Synthesis of biogenic silver nanoparticles (bAgNPs) using leaf extract of Mirabilis jalapa and evaluation of anti-vibriocidal, anti-oxidant properties and cytotoxicity

Synthesis of biogenic silver nanoparticles (bAgNPs) using leaf extract of Mirabilis jalapa and evaluation of anti-vibriocidal, anti-oxidant properties and cytotoxicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis of biogenic silver nanoparticles (bAgNPs) using leaf extract of Mirabilis jalapa and evaluation of anti-vibriocidal, anti-oxidant properties and cytotoxicity Vijaya Nirmala Pangi, Abhinash Marukurti, Alavala Matta Reddy, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2220633/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Feb, 2023 Read the published version in BioNanoScience → Version 1 posted 7 You are reading this latest preprint version Abstract Acute hepatopancreatic necrosis disease 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 the antibiotic resistance in pathogens. In the present study, biogenic silver nanoparticles (bAgNPs) are synthesized by reducing AgNO 3 using 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 silver nanoparticles was confirmed by the appearance of dark brown colored solution and with a maximum absorption peak at 434nm. The characterization of bAgNPs using FESEM, TEM, XRD, FTIR, and DLS has confirmed that the nanoparticles are crystalline, spherical in shape with an approximate diameter of 50nm, and have capping agents. The diameter of microbial growth inhibition zones for Vp and Vh are 26mm and 23mm respectively. Further, the MIC values for Vp and Vh are 31.25µg/mL and 93.75µg/mL respectively. The DPPH and FRAP assays showed substantial anti-oxidant activity with IC 50 values of 67.39µg/mL and 5.509µg/mL respectively. MTT assay to check cytotoxicity effect of bAgNPs on Vero cells resulted very less toxicity at maximum concentration tested with an IC 50 value of 293.5µg/mL. Therefore, the biogenic silver nanoparticles synthesized from leaves of MJ showed effective anti-vibriocidal and anti-oxidant properties with negligible cytotoxic effect. Aquaculture Shrimp Vibriosis Vibrio parahaemolyticus Vibrio harveyi Antimicrobial-resistance Silver nanoparticles Mirabilis jalapa Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Shrimp farming is accounted for multibillion-dollar industry worldwide that continues to show significant development (Srinivas and Venkatrayulu 2019). Penaeid shrimp culture is dominated by Litopenaeus vannamei (Pacific white leg shrimp), Penaeus monodon (tiger shrimp) and Macrobrachium rosenbergii (Giant fresh water prawn) etc., species because of their fast growth rate, adaptation to wide salinity ranges and high yield (Santos et al. 2020 ) (Jayasankar 2018 ) (Ngasotter et al. 2020 ). The high profitable value of shrimp has created excellent employment opportunities which plays a significant role in supporting their economic livelihoods of people working in aquaculture sector (Salunke et al 2020 ). Penaeid shrimps are more susceptible to diseases due to high stocking density, excess feed and inappropriate maintenance of physicochemical parameters in culture ponds (Istiqomah et al.2020) (Karunasagar and Ababouch 2012). Vibriosis is a major bacterial disease affects penaeid shrimp caused by an opportunistic gram negative vibrio bacteria of vibrionaceae family (Novriadi, 2016). There are several vibrio species cause shrimp vibriosis among them Vibrio parahaemolyticus and Vibrio harveyi are the two major pathogens which are highly prevalent among others and causes Acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS) and luminescent vibriosis respectively, resulting in massive mortality and billion dollar economic loss in shrimp aquaculture sector (Istiqomah et al.2020) (Novriadi, 2016). The shrimp affected with Vibriosis are identified with symptoms such as stomach lethargy, discolouration of hepatopancreas and hemocytic infiltration etc. (Letchumanan et al. 2015 ). Vibrio parahaemolyticus exhibit virulence to the shrimp by producing a binary toxins Photorhabdus insect-related A (Pir A) and Pir B which are present on an extra chromosomal plasmid pVA1, whereas the virulence of Vibrio harveyi caused by Extra cellular products (ECP’s) which are lethal to the shrimp (Letchumanan et al. 2016). There are several traditional treatment methods are available to control the shrimp vibriosis . Antibiotics such as Azithromycin, Oxytetracycline, oxolinic acid, and florfenicol etc. are used to inhibit both Vibrio parahaemolyticus and Vibrio harveyi but inappropriate and extensive use of antibiotics leads to the development of antibiotic resistance to almost all the antibiotics and also leaving adverse impact on environment (Holmström et al. 2017). There is an appropriate solution to eliminate the antibiotic resistance in pathogens is to replace the antibiotics with biogenic, eco-friendly methods to achieve the sustainable aquaculture practices (Boyd et al. 2020 ). One of the eco-friendly technologies to reduce the adverse environmental impact is Nanotechnology. Nanotechnology is the novel science which deals with nanomaterial which has larger surface area to volume ratio and it is one of the powerful emerging science (Ahmad et al., 2019 ). Metal nanoparticles such as silver, gold, titanium and magnetic nanoparticles etc. have shown anti-microbial, anti-fungal, anti-viral, anti-tumour and anti-parasitic etc. activities. Since 1000 B.C silver has been used as antiseptic agent and also has the antibacterial and antitumor properties (Zhang et al., 2015 ). Silver nanoparticles (AgNPs) are the nanometer sized silver particles with few to 100nm diameter size are produced by physical, chemical and biological methods (Sivolella et al., 2012 ). Gamma irradiation, ultrasonic irradiation, evaporation-condensation are physical methods to synthesize AgNPs. In chemical methods sodium borohydride, polyol etc. compounds are used as reducing agents for synthesis of AgNPs. Secondary metabolites of plants, algae, fungi and bacteria are used as reducing agents to synthesize the biogenic or green AgNPs, that possess less toxicity than physically and chemically synthesized AgNPs (Van Hengel et al., 2020 ) (Samanta et al. 2019 ). In recent decades biogenic silver nanoparticles have gained great attention of the researchers and opened new insights in science and technology which are reported more efficient anti-bacterial activity than chemical or physically synthesized AgNPs (Ahmad et al. 2019 ) (Journal et al. 2017 ). A thorough study of literature have been carried out to find appropriate methods using biogenic or green AgNPs against shrimp vibriosis, but unexpectedly very few studies were done on the effect of biogenic AgNPs on shrimp vibriosis. There is a need to synthesize biogenic or green AgNPs using different plant species or other biological sources in order to meet the demand of the aquaculture farmers who are depending exclusively for livelihood on the shrimp aquaculture (Geetha et al., 2020 ). The present study proposed to synthesize biogenic AgNPs using Mirabilis jalapa aqueous leaf extract as reducing agent and assessment of anti-vibriocidal activity on Vibrio parahaemolyticus and Vibrio harveyi . The Antioxidant activities (DPPH, FRAP) and Vero cell cytotoxicity were also evaluated. Materials And Methods Preparation of leaf aqueous extract: In the present study, plant Mirabilis jalapa L. of Nyctaginaceae , perennial herb with bushy in nature was selected for the synthesis of biogenic silver nanoparticles and leaves were collected from Rajahmundry, East Godavari, Andhra Pradesh, India. Aqueous leaf extract was prepared using decoction method according to the Singh et al. 2012 . Leaves were subjected to shade drying under sunlight for 2–3 days and grounded into fine powder. 5% of leaf powder was dissolved in distilled water, boiled for 60 minutes at 50 o C and the resultant solution was filtered using Whatman number 1 filter paper and final aqueous leaf extract was stored in screwed bottle at 4 o C temperature until further analysis (Singh et al. 2012 ). Biogenic synthesis of Silver nanoparticles (bAgNPs) : Biogenic synthesis of silver nanoparticles was done according to the protocol of Chand et al. 2020 . Aqueous leaf extract of Mirabilis jalapa was added to the 10mM of Silver Nitrate (AgNO 3 ) solution in 1:9 ratio was prepared with distilled water. The AgNO 3 solution and aqueous leaf extract were added and kept in constant magnetic stirrer up to 4 hours. The synthesis of silver nanoparticles was confirmed by changing of colour from yellowish green to dark brown. The solution was incubated for 24 hours in dark chamber and after incubation, the solution was centrifuged at 12000 rpm for 10 minutes and pellet was washed with distilled water for 2–3 times and dried into powder using hot air oven. Characterization of biogenic silver nanoparticles (bAgNPs): UV–Vis spectroscopy technique was used to confirm the formation of nanoparticles from the precursor 10mM AgNO 3 solution reduced by leaf aqueous extract. The absorbance spectrum of the sample was obtained in the range of 200–800 nm wavelength, using UV–Vis spectrometer Shimadzu-UV 1800 at different time intervals such as 0 hour, 2hours and 4hours.Fourier-transform infrared spectroscopy (FTIR) analysis was performed to analyse and identify the functional groups responsible for the reduction and stabilization of biogenic silver nanoparticles using ALPHA II, a compact FT-IR spectrometer (Bruker) in the wavelength range 4000–400 cm − 1 . X-ray diffraction (XRD) method is an efficient technique to identify the crystalline phase of silver nanoparticles 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 o to 90 o in 2θ angles. The obtained data was compared with the International centre for diffraction studies (ICDS) library to account for the crystalline structure. The morphology and shape of the silver nanoparticles were examined using Field Emission (FEI) Quanta 200 FEG MKII scanning electron microscope (SEM). The resolution of FESEM is 1.5 nm and with high output thermal field emission (> 100nA beam current). The FESEM has backscatter (BSE) detector with high sensitivity (18 mm) for atomic number contrast. TEM analysis was also used to determine the morphology, size and shape of the silver nanoparticles. 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. The hydrodynamic size and zeta potential of the bAgNPs was confirmed by using the Zeta sizer nano ZS90 with 90 degree scattering optics. Anti-Vibriocidal efficiency of biogenic silver nanoparticles (bAgNPs): The biogenic AgNP’s synthesized from the aqueous leaf extract of Mirabilis jalapa was tested for anti-vibriocidal activity on Vibrio parahaemolyticus (MTCC No 451) and Vibrio harveyi (ATCC No 334). The anti-vibriocidal activities of the bAgNPs were determined by the well diffusion assay and micro broth dilution method using marine nutrient agar (MNA) medium. In this method, 10 5 CFU/mL of pathogenic organisms, Vibrio parahaemolyticus and Vibrio harveyi were taken from pure cultures and are swabbed on Marine nutrient agar (MNB) plates using sterile cotton swab. An approximately six wells with depth of 2.5mm 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 20mg/mL) was added. WL2 to WL6 were added with 25 µl, 50 µl, 75 µl and 100 µl of bAgNPs (20mg/mL) respectively and incubated at 37 o C for 24 hours. The experiment was carried out in triplicates under aseptic conditions. The mean ± SD values of zone of inhibition were calculated (Ravichandran et al. 2019 ). Micro dilution broth was performed to determine the minimum inhibitory concentration (MIC) of bAgNPs according to the Loo et al. 2018 . This assay was carried out in 96-well micro titer plate using 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 titer plate. 100 µl of bAgNP’s stock solution (20mg/mL) was added to the column 1 & 2, and column-11 and 12 served as negative control. Except negative control, 50µl (10 5 CFU / mL) of each pathogen were added to the all other columns and plates were incubated at 37 o C for 24 hours. After incubation the wells were observed for bacterial growth. Formation of turbidity indicates the growth of bacteria. The experiment was run in triplicates and The mean ± SD values of MIC was calculated (Loo et al. 2018 ). Evaluation of Anti-oxidant activity: 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay Free radical scavenging activity of biogenic silver nanoparticles was determined using DPPH assay. Biogenic AgNPs prepared at different concentrations – 20, 40, 60, 80 and 100 µg/mL were agitated thoroughly and added to 2mL of 3 x 10 − 5 M DPPH in methanol was added to all test tubes. The solutions were incubated at 37 o C in dark chamber for about 2 hours and the absorbance was recorded at 517nm wavelength using UV-Vis spectroscopy. The DPPH scavenging activity of each concentration was calculated by given below formula (Sumbal et al. 2017). Ascorbic acid was used as standard. DPPH scavenging effect % Inhibition = A 0 -A 1 /A 0 × 100 Where A 0 = the absorbance of control. A 1 = the absorbance of standard Ferric reduction anti-oxidant power (FRAP) assay FRAP assay was done according to the Sumbal et al. 2019 . This method is depend upon the ability of bAgNPs to reduce Fe + 3 to Fe + 2 in the presence of 2, 4, 6-tripyridyl-s-triazine (TPTZ) and formation of intense blue complex (Fe + 2 – TPTZ). Biogenic AgNPs prepared at different concentrations – 20, 40, 60, 80 and 100 µg/mL were agitated thoroughly and added to 2mL of FRAP reagent. The solutions were incubated at 37 o C in dark chamber for about 2 hours and the absorbance was recorded at 593nm wavelength using UV-Vis spectroscopy. The ferric reduction capacity of each concentration was calculated by given below formula. Ascorbic acid was used as standard. % Inhibition of FRAP = A 0 -A 1 /A 0 × 100 Where A 0 = the absorbance of control. 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) medium was discarded from Vero cell sub culture and trypsinized separately. To this disaggregated cells in the flask 25mL of DMEM with 10% FCS (fetal calf serum) was added. The cells were homogenized in the suspended medium. One mL of homogenized suspension was added to each well in a 24 wells culture plate along with the addition of 0, 50, 100, 150, 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. After incubation, the treated cells were washed in phosphate buffered saline of pH 7.4 and cultured plate was loaded with 20 µl of MTT reagent and incubated at room temperature for about 3 hours. After incubation, the content was removed from the wells and 100µl Sodium dodecyl sulphate (SDS) in Dimethyl sulfoxide (DMSO) was added to the all wells to dissolve formazan crystals and absorbance was read at 540nm with Lark LIPR-96 micro titre ELISA reader. The percentage of cell viability and IC 50 values were calculated using absorbance values (Sumbal et al. 1993). Results And Discussion Biogenic synthesis and characterization of bAgNPs: The synthesis of bAgNPs was confirmed by observing the change in the color of solution from yellowish green to dark brown color. This color transition is due to the excitation of Surface Plasmon Vibrations of silver nanoparticles and was primarily confirmed by UV-Vis spectroscopy at different time intervals (0, 2 and 4 hours) and the absorbance peak was observed at 434 and 451 wavelengths, after 2 hours and 4 hours of synthesis respectively, which indicating the formation of biogenic silver nanoparticles (Fig. 1 ). Green AgNPs synthesized from Prosopis farcta fruit extract showed absorbance peak around 475nm wavelength (Salari et al., 2019 ). Another study showed that green AgNPs synthesized using Brassica oleracea leaf extract gave absorbance peak at 415nm wavelength (Ansar et al., 2020 ). The smaller the wavelength peak, the lesser the size of silver nanoparticles and more effective in its biological activity (Salari et al. 2019 ). The XRD is a primary technique to determine the crystalline nature of silver nanoparticles (Rafique et al., 2017 ) by predicting spectrum of 2θ value ranges from 10 o to 90 o . The XRD pattern of bAgNPs in this study revealed intense peaks at 111, 200, 220 and 311 which strongly reflected the Bragg’s reflection with face centred cubic (fcc) and confirming the crystalline nature of bAgNPs sample using ICDD (Crystallographic sheet 04-0783) shown in Fig. 2 . The peaks which are unassigned in XRD pattern are due to the crystallization of bioorganic phase that occurs on the surface of silver nanoparticles. The intense peaks 111, 220 corresponds to the ultra-small nanoparticles and face centre cubic (fcc) (Mourdikoudis et al., 2018 ). XRD of Biogenic silver nanoparticles synthesized using sheep’s blood ( Ovis aries ) showed intense peaks 111, 200, 220 and 311 which is similar to the present study (Kakakhel et al., 2021 ). Silver nanoparticles 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 (Kandasamy and Alikunhi 2013 ). The phytoconstituents present in the plant extract have dual role which act as both reducing and capping agents. The presence of functional groups in the synthesized biogenic silver nanoparticles was confirmed through FTIR analysis in the spectral range of 400–4000 cm − 1 and depicted in Fig. 3 . The observed peak broadening and noise were probably macromolecules present in the plant extract which may be responsible for the reduction of silver nanoparticles. The FTIR band at 1008.50 cm − 1 corresponds to Flouro halogen group (C-F stretching) which may be attached to the reducing or stabilizing agent, band at 1416.50 cm − 1 corresponds to C-H bending (Alkane), band at 1636.95 cm − 1 corresponds to C = O ( ketones) and band at 3298.60 cm − 1 corresponds to O-H stretching (alcohol). Gopalakrishnan et al., 2019 synthesized green silver nanoparticles using aqueous leaf extract of Mirabilis jalapa shown two peaks 3323 cm − 1 and 1620 cm − 1 corresponds to OH stretching and C = O ketones. Sumbal et al., 2019 found OH stretching ( 3283.69 cm − 1 ), C-N group or alkyne (2122.60 cm − 1 ), C = C or NH group ( 1643.20 cm − 1 ) and C-O (1318.89 cm − 1) corresponds to the alcohols, amides and amine functional groups in the aqueous leaf extract of Mirabilis jalapa. 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. 4 a). The TEM images of the biogenic silver nanoparticles revealed variable shapes and most of are with spherical shapes with particle size of 50nm and above (Fig. 4 b) which was further confirmed by the Zeta size analysis. The TEM analysis of bAgNPs synthesized from Prosopis farcta fruit extract showed 60nm sized bAgNPs with corresponds to the UV-Vis absorbance wavelength around 475 nm, higher than the UV absorbance and size of bAgNPs of the present study (Salari et al., 2019 ). bAgNPs synthesized from leaf extract of Erythrina suberosa TEM analysis showed the size of nanoparticle between 15-34nm with corresponds to the UV-Vis absorbance peak at ~ 428nm (Mohanta et al., 2017 ). This clearly shows that, smaller the absorbance of UV-Vis spectroscopy then lesser the size of the nanoparticle. In the present study, TEM analysis of bAgNPs showed the particle size was around 50nm and a UV-Vis absorbance peak at 434nm (Fig. 4 ). FESEM analysis was performed to determine the morphology of the bAgNPs. Figure 5 shows the SEM micrograph formed by the secondary electrons (SE) of silver nanoparticles. The FESEM analysis showed well dispersed bAgNPs at 0.5µm and most nanoparticles are with aggregated and spherical in shape. Green AgNPs synthesized from methanolic extracts of Ipomoea carnea shown spherical shaped and uniformly dispersed nanoparticles in FE SEM analysis (Singh, 2021 ) and gAgNPs synthesized from Pseudoduganella eburnean shown rod shaped and uniformly dispersed nanoparticles (Huq, 2020 ). In contrast FE SEM analysis of the present study reported the bAgNPs with Spherical and non-homogenous shapes and aggregated in form (Fig. 5 ). The particle size and particle dispersion Index (PdI) was analysed using dynamic light scattering (DLS) which showed the size of biogenic synthesized silver nanoparticles to be > 50nm (Fig. 6a) and particle dispersion Index (PdI) was 0.202. The Z average (d.nm) is found to be 269. The zeta potential value of biogenic synthesized silver nanoparticles was found to be -24.9mV which indicating the 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 nanoparticles in aqueous suspension (Mat Yusuf et al., 2020 ). 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 (Mat Yusuf et al., 2020 ). Anti-vibriocidal activity: Over the years, antibiotic resistant strains of Vibrio species have emerged in the environment due to 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 the chemotherapeutic agents (Letchumanan et al. 2015 ). Vibrio parahaemolyticus and Vibrio harveyi are more prevalent pathogens among the Vibrio species which cause vibriosis to shrimp (Campus 2018) (Mastan and Begum 2016 ). In recent decades, silver nanoparticles gained a lot of attention in different research and commercial sectors due to its targeted specific action and anti-microbial activity. Application of AgNPs in commercial aquaculture sectors have been increased to combat the various disease causing pathogens (Morones et al.2005) (Shankar et al.2004). In this study well diffusion assay was performed to determine the anti-vibriocidal activity of biogenic silver nanoparticles (bAgNPs) and exhibited the maximum inhibition zone of 26.2 ± 0.54mm and 28.375 ± 0.47 mm wide respectively. The mean ± SD values of zone of inhibitions of Vibrio parahaemolyticus and Vibrio harveyi are depicted in Figs. 6 & 7 respectively. In contrast the control (antibiotic) used in this study showed inhibition zone of 23.1 ± 0.49 mm and 13.125 ± 0.29 mm wide of the both species respectively which are less than zone of inhibition of bAgNPs. Micro dilution broth (MBS) was done to determine the Minimum Inhibitory Concentration (MIC) of green silver nanoparticles on Vibrio parahaemolyticus and Vibrio harveyi. The MIC values of bAgNPs against the growth of Vibrio parahaemolyticus and Vibrio harveyi are 31.25 ± 0 and 93.75 ± 44.19 µg/ mL respectively (Fig. 8 ).. In contrast to the present study, colloidal silver nanoparticles synthesized by photo-assisted reduction method obtained two different sized nanoparticles 16.62nm and 22.22nm that showed 42.1mm and 43.12mm of zone of inhibition against Vibrio harveyi (Nafisi et al. 2017 ). Green silver nanoparticles synthesized by extracellular products of Bacillus subtilis produced size range of 10–25 nm showed zone of inhibition 21.25nm and 19.27nm on Vibrio parahaemolyticus and Vibrio harveyi respectively (Sivaramasamy and Zhiwei 2016 ). Green silver nanoparticles synthesized from leaf extract of Prosopis chilensis obtained size ranges from 5 to 25 nm with zone of inhibition 16nm and 19nm against Vibrio harveyi and Vibrio parahaemolyticus respectively (Kandasamy and Alikunhi 2013 ). 10mg of silver nanoparticles synthesized from tea leaf extract ( Camellia sinensis ) inhibited 70% of Vibrio harveyi growth in culture media (Vaseeharan et al. 2010 ). Biogenic silver nanoparticles synthesized from Cheatomorpha antennia shown anti-vibriocidal activity against Vibrio harveyi and exhibited 17.8mm zone of inhibition (Thanigaivel et al., 2021 ). 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 has been developed to determine the antioxidant capacity of both natural and synthetic substances (Bedlovicová et al., 2020 ). In this study DPPH and FRAP were used to evaluate the antioxidant activity of biogenic silver nanoparticles synthesized from aqueous leaf extract of Mirabilis jalapa . DPPH assay: DPPH free radical scavenging activity was done for the assessment of antioxidant potential of green silver nanoparticles. The purple solution DPPH containing solution turned yellow after the addition of bAgNPs, which indicates the free radical scavenging activity. Figure-9 shows maximum inhibition at 500 µl/mL with 60.77% and the Inhibitory concentration 50 (IC 50 ) value was determined using the formula IC 50 (0.5-b)/a, which was 67.3987 ± 202 µl/mL, indicating the concentration of bAgNPs required to inhibit the 50% of DPPH concentration. FRAP assay: In FRAP assay, the maximum inhibitory percentage of bAgNPs required to reduce Fe3 + to Fe2 + ranges from 3.82 ± 0.65 to 22.92 ± 0.54%. The IC 50 value of bAgNPs was 5.509 ± 152 µl/mL, indicates at this concentration 50% of Fe3 + is reduced to Fe2 + and is depicted in Fig. 10 . The significant anti-oxidant ability of bAgNPs was carried out by DPPH and FRAP assays showed IC 50 value 67.39 µg/mL and 5.50 µg/mL respectively. Green silver nanoparticles synthesized from leaf extract of Erythrina suberosa shown significant free radical scavenging activity with IC 50 value of 30.04 µg/mL (Mohanta et al., 2017 ). Green synthesized silver nanoparticles from Prosopis farcta shown significant DPPH free radical scavenging activity with IC 50 value of 70 µg/mL which is higher than the present study (Salari et al., 2019 ). Green silver nanoparticles synthesized from Cestrum nocturnum shown 29.55% of anti-oxidant activity at 100 µg/mL, (Keshari et al. 2020 ), which is higher than our present study. Green silver nanoparticles synthesized from Caesalpinia sappan aqueous extract shown Ferric oxide anti-oxidant activity with 78.7 µg/mL (Suwan et al., 2018 ) and gAgNPs synthesized from Nothapodytes nimmoniana fruit extract studied for FRAP assay shown 214.89 µg/mL, (Mahendran and Ranjitha Kumari, 2016) and the concentration is higher than the present study. Vero cell cytotoxicity: The major consequence of nanoparticles is safety aspects. With the increasing applications of nanoparticles, there are possible adverse effects on environment and organisms. It is necessary to extensively evaluate the nanoparticles toxicity in both in vitro and in vivo (Saranya et al., 2017 ). In this study, the toxicity of biogenic silver nanoparticles 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 Mirabilis jalapa against Vero cells showed minimal inhibition towards tested cell line. However, the increased concentration of bAgNPs, the cell viability decreased. The maximum cytotoxicity 69.49% of bAgNPs observed when 200 µg/ mL of bAgNPs were used against Vero cell lines. 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 & 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 73.87% of cell viability at maximum concentration of 200µg/mL and the IC 50 value of bAgNPs on Vero cell lines is 293.5 µg/mL. Biogenic AgNPs synthesized from Cassia fistula shown 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 (Remya et al., 2015 ). Colloidal silver nanoparticles 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 indicating the less concentration of colloidal silver nanoparticles cause 50% of cell death. Green AgNPs synthesized from leaf extract of copperrod plant ( Peltophoroum pterocarpum ) and studied cytotoxicity on Vero cell lines showed IC 50 value of 90 µg/mL (Pannerselvam et al., 2021 ). Green silver nanoparticles synthesized from Alysicarpus monilifer showed very limited toxicity of 25 µg/mL on Vero cell lines after 72 hours of incubation (Kasithevar et al., 2017 ). Conclusion The present work describes the synthesis of biogenic silver nanoparticles using aqueous leaf extract of Mirabilis jalapa . The leaf extract have bioactive compounds such as flavonoids, phenols, terpenoids, tannins, and alkaloids etc. which act as reducing, stabilizing and capping agents for the synthesis of biogenic silver nanoparticles. The formation bAgNPs was confirmed by UV-Vis spectroscopy, XRD, FTIR, TEM and FE SEM, Zeta size and Zeta potential showed spherical, cone shaped with around 50nm and above sized nanoparticles. The bAgNPs synthesized in this study have shown significant anti-vibriocidal activity on Vibrio parahaemolyticus and Vibrio harveyi which are more prevalent pathogens causes Vibriosis to the shrimp. The zone of inhibition for both pathogens 26nm and 28nm are respectively at 100µg/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 concentration of bAgNPs are required to inhibit the Vibrio harveyi than Vibrio parahaemolyticus . Since, bAgNPs has free radical scavenging and ferric oxide reducing anti-oxidant activities. This is due to functional groups present on bAgNPs surface as capping agents. The cytotoxicity of bAgNPs on Vero cell lines has shown lesser toxicity at maximum concentration of 200µg/mL. These biogenic silver nanoparticles might be used as anti-microbial agents in future due to eco-friendly, less toxicity and highly effective against the various pathogenic organisms. Abbreviations AHPND – Acute Hepatopancreatic Necrosis Disease EMS – Early Mortality Syndrome Pir – Photorhabdus insect related bAgNPs – Biogenic silver nanoparticles FTIR – Fourier Transform Infrared Spectroscopy XRD – X-ray diffraction SEM – Scanning Electron Microscope TEM – Transmission Electron Microscope DLS – Dynamic Light Scattering Declarations Ethics approval and consent to participate Not Applicable Consent for Publication Not Applicable Competing interests The authors declared no competing interest Funding This work was supported financially by Regional Centre for Biotechnology, Department of Biotechnology, Faridabad, Junior Research Fellowship (Grant No. DBT/2020/ANU/1330). Acknowledgments The authors also thankful to School of Life and Health Sciences, Adikavi Nannaya University, Rajahmundry for providing necessary facilities for conducting this research work. Authors Contributions PVN were involved in supervision and conceptualization of project. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2220633","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":148428517,"identity":"fa4c1b2a-a4d6-4450-a1ff-75def5408e21","order_by":0,"name":"Vijaya Nirmala 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3","display":"","copyAsset":false,"role":"figure","size":54355,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of bAgNPs synthesized from leaf aqueous extract of \u003cem\u003eMirabilis jalapa\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/084c5819c90f2a88f2ed404e.png"},{"id":28615359,"identity":"b7d2f31d-a411-4bbe-ada4-15e9bb532108","added_by":"auto","created_at":"2022-11-03 14:29:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":652769,"visible":true,"origin":"","legend":"\u003cp\u003e(a) SAED pattern of bAgNPs (b) Micrograph of biogenic silver nanoparticles showing the size and shape with TEM analysis\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/fcb98990098f6e664cde4174.png"},{"id":28615355,"identity":"89b4918d-6cf7-49a8-aa48-781a310c0a71","added_by":"auto","created_at":"2022-11-03 14:29:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":224517,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrograph of biogenic silver nanoparticles\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/2f63d0f24322e016797fdaec.png"},{"id":28615846,"identity":"aca6a732-1680-4bc2-a8ef-ac580a7506b9","added_by":"auto","created_at":"2022-11-03 14:37:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":130762,"visible":true,"origin":"","legend":"\u003cp\u003e(a). The average size of biogenic AgNPs synthesized from \u003cem\u003eMirabilis jalapa\u003c/em\u003e by Zeta sizer (b) Zeta potential of bAgNPs.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/5f7a94585c3418f10ee49972.png"},{"id":28615352,"identity":"e81c0f77-1544-4e29-a31a-d03c648798ef","added_by":"auto","created_at":"2022-11-03 14:29:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":20927,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of bAgNPs against \u003cem\u003eVibrio parahaemolyticus \u003c/em\u003eand \u003cem\u003eVibrio harveyi.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/1a4fd787f1af7d95b3a41636.png"},{"id":28615367,"identity":"0fb9cbff-442b-4e36-9971-107a54a37902","added_by":"auto","created_at":"2022-11-03 14:29:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":15283,"visible":true,"origin":"","legend":"\u003cp\u003eMinimum Inhibitory concentrations of bAgNPs on\u003cem\u003e Vibrio parahaemolyticus \u003c/em\u003eand \u003cem\u003eVibrio harveyi\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/cc993d9a2f56fa875af63a7e.png"},{"id":28615349,"identity":"bbb41518-0a66-4942-9e92-ca7d47dd2527","added_by":"auto","created_at":"2022-11-03 14:29:22","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":26374,"visible":true,"origin":"","legend":"\u003cp\u003eDPPH scavenging activity of Ascorbic acid and bAgNPs (μg/mL)\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/39fdc87e570d79c303a68ead.png"},{"id":28615848,"identity":"a0e6b177-c150-4aae-a861-60acaca3a4f3","added_by":"auto","created_at":"2022-11-03 14:37:23","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":27243,"visible":true,"origin":"","legend":"\u003cp\u003eFRAP assay of Ascorbic acid and bAgNPs\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/c96b1a9a9e2eeced5acda27b.png"},{"id":28615847,"identity":"a3445aa6-086a-4d70-a90b-fcbd6ad31060","added_by":"auto","created_at":"2022-11-03 14:37:22","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":33038,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity of bAgNPs on Vero cell line viability.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/979690b4854787e10b586545.png"},{"id":28615375,"identity":"5caa60b1-658d-4765-ac78-ae319144c90f","added_by":"auto","created_at":"2022-11-03 14:29:23","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":371120,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effect of biogenic AgNPs against Vero cell lines.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/5d9b0d75ea1e066f85e226b0.png"},{"id":44719845,"identity":"c5df564f-8ac8-4a75-a71e-4227a55458ac","added_by":"auto","created_at":"2023-10-16 19:02:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1957645,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2220633/v1/6b6243a1-1d71-4230-ba5e-f874f65533b1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis of biogenic silver nanoparticles (bAgNPs) using leaf extract of Mirabilis jalapa and evaluation of anti-vibriocidal, anti-oxidant properties and cytotoxicity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eShrimp farming is accounted for multibillion-dollar industry worldwide that continues to show significant development (Srinivas and Venkatrayulu 2019). Penaeid shrimp culture is dominated by \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e (Pacific white leg shrimp), \u003cem\u003ePenaeus monodon\u003c/em\u003e (tiger shrimp) and \u003cem\u003eMacrobrachium rosenbergii\u003c/em\u003e (Giant fresh water prawn) etc., species because of their fast growth rate, adaptation to wide salinity ranges and high yield (Santos et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Jayasankar \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) (Ngasotter et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The high profitable value of shrimp has created excellent employment opportunities which plays a significant role in supporting their economic livelihoods of people working in aquaculture sector (Salunke et al \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Penaeid shrimps are more susceptible to diseases due to high stocking density, excess feed and inappropriate maintenance of physicochemical parameters in culture ponds (Istiqomah et al.2020) (Karunasagar and Ababouch 2012).\u003c/p\u003e \u003cp\u003eVibriosis is a major bacterial disease affects penaeid shrimp caused by an opportunistic gram negative \u003cem\u003evibrio\u003c/em\u003e bacteria of \u003cem\u003evibrionaceae\u003c/em\u003e family (Novriadi, 2016). There are several vibrio species cause shrimp vibriosis among them \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e are the two major pathogens which are highly prevalent among others and causes Acute hepatopancreatic necrosis disease (AHPND) or early mortality syndrome (EMS) and luminescent vibriosis respectively, resulting in massive mortality and billion dollar economic loss in shrimp aquaculture sector (Istiqomah et al.2020) (Novriadi, 2016). The shrimp affected with Vibriosis are identified with symptoms such as stomach lethargy, discolouration of hepatopancreas and hemocytic infiltration etc. (Letchumanan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e exhibit virulence to the shrimp by producing a binary toxins Photorhabdus insect-related A (Pir A) and Pir B which are present on an extra chromosomal plasmid pVA1, whereas the virulence of \u003cem\u003eVibrio harveyi\u003c/em\u003e caused by Extra cellular products (ECP\u0026rsquo;s) which are lethal to the shrimp (Letchumanan et al. 2016).\u003c/p\u003e \u003cp\u003eThere are several traditional treatment methods are available to control the shrimp \u003cem\u003evibriosis\u003c/em\u003e. Antibiotics such as Azithromycin, Oxytetracycline, oxolinic acid, and florfenicol etc. are used to inhibit both \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e but inappropriate and extensive use of antibiotics leads to the development of antibiotic resistance to almost all the antibiotics and also leaving adverse impact on environment (Holmstr\u0026ouml;m et al. 2017). There is an appropriate solution to eliminate the antibiotic resistance in pathogens is to replace the antibiotics with biogenic, eco-friendly methods to achieve the sustainable aquaculture practices (Boyd et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne of the eco-friendly technologies to reduce the adverse environmental impact is Nanotechnology. Nanotechnology is the novel science which deals with nanomaterial which has larger surface area to volume ratio and it is one of the powerful emerging science (Ahmad et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Metal nanoparticles such as silver, gold, titanium and magnetic nanoparticles etc. have shown anti-microbial, anti-fungal, anti-viral, anti-tumour and anti-parasitic etc. activities. Since 1000 B.C silver has been used as antiseptic agent and also has the antibacterial and antitumor properties (Zhang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Silver nanoparticles (AgNPs) are the nanometer sized silver particles with few to 100nm diameter size are produced by physical, chemical and biological methods (Sivolella et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Gamma irradiation, ultrasonic irradiation, evaporation-condensation are physical methods to synthesize AgNPs. In chemical methods sodium borohydride, polyol etc. compounds are used as reducing agents for synthesis of AgNPs. Secondary metabolites of plants, algae, fungi and bacteria are used as reducing agents to synthesize the biogenic or green AgNPs, that possess less toxicity than physically and chemically synthesized AgNPs (Van Hengel et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Samanta et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In recent decades biogenic silver nanoparticles have gained great attention of the researchers and opened new insights in science and technology which are reported more efficient anti-bacterial activity than chemical or physically synthesized AgNPs (Ahmad et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (Journal et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA thorough study of literature have been carried out to find appropriate methods using biogenic or green AgNPs against shrimp vibriosis, but unexpectedly very few studies were done on the effect of biogenic AgNPs on shrimp vibriosis. There is a need to synthesize biogenic or green AgNPs using different plant species or other biological sources in order to meet the demand of the aquaculture farmers who are depending exclusively for livelihood on the shrimp aquaculture (Geetha et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study proposed to synthesize biogenic AgNPs using \u003cem\u003eMirabilis jalapa\u003c/em\u003e aqueous leaf extract as reducing agent and assessment of anti-vibriocidal activity on \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e. The Antioxidant activities (DPPH, FRAP) and Vero cell cytotoxicity were also evaluated.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of leaf aqueous extract:\u003c/h2\u003e \u003cp\u003eIn the present study, plant \u003cem\u003eMirabilis jalapa L.\u003c/em\u003e of \u003cem\u003eNyctaginaceae\u003c/em\u003e, perennial herb with bushy in nature was selected for the synthesis of biogenic silver nanoparticles and leaves were collected from Rajahmundry, East Godavari, Andhra Pradesh, India. Aqueous leaf extract was prepared using decoction method according to the Singh et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e. Leaves were subjected to shade drying under sunlight for 2\u0026ndash;3 days and grounded into fine powder. 5% of leaf powder was dissolved in distilled water, boiled for 60 minutes at 50\u003csup\u003eo\u003c/sup\u003eC and the resultant solution was filtered using Whatman number 1 filter paper and final aqueous leaf extract was stored in screwed bottle at 4\u003csup\u003eo\u003c/sup\u003eC temperature until further analysis (Singh et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiogenic synthesis of Silver nanoparticles (bAgNPs)\u003c/b\u003e: Biogenic synthesis of silver nanoparticles was done according to the protocol of Chand et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e. Aqueous leaf extract of Mirabilis jalapa was added to the 10mM of Silver Nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e) solution in 1:9 ratio was prepared with distilled water. The AgNO\u003csub\u003e3\u003c/sub\u003e solution and aqueous leaf extract were added and kept in constant magnetic stirrer up to 4 hours. The synthesis of silver nanoparticles was confirmed by changing of colour from yellowish green to dark brown. The solution was incubated for 24 hours in dark chamber and after incubation, the solution was centrifuged at 12000 rpm for 10 minutes and pellet was washed with distilled water for 2\u0026ndash;3 times and dried into powder using hot air oven.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of biogenic silver nanoparticles (bAgNPs):\u003c/h2\u003e \u003cp\u003eUV\u0026ndash;Vis spectroscopy technique was used to confirm the formation of nanoparticles from the precursor 10mM AgNO\u003csub\u003e3\u003c/sub\u003e solution reduced by leaf aqueous extract. The absorbance spectrum of the sample was obtained in the range of 200\u0026ndash;800 nm wavelength, using UV\u0026ndash;Vis spectrometer Shimadzu-UV 1800 at different time intervals such as 0 hour, 2hours and 4hours.Fourier-transform infrared spectroscopy (FTIR) analysis was performed to analyse and identify the functional groups responsible for the reduction and stabilization of biogenic silver nanoparticles using ALPHA II, a compact FT-IR spectrometer (Bruker) in the wavelength range 4000\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. X-ray diffraction (XRD) method is an efficient technique to identify the crystalline phase of silver nanoparticles and it was conducted by XPERT-PRO using monochromatic Cu ka radiation (k\u0026thinsp;=\u0026thinsp;1.5406 A˚) operated at 40 kV and 30 mA from 10\u003csup\u003eo\u003c/sup\u003e to 90\u003csup\u003eo\u003c/sup\u003e in 2θ angles. The obtained data was compared with the International centre for diffraction studies (ICDS) library to account for the crystalline structure. The morphology and shape of the silver nanoparticles were examined using Field Emission (FEI) Quanta 200 FEG MKII scanning electron microscope (SEM). The resolution of FESEM is 1.5 nm and with high output thermal field emission (\u0026gt;\u0026thinsp;100nA beam current). The FESEM has backscatter (BSE) detector with high sensitivity (18 mm) for atomic number contrast. TEM analysis was also used to determine the morphology, size and shape of the silver nanoparticles. 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. The hydrodynamic size and zeta potential of the bAgNPs was confirmed by using the Zeta sizer nano ZS90 with 90 degree scattering optics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnti-Vibriocidal efficiency of biogenic silver nanoparticles (bAgNPs):\u003c/h2\u003e \u003cp\u003eThe biogenic AgNP\u0026rsquo;s synthesized from the aqueous leaf extract of \u003cem\u003eMirabilis jalapa\u003c/em\u003e was tested for anti-vibriocidal activity on \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (MTCC No 451) and \u003cem\u003eVibrio harveyi\u003c/em\u003e (ATCC No 334). The anti-vibriocidal activities of the bAgNPs were determined by the well diffusion assay and micro broth dilution method using marine nutrient agar (MNA) medium. In this method, 10\u003csup\u003e5\u003c/sup\u003e CFU/mL of pathogenic organisms, \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e were taken from pure cultures and are swabbed on Marine nutrient agar (MNB) plates using sterile cotton swab. An approximately six wells with depth of 2.5mm 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 \u0026micro;l of antibiotic (Azithromycin 20mg/mL) was added. WL2 to WL6 were added with 25 \u0026micro;l, 50 \u0026micro;l, 75 \u0026micro;l and 100 \u0026micro;l of bAgNPs (20mg/mL) respectively and incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24 hours. The experiment was carried out in triplicates under aseptic conditions. The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD values of zone of inhibition were calculated (Ravichandran et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicro dilution broth was performed to determine the minimum inhibitory concentration (MIC) of bAgNPs according to the Loo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e. This assay was carried out in 96-well micro titer plate using standard broth dilution method. The bacterial inoculums of two pathogens were adjusted to the concentration of 10\u003csup\u003e5\u003c/sup\u003e CFU / mL. Column-1 is taken as negative control and filled with 100 \u0026micro;l of Maine nutrient broth in micro titer plate. 100 \u0026micro;l of bAgNP\u0026rsquo;s stock solution (20mg/mL) was added to the column 1 \u0026amp; 2, and column-11 and 12 served as negative control. Except negative control, 50\u0026micro;l (10\u003csup\u003e5\u003c/sup\u003e CFU / mL) of each pathogen were added to the all other columns and plates were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 24 hours. After incubation the wells were observed for bacterial growth. Formation of turbidity indicates the growth of bacteria. The experiment was run in triplicates and The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD values of MIC was calculated (Loo et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of Anti-oxidant activity:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003e2,2-diphenyl-1-picrylhydrazyl (DPPH) assay\u003c/strong\u003e \u003cp\u003eFree radical scavenging activity of biogenic silver nanoparticles was determined using DPPH assay. Biogenic AgNPs prepared at different concentrations \u0026ndash; 20, 40, 60, 80 and 100 \u0026micro;g/mL were agitated thoroughly and added to 2mL of 3 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M DPPH in methanol was added to all test tubes. The solutions were incubated at 37\u003csup\u003eo\u003c/sup\u003eC in dark chamber for about 2 hours and the absorbance was recorded at 517nm wavelength using UV-Vis spectroscopy. The DPPH scavenging activity of each concentration was calculated by given below formula (Sumbal et al. 2017). Ascorbic acid was used as standard.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eDPPH scavenging effect % Inhibition\u0026thinsp;=\u0026thinsp;A\u003csub\u003e0\u003c/sub\u003e -A\u003csub\u003e1\u003c/sub\u003e /A\u003csub\u003e0\u003c/sub\u003e \u0026times; 100\u003c/p\u003e \u003cp\u003eWhere A\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;the absorbance of control.\u003c/p\u003e \u003cp\u003eA\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;the absorbance of standard\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFerric reduction anti-oxidant power (FRAP) assay\u003c/strong\u003e \u003cp\u003eFRAP assay was done according to the \u003cem\u003eSumbal et al. 2019\u003c/em\u003e. This method is depend upon the ability of bAgNPs to reduce Fe\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e to Fe\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e in the presence of 2, 4, 6-tripyridyl-s-triazine (TPTZ) and formation of intense blue complex (Fe\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e \u0026ndash; TPTZ). Biogenic AgNPs prepared at different concentrations \u0026ndash; 20, 40, 60, 80 and 100 \u0026micro;g/mL were agitated thoroughly and added to 2mL of FRAP reagent. The solutions were incubated at 37\u003csup\u003eo\u003c/sup\u003eC in dark chamber for about 2 hours and the absorbance was recorded at 593nm wavelength using UV-Vis spectroscopy. The ferric reduction capacity of each concentration was calculated by given below formula. Ascorbic acid was used as standard.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e% Inhibition of FRAP\u0026thinsp;=\u0026thinsp;A\u003csub\u003e0\u003c/sub\u003e -A\u003csub\u003e1\u003c/sub\u003e /A\u003csub\u003e0\u003c/sub\u003e \u0026times; 100\u003c/p\u003e \u003cp\u003eWhere A\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;the absorbance of control.\u003c/p\u003e \u003cp\u003eA\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;the absorbance of standard\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity assay:\u003c/h2\u003e \u003cp\u003eThe 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\u0026rsquo;s Modified Eagle Medium (DMEM) medium was discarded from Vero cell sub culture and trypsinized separately. To this disaggregated cells in the flask 25mL of DMEM with 10% FCS (fetal calf serum) was added. The cells were homogenized in the suspended medium. One mL of homogenized suspension was added to each well in a 24 wells culture plate along with the addition of 0, 50, 100, 150, 200 \u0026micro;g/mL concentration bAgNPs in each column respectively and were incubated at 37\u0026deg;C in a humidified CO\u003csub\u003e2\u003c/sub\u003e incubator and allowed to reach 80% confluence.\u003c/p\u003e \u003cp\u003eAfter incubation, the treated cells were washed in phosphate buffered saline of pH 7.4 and cultured plate was loaded with 20 \u0026micro;l of MTT reagent and incubated at room temperature for about 3 hours. After incubation, the content was removed from the wells and 100\u0026micro;l Sodium dodecyl sulphate (SDS) in Dimethyl sulfoxide (DMSO) was added to the all wells to dissolve formazan crystals and absorbance was read at 540nm with Lark LIPR-96 micro titre ELISA reader. The percentage of cell viability and IC\u003csub\u003e50\u003c/sub\u003e values were calculated using absorbance values (Sumbal et al. 1993).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003eBiogenic synthesis and characterization of bAgNPs:\u003c/h2\u003e\n \u003cp\u003eThe synthesis of bAgNPs was confirmed by observing the change in the color of solution from yellowish green to dark brown color. This color transition is due to the excitation of Surface Plasmon Vibrations of silver nanoparticles and was primarily confirmed by UV-Vis spectroscopy at different time intervals (0, 2 and 4 hours) and the absorbance peak was observed at 434 and 451 wavelengths, after 2 hours and 4 hours of synthesis respectively, which indicating the formation of biogenic silver nanoparticles (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Green AgNPs synthesized from \u003cem\u003eProsopis farcta\u003c/em\u003e fruit extract showed absorbance peak around 475nm wavelength (Salari et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Another study showed that green AgNPs synthesized using \u003cem\u003eBrassica oleracea\u003c/em\u003e leaf extract gave absorbance peak at 415nm wavelength (Ansar et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The smaller the wavelength peak, the lesser the size of silver nanoparticles and more effective in its biological activity (Salari et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe XRD is a primary technique to determine the crystalline nature of silver nanoparticles (Rafique et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) by predicting spectrum of 2\u0026theta; value ranges from 10\u003csup\u003eo\u003c/sup\u003e to 90\u003csup\u003eo\u003c/sup\u003e. The XRD pattern of bAgNPs in this study revealed intense peaks at 111, 200, 220 and 311 which strongly reflected the Bragg\u0026rsquo;s reflection with face centred cubic (fcc) and confirming the crystalline nature of bAgNPs sample using ICDD (Crystallographic sheet 04-0783) shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The peaks which are unassigned in XRD pattern are due to the crystallization of bioorganic phase that occurs on the surface of silver nanoparticles. The intense peaks 111, 220 corresponds to the ultra-small nanoparticles and face centre cubic (fcc) (Mourdikoudis et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). XRD of Biogenic silver nanoparticles synthesized using sheep\u0026rsquo;s blood (\u003cem\u003eOvis aries\u003c/em\u003e) showed intense peaks 111, 200, 220 and 311 which is similar to the present study (Kakakhel et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Silver nanoparticles synthesized from \u003cem\u003eProsopis chilensis\u003c/em\u003e leaf extract XRD analysis showed four intense peaks 111, 200, 220 and 311 which strongly support Bragg\u0026rsquo;s reflection and fcc (Kandasamy and Alikunhi \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe phytoconstituents present in the plant extract have dual role which act as both reducing and capping agents. The presence of functional groups in the synthesized biogenic silver nanoparticles was confirmed through FTIR analysis in the spectral range of 400\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and depicted in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The observed peak broadening and noise were probably macromolecules present in the plant extract which may be responsible for the reduction of silver nanoparticles. The FTIR band at 1008.50 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to Flouro halogen group (C-F stretching) which may be attached to the reducing or stabilizing agent, band at 1416.50 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to C-H bending (Alkane), band at 1636.95 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to C\u0026thinsp;=\u0026thinsp;O ( ketones) and band at 3298.60 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to O-H stretching (alcohol). Gopalakrishnan et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e synthesized green silver nanoparticles using aqueous leaf extract of \u003cem\u003eMirabilis jalapa\u003c/em\u003e shown two peaks 3323 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1620 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to OH stretching and C\u0026thinsp;=\u0026thinsp;O ketones. \u003cem\u003eSumbal et al., 2019\u003c/em\u003e found OH stretching ( 3283.69 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), C-N group or alkyne (2122.60 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), C\u0026thinsp;=\u0026thinsp;C or NH group ( 1643.20 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and C-O (1318.89 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e1) corresponds to the alcohols, amides and amine functional groups in the aqueous leaf extract of \u003cem\u003eMirabilis jalapa.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eSelected 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. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). The TEM images of the biogenic silver nanoparticles revealed variable shapes and most of are with spherical shapes with particle size of 50nm and above (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb) which was further confirmed by the Zeta size analysis. The TEM analysis of bAgNPs synthesized from \u003cem\u003eProsopis farcta\u003c/em\u003e fruit extract showed 60nm sized bAgNPs with corresponds to the UV-Vis absorbance wavelength around 475 nm, higher than the UV absorbance and size of bAgNPs of the present study (Salari et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). bAgNPs synthesized from leaf extract of \u003cem\u003eErythrina suberosa\u003c/em\u003e TEM analysis showed the size of nanoparticle between 15-34nm with corresponds to the UV-Vis absorbance peak at ~\u0026thinsp;428nm (Mohanta et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). This clearly shows that, smaller the absorbance of UV-Vis spectroscopy then lesser the size of the nanoparticle. In the present study, TEM analysis of bAgNPs showed the particle size was around 50nm and a UV-Vis absorbance peak at 434nm (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFESEM analysis was performed to determine the morphology of the bAgNPs. Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the SEM micrograph formed by the secondary electrons (SE) of silver nanoparticles. The FESEM analysis showed well dispersed bAgNPs at 0.5\u0026micro;m and most nanoparticles are with aggregated and spherical in shape. Green AgNPs synthesized from methanolic extracts of \u003cem\u003eIpomoea carnea\u003c/em\u003e shown spherical shaped and uniformly dispersed nanoparticles in FE SEM analysis (Singh, \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) and gAgNPs synthesized from \u003cem\u003ePseudoduganella eburnean\u003c/em\u003e shown rod shaped and uniformly dispersed nanoparticles (Huq, \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast FE SEM analysis of the present study reported the bAgNPs with Spherical and non-homogenous shapes and aggregated in form (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe particle size and particle dispersion Index (PdI) was analysed using dynamic light scattering (DLS) which showed the size of biogenic synthesized silver nanoparticles to be \u0026gt;\u0026thinsp;50nm (Fig. 6a) and particle dispersion Index (PdI) was 0.202. The Z average (d.nm) is found to be 269. The zeta potential value of biogenic synthesized silver nanoparticles was found to be -24.9mV which indicating the 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 nanoparticles in aqueous suspension (Mat Yusuf et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the computed PDI value of the bAgNPs was found to be in the range of 0\u0026ndash;1, indicating that the bAgNPs were in a monodisperse phase with minimal particle aggregation (Mat Yusuf et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003eAnti-vibriocidal activity:\u003c/h2\u003e\n \u003cp\u003eOver the years, antibiotic resistant strains of \u003cem\u003eVibrio\u003c/em\u003e species have emerged in the environment due to 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 the chemotherapeutic agents (Letchumanan et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e are more prevalent pathogens among the \u003cem\u003eVibrio\u003c/em\u003e species which cause \u003cem\u003evibriosis\u003c/em\u003e to shrimp (Campus 2018) (Mastan and Begum \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). In recent decades, silver nanoparticles gained a lot of attention in different research and commercial sectors due to its targeted specific action and anti-microbial activity. Application of AgNPs in commercial aquaculture sectors have been increased to combat the various disease causing pathogens (Morones et al.2005) (Shankar et al.2004).\u003c/p\u003e\n \u003cp\u003eIn this study well diffusion assay was performed to determine the anti-vibriocidal activity of biogenic silver nanoparticles (bAgNPs) and exhibited the maximum inhibition zone of 26.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54mm and 28.375\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 mm wide respectively. The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD values of zone of inhibitions of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e are depicted in Figs. 6 \u0026amp; \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e respectively. In contrast the control (antibiotic) used in this study showed inhibition zone of 23.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 mm and 13.125\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mm wide of the both species respectively which are less than zone of inhibition of bAgNPs.\u003c/p\u003e\n \u003cp\u003eMicro dilution broth (MBS) was done to determine the Minimum Inhibitory Concentration (MIC) of green silver nanoparticles on \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi.\u003c/em\u003e The MIC values of bAgNPs against the growth of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e are 31.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0 and 93.75\u0026thinsp;\u0026plusmn;\u0026thinsp;44.19 \u0026micro;g/ mL respectively (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e)..\u003c/p\u003e\n \u003cp\u003eIn contrast to the present study, colloidal silver nanoparticles synthesized by photo-assisted reduction method obtained two different sized nanoparticles 16.62nm and 22.22nm that showed 42.1mm and 43.12mm of zone of inhibition against \u003cem\u003eVibrio harveyi\u003c/em\u003e (Nafisi et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Green silver nanoparticles synthesized by extracellular products of \u003cem\u003eBacillus subtilis\u003c/em\u003e produced size range of 10\u0026ndash;25 nm showed zone of inhibition 21.25nm and 19.27nm on \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e respectively (Sivaramasamy and Zhiwei \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Green silver nanoparticles synthesized from leaf extract of \u003cem\u003eProsopis chilensis\u003c/em\u003e obtained size ranges from 5 to 25 nm with zone of inhibition 16nm and 19nm against \u003cem\u003eVibrio harveyi\u003c/em\u003e and \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e respectively (Kandasamy and Alikunhi \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). 10mg of silver nanoparticles synthesized from tea leaf extract (\u003cem\u003eCamellia sinensis\u003c/em\u003e) inhibited 70% of \u003cem\u003eVibrio harveyi\u003c/em\u003e growth in culture media (Vaseeharan et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Biogenic silver nanoparticles synthesized from \u003cem\u003eCheatomorpha antennia\u003c/em\u003e shown anti-vibriocidal activity against \u003cem\u003eVibrio harveyi\u003c/em\u003e and exhibited 17.8mm zone of inhibition (Thanigaivel et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAnti-oxidant activity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAntioxidants 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 has been developed to determine the antioxidant capacity of both natural and synthetic substances (Bedlovicov\u0026aacute; et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study DPPH and FRAP were used to evaluate the antioxidant activity of biogenic silver nanoparticles synthesized from aqueous leaf extract of \u003cem\u003eMirabilis jalapa\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eDPPH assay:\u003c/strong\u003eDPPH free radical scavenging activity was done for the assessment of antioxidant potential of green silver nanoparticles. The purple solution DPPH containing solution turned yellow after the addition of bAgNPs, which indicates the free radical scavenging activity. Figure-9 shows maximum inhibition at 500 \u0026micro;l/mL with 60.77% and the Inhibitory concentration 50 (IC\u003csub\u003e50\u003c/sub\u003e) value was determined using the formula IC\u003csub\u003e50\u003c/sub\u003e (0.5-b)/a, which was 67.3987\u0026thinsp;\u0026plusmn;\u0026thinsp;202 \u0026micro;l/mL, indicating the concentration of bAgNPs required to inhibit the 50% of DPPH concentration.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFRAP assay:\u003c/strong\u003eIn FRAP assay, the maximum inhibitory percentage of bAgNPs required to reduce Fe3\u003csup\u003e+\u003c/sup\u003e to Fe2\u003csup\u003e+\u003c/sup\u003e ranges from 3.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65 to 22.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54%. The IC\u003csub\u003e50\u003c/sub\u003e value of bAgNPs was 5.509\u0026thinsp;\u0026plusmn;\u0026thinsp;152 \u0026micro;l/mL, indicates at this concentration 50% of Fe3\u003csup\u003e+\u003c/sup\u003e is reduced to Fe2\u003csup\u003e+\u003c/sup\u003e and is depicted in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe significant anti-oxidant ability of bAgNPs was carried out by DPPH and FRAP assays showed IC\u003csub\u003e50\u003c/sub\u003e value 67.39 \u0026micro;g/mL and 5.50 \u0026micro;g/mL respectively. Green silver nanoparticles synthesized from leaf extract of \u003cem\u003eErythrina suberosa\u003c/em\u003e shown significant free radical scavenging activity with IC\u003csub\u003e50\u003c/sub\u003e value of 30.04 \u0026micro;g/mL (Mohanta et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Green synthesized silver nanoparticles from \u003cem\u003eProsopis farcta\u003c/em\u003e shown significant DPPH free radical scavenging activity with IC\u003csub\u003e50\u003c/sub\u003e value of 70 \u0026micro;g/mL which is higher than the present study (Salari et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Green silver nanoparticles synthesized from \u003cem\u003eCestrum nocturnum\u003c/em\u003e shown 29.55% of anti-oxidant activity at 100 \u0026micro;g/mL, (Keshari et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), which is higher than our present study. Green silver nanoparticles synthesized from \u003cem\u003eCaesalpinia sappan\u003c/em\u003e aqueous extract shown Ferric oxide anti-oxidant activity with 78.7 \u0026micro;g/mL (Suwan et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) and gAgNPs synthesized from \u003cem\u003eNothapodytes nimmoniana\u003c/em\u003e fruit extract studied for FRAP assay shown 214.89 \u0026micro;g/mL, (Mahendran and Ranjitha Kumari, 2016) and the concentration is higher than the present study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003eVero cell cytotoxicity:\u003c/h2\u003e\n \u003cp\u003eThe major consequence of nanoparticles is safety aspects. With the increasing applications of nanoparticles, there are possible adverse effects on environment and organisms. It is necessary to extensively evaluate the nanoparticles toxicity in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Saranya et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, the toxicity of biogenic silver nanoparticles was evaluated on Vero cell lines (African green monkey kidney cell lines) which are non-cancerous cell lines.\u003c/p\u003e\n \u003cp\u003eThe \u003cem\u003ein-vitro\u003c/em\u003e cytotoxicity activity results of the bAgNPs synthesized from leaf extract of \u003cem\u003eMirabilis jalapa\u003c/em\u003e against Vero cells showed minimal inhibition towards tested cell line. However, the increased concentration of bAgNPs, the cell viability decreased. The maximum cytotoxicity 69.49% of bAgNPs observed when 200 \u0026micro;g/ mL of bAgNPs were used against Vero cell lines. 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. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e \u0026amp; \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e). The IC\u003csub\u003e50\u003c/sub\u003e concentration of the tested sample against Vero cells was 293.5 \u0026micro;g/ mL.\u003c/p\u003e\n \u003cp\u003eMTT assay was done in the present study to evaluate the cytotoxic effect of bAgNPs on Vero cell lines, showed 73.87% of cell viability at maximum concentration of 200\u0026micro;g/mL and the IC\u003csub\u003e50\u003c/sub\u003e value of bAgNPs on Vero cell lines is 293.5 \u0026micro;g/mL. Biogenic AgNPs synthesized from \u003cem\u003eCassia fistula\u003c/em\u003e shown Vero cell line cell death was 89.7% at 1000 \u0026micro;g/mL and IC\u003csub\u003e50\u003c/sub\u003e value is 66.34 \u0026micro;g/mL, which is less than our current study (Remya et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Colloidal silver nanoparticles synthesized using silver nitrate and citric acid showed mortality of 68.54% at 1000 \u0026micro;g/mL on Vero cell lines and IC\u003csub\u003e50\u003c/sub\u003e value 10.68 \u0026micro;g/mL, which indicating the less concentration of colloidal silver nanoparticles cause 50% of cell death. Green AgNPs synthesized from leaf extract of copperrod plant (\u003cem\u003ePeltophoroum pterocarpum\u003c/em\u003e) and studied cytotoxicity on Vero cell lines showed IC\u003csub\u003e50\u003c/sub\u003e value of 90 \u0026micro;g/mL (Pannerselvam et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Green silver nanoparticles synthesized from \u003cem\u003eAlysicarpus monilifer\u003c/em\u003e showed very limited toxicity of 25 \u0026micro;g/mL on Vero cell lines after 72 hours of incubation (Kasithevar et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present work describes the synthesis of biogenic silver nanoparticles using aqueous leaf extract of \u003cem\u003eMirabilis jalapa\u003c/em\u003e. The leaf extract have bioactive compounds such as flavonoids, phenols, terpenoids, tannins, and alkaloids etc. which act as reducing, stabilizing and capping agents for the synthesis of biogenic silver nanoparticles. The formation bAgNPs was confirmed by UV-Vis spectroscopy, XRD, FTIR, TEM and FE SEM, Zeta size and Zeta potential showed spherical, cone shaped with around 50nm and above sized nanoparticles. The bAgNPs synthesized in this study have shown significant anti-vibriocidal activity on \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003eVibrio harveyi\u003c/em\u003e which are more prevalent pathogens causes \u003cem\u003eVibriosis\u003c/em\u003e to the shrimp. The zone of inhibition for both pathogens 26nm and 28nm are respectively at 100\u0026micro;g/mL. Minimum Inhibitory Concentration (MIC) was determined as 31.25 and 187.5 \u0026micro;g/mL for both Vibrio species respectively and it was observed that, more concentration of bAgNPs are required to inhibit the \u003cem\u003eVibrio harveyi\u003c/em\u003e than \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e. Since, bAgNPs has free radical scavenging and ferric oxide reducing anti-oxidant activities. This is due to functional groups present on bAgNPs surface as capping agents. The cytotoxicity of bAgNPs on Vero cell lines has shown lesser toxicity at maximum concentration of 200\u0026micro;g/mL. These biogenic silver nanoparticles might be used as anti-microbial agents in future due to eco-friendly, less toxicity and highly effective against the various pathogenic organisms.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eAHPND\u003c/strong\u003e \u0026ndash; Acute Hepatopancreatic Necrosis Disease\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEMS\u003c/strong\u003e \u0026ndash; Early Mortality Syndrome\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePir\u003c/strong\u003e \u0026ndash; Photorhabdus insect related\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ebAgNPs\u003c/strong\u003e \u0026ndash; Biogenic silver nanoparticles\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTIR\u003c/strong\u003e \u0026ndash; Fourier Transform Infrared Spectroscopy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXRD\u003c/strong\u003e \u0026ndash; X-ray diffraction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e \u0026ndash; Scanning Electron Microscope\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTEM\u003c/strong\u003e \u0026ndash; Transmission Electron Microscope\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDLS \u0026ndash;\u0026nbsp;\u003c/strong\u003eDynamic Light Scattering\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no competing interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported financially by Regional Centre for Biotechnology, Department of Biotechnology, Faridabad, Junior Research Fellowship (Grant No. DBT/2020/ANU/1330).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors also thankful to School of Life and Health Sciences, Adikavi Nannaya University, Rajahmundry for providing necessary facilities for conducting this research work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePVN were involved in supervision and conceptualization of project. AM and AMR were responsible for methodology, investigation, resources and writing the original draft manuscript. SRM were involved in data curation, editing and reviewing of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to PVN\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhmad, S., Munir, S., Zeb, N., Ullah, A., Ali, J., Bilal, M., Omer, M., Alamzeb, M., Salman, S.M., Ali, S., 2019. Green nanotechnology : a review on green synthesis of silver nanoparticles \u0026mdash; an ecofriendly approach.\u003c/li\u003e\n \u003cli\u003eAnsar, S., Tabassum, H., Aladwan, N.S.M., Ali, M.N., 2020. Eco friendly silver nanoparticles synthesis by Brassica oleracea and its antibacterial , anticancer and antioxidant properties. Sci. 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Med. 11, 1949\u0026ndash;1959. https://doi.org/10.1016/j.nano.2015.07.016\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aquaculture, Shrimp Vibriosis, Vibrio parahaemolyticus, Vibrio harveyi, Antimicrobial-resistance, Silver nanoparticles, Mirabilis jalapa","lastPublishedDoi":"10.21203/rs.3.rs-2220633/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2220633/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute hepatopancreatic necrosis disease and luminescent vibriosis are two major bacterial diseases of penaeid shrimp which are caused by gram negative pathogenic bacteria \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (\u003cem\u003eVp\u003c/em\u003e) and \u003cem\u003eVibrio harveyi \u003c/em\u003e(\u003cem\u003eVh\u003c/em\u003e) 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 the antibiotic resistance in pathogens. In the present study, biogenic silver nanoparticles (bAgNPs) are synthesized by reducing AgNO\u003csub\u003e3\u003c/sub\u003e using aqueous extract of \u003cem\u003eMirabilis jalapa\u003c/em\u003e (MJ) leaves. The anti-oxidant, cytotoxic and anti-vibriocidal activity of bAgNPs against \u003cem\u003eVp \u003c/em\u003eand \u003cem\u003eVh\u003c/em\u003e are evaluated. The formation of silver nanoparticles was confirmed by the appearance of dark brown colored solution and with a maximum absorption peak at 434nm. The characterization of bAgNPs using FESEM, TEM, XRD, FTIR, and DLS has confirmed that the nanoparticles are crystalline, spherical in shape with an approximate diameter of 50nm, and have capping agents. The diameter of microbial growth inhibition zones for \u003cem\u003eVp\u003c/em\u003e and \u003cem\u003eVh \u003c/em\u003eare 26mm and 23mm respectively. Further, the MIC values for \u003cem\u003eVp \u003c/em\u003eand Vh\u003cem\u003e \u003c/em\u003eare 31.25µg/mL and 93.75µg/mL respectively. The DPPH and FRAP\u003cstrong\u003e \u003c/strong\u003eassays showed substantial anti-oxidant activity with IC\u003csub\u003e50\u003c/sub\u003e values of 67.39µg/mL and 5.509µg/mL respectively. MTT assay to check cytotoxicity effect of bAgNPs on Vero cells resulted very less toxicity at maximum concentration tested with an IC\u003csub\u003e50\u003c/sub\u003e value of 293.5µg/mL. Therefore, the biogenic silver nanoparticles synthesized from leaves of MJ\u003cem\u003e \u003c/em\u003eshowed effective anti-vibriocidal and anti-oxidant properties with negligible cytotoxic effect.\u003c/p\u003e","manuscriptTitle":"Synthesis of biogenic silver nanoparticles (bAgNPs) using leaf extract of Mirabilis jalapa and evaluation of anti-vibriocidal, anti-oxidant properties and cytotoxicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-03 14:29:15","doi":"10.21203/rs.3.rs-2220633/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-11-18T09:05:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-11-13T12:29:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b7f8760f-b179-4b02-89f2-517c0a3ab396","date":"2022-11-08T02:28:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-11-07T18:31:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-11-02T13:29:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-11-01T00:39:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2022-10-31T07:27:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6ed179fb-37ba-49c4-8acd-aa23a9684f1a","owner":[],"postedDate":"November 3rd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T19:00:07+00:00","versionOfRecord":{"articleIdentity":"rs-2220633","link":"https://doi.org/10.1007/s12668-023-01060-x","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2023-02-18 18:56:57","publishedOnDateReadable":"February 18th, 2023"},"versionCreatedAt":"2022-11-03 14:29:15","video":"","vorDoi":"10.1007/s12668-023-01060-x","vorDoiUrl":"https://doi.org/10.1007/s12668-023-01060-x","workflowStages":[]},"version":"v1","identity":"rs-2220633","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2220633","identity":"rs-2220633","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}