of inhibition at 21.25 nm and 19.27 nm on Vp and Vh respectively [44]. The green AgNPs synthesized from leaf extract of Prosopis chilensis obtained size ranges from 5 to 25 nm with a zone of inhibition at 16 nm and 19 nm against Vh and Vp respectively [35]. Ten milligrammes of AgNPs synthesized from tea leaf extract (Camellia sinensis) inhibited 70% of Vh growth in culture media [45]. The bAgNPs synthesized from Cheatomorpha antennia showed anti-vibriocidal activity against Vh and exhibited a 17.8-mm zone of inhibition [46].
The anti-microbial mechanism and action of AgNPs are yet to be understood. Several hypothetical mechanisms were proposed from different studies based on structural and morphological changes of microorganisms that occurred when treated with AgNPs. The AgNPs effectively inhibit gram-negative bacteria than gram-positive bacteria due to the absence of a peptidoglycan cell wall. Several studies reported that AgNPs induce the generation of reactive oxygen or reactive nitrogen species which cause oxidative stress: it triggers the conformational changes of biomolecules within the microorganisms. AgNPs also interact with bacterial cell membrane proteins and increase the membrane permeability which leads to cellular components and activates apoptosis [47][48][49].

Anti-oxidant Activity
Antioxidants are natural or synthetic compounds which delay, remove, or prevent the damage of a cell caused by free radicals or species which caused oxidative stress in cells. The antioxidant substance must be in low concentration and ability to scavenge free radicals or reactive oxygen/ nitrogen species. In recent years, several spectrophotometric methods have been developed to determine the antioxidant capacity of both natural and synthetic substances [50]. In this study, DPPH and FRAP were used to evaluate the antioxidant activity of bAgNPs synthesized from the aqueous leaf extract of MJ.
The significant anti-oxidant ability of bAgNPs was carried out by DPPH and FRAP assays showing IC 50 values of 67.39 µg/mL and 5.50 µg/mL respectively. The green AgNPs synthesized from leaf extract of Erythrina suberosa showed significant free radical scavenging activity with an IC 50 value of 30.04 µg/mL [38]. The green synthesized AgNPs from Prosopis farcta showed significant DPPH free radical scavenging activity with an IC 50 value of 70 µg/mL which is higher than the present study  [30]. Green AgNPs synthesized from Cestrum nocturnum showed 29.55% of anti-oxidant activity at 100 µg/mL, [51], which is higher than our present study. The AgNPs synthesized from Caesalpinia sappan aqueous extract showed ferric oxide anti-oxidant activity with 78.7 µg/ mL [52] and gAgNPs synthesized from Nothapodytes nimmoniana fruit extract studied for FRAP assay showed 214.89 µg/mL, [53] and the concentration is higher than the present study.

Cytotoxicity Assay
The major consequence of AgNPs is safety aspects. With the increasing applications of nanoparticles, there are possible adverse effects on the environment and organisms. It is necessary to extensively evaluate the nanoparticle toxicity in both in vitro and in vivo [54]. In this study, the toxicity of bAgNPs was evaluated on Vero cell lines (African green monkey kidney cell lines) which are non-cancerous cell lines. The in vitro cytotoxicity activity results of the bAgNPs synthesized from leaf extract of MJ against Vero cells showed minimal inhibition towards the tested cell line. However, with the increased concentration of bAgNPs, the cell viability decreased. The maximum cytotoxicity of bAgNPs in this study was observed at 200 µg/mL concentration and cell viability was 64.55% at this concentration. It was proven that the moderate cytotoxicity effect of the test sample showed no cell disintegration after 48 h of treatment against the selected tested cell lines even at higher concentrations (Figs. 11 and 12). The IC 50 concentration of the tested sample against Vero cells was 293.5 µg/ mL.
MTT assay was done in the present study to evaluate the cytotoxic effect of bAgNPs on Vero cell lines, showed % of cell viability at different concentrations which are 98.06% (12.5 µg/mL), 88.59% (25 µg/mL), 78.46% (50 µg/ mL), 70.43% (100 µg/mL), and 64.55% (200 µg/mL). The IC 50 value of bAgNPs on Vero cell lines was found to be 293.5 µg/mL. The bAgNPs synthesized from Cassia fistula showed Vero cell line cell death was 89.7% at 1000 µg/ mL and IC 50 value is 66.34 µg/mL, which is less than our current study [55]. Colloidal AgNPs synthesized using silver nitrate and citric acid showed mortality of 68.54% at 1000 µg/mL on Vero cell lines and IC 50 value 10.68 µg/ mL, which indicates the less concentration of colloidal AgNPs cause 50% of cell death. Green AgNPs synthesized from leaf extract of the copper rod plant (Peltophoroum pterocarpum) and studied cytotoxicity on Vero cell lines showed an IC 50 value of 90 µg/mL [56]. Green AgNPs synthesized from Alysicarpus monilifer showed very limited toxicity of 25 µg/mL on Vero cell lines after 72 h of incubation [57].

Conclusion
The present work describes the synthesis of bAgNPs using the aqueous leaf extract of MJ. The leaf extract has bioactive compounds such as flavonoids, phenols, terpenoids, tannins, alkaloids, etc. which act as reducing, stabilizing, and capping agents for the synthesis of bAgNPs. The formation bAgNPs was confirmed by UV-Vis spectroscopy, XRD, FTIR, TEM, and FESEM and EDX; zeta size and zeta potential showed a spherical, cone shape with around 50 nm and above-sized AgNPs. The bAgNPs synthesized in this study have shown significant anti-vibriocidal activity on Vp and Vh which are more prevalent pathogens that cause Vibriosis in shrimp. The zone of inhibition for both pathogens 26 nm and 28 nm are respectively at 100 μg/ mL. Minimum inhibitory concentration (MIC) was determined as 31.25 and 187.5 μg/mL for both Vibrio species respectively and it was observed that more concentrations of bAgNPs are required to inhibit the Vp and Vh. Since bAgNPs have free radical scavenging and ferric oxidereducing anti-oxidant activities. This is due to functional groups present on the bAgNP surface as capping agents. The cytotoxicity of bAgNPs on Vero cell lines has shown lesser toxicity at a maximum concentration of 200 μg/mL. These bAgNPs might be used as anti-microbial agents in the future due to being eco-friendly, less toxic, and highly effective against various pathogenic organisms.

Funding Statement
This work was supported financially by the Regional Centre for Biotechnology, Department of Biotechnology, Faridabad, Junior Research Fellowship (Grant No. DBT/2020/ANU/1330).