Ultra-sonication enhanced green synthesis of silver nanoparticles using Barleria buxifolia leaf extract and its possible application


 A simple and eco-friendly method for the green synthesis of silver nanoparticles (AgNPs) by ultrasound-assisted strategy using Barleria buxifolia leaf extract as a reducing and capping agent was established in this study. The obtained AgNPs were characterized. UV-vis spectrum, Fourier transform infrared spectroscopy (FTIR), scanning and transmission electron microscopy (SEM and TEM), Energy Dispersive X-Ray Analyzer (EDX), X-ray diffraction, dynamic light scattering (DLS) analysis showed that the obtained AgNPs were mono dispersed spheres with uniform size of 80 nm. UV-vis spectroscopy, FTIR, and XRD analysis indicated that the surface of the obtained AgNPs was covered with organic molecules in plant extracts. The results of ABTS assays showed that high antioxidant activity was seen in the obtained AgNPs. Green synthesized AgNPs showed potent antibacterial and anti-biofilm activity against tested pathogens. Cytotoxicity assay showed that the obtained AgNPs were significantly cytotoxic to cancer cell line (MCF-7). In addition, the AgNPs synthesized in this paper can also photo catalytically degrade methylene blue dye under visible light. The potent bioactivity exhibited by the green synthesized silver nanoparticles leads towards the multiple use as antioxidant, antibacterial, anti-biofilm, cytotoxic as well as photo catalytic agent.


Introduction
In modern science, metal nanoparticles are fascinating materials with speci c properties and wide application in a variety of elds, including pharmaceuticals, catalysis, food and agriculture, electronics, chemical units, cosmetics, mechanics and optics, etc. [1,2,3,4,5]. Generally metal nanomaterials synthesized by physical, mechanical, electrochemical, microwave-assisted, hydrothermal and green chemistry methods are favored [6,7]. However, most of the physiochemical methods are extremely costly, non-degradable, and the use of toxic chemicals has contributed to multiple biological threats [8,9,10].
As a kind of metal nanoparticles with good physical and chemical properties and biocompatibility, silver nanoparticles (AgNPs) have been widely studied. [21,22]. The green synthesis of AgNPs through ecofriendly microbes and plant has in recent times grown to be trendy in recently. According to the reports of Gardea et al. and J. Das, P. Velusamy [23,24], the synthesis of nanoparticles using plant extract is very simple and easy to handle compared to microbes. In these organic strategies for the synthesis of NPs, plant extracts act as reducing and capping agents. So far, Ag NPs had been e ciently and swiftly synthesized by numerous plant extract such as, Artemisia nilagirica [25], Iresine herbstii [ 26], Vaccinium macrocarpon [27], Parthenium hysterophorous [28], Urticadioica Linn. [1], Mimusops elengi, Linn. [29], Azadirachta indica [30], Lonicera hypoglauca [8], Durio zibethinus [7]. In addition, many scientists examined the potential properties like antioxidant, antimicrobial, antiviral, anticancer, antimalarial properties and photo catalytic effects of green synthesized AgNPs by plant extracts [31,32]. Barleria buxifolia is one of the family of acanthaceae and it have been types of secondary metabolites in the leaf, ower and root parts. The roots and leaves have been used as a traditional herbal medicine in India to deal with cough, bronchitis, in ammation and antibacterial activity against human pathogens [33,34,35].
With our expertise, till date there is no report on synthesis of AgNPs using B. buxifolia. In this direction, herein our proposed work to develop a novel approach for the biosynthesis of silver nanoparticles the usage of leaf extract of B. buxifolia through ultra-sonication process.
The present experimental investigation reports the green synthesis of silver nanoparticles using B. buxifolia leaf extract with the aid of ultra-sonication process has been mentioned. Herein we utilize B. buxifolia leaf extract were functions as both reducing and stabilizing agents during AgNPs synthesis. Obtained AgNPs were characterized by using UV-vis spectrum, FTIR, SEM and TEM, XRD, EDX, DLS and check its antioxidant properties using ABTS assay. Also we evaluated antibacterial, anti-bio lm and EPS reduction activity of AgNPs against pathogens such as Escherichia coli, pseudomonas aeruginosa, Salmonella enterica, Shigella spp. Furthermore, the synthesized AgNPs catalytic activity in the reduction of MB and their cytotoxicity toward MCF-7 breast cancer cell line has also been studied.

Materials And Methods
All the analytical reagents used in the study were of analytical grade and were purchased from Merck, were then incubated for 6 min and the absorbance was taken at 734 nm. The change in absorbance with respective control (containing ABTS solution without antioxidants, expressed as 100% free radicals) was calculated as percentage free radical scavenging. Ascorbic acid was used as positive control [37].
Where A control is the absorbance of the ABTS solution and A sample is the absorbance of the test sample.

Evaluation of antibacterial activity
Antagonistic activity of the AgNPs, leaf extract biomass and amikacin have been assessed using Muller-Hinton agar well diffusion approach towards MDR clinical pathogens i.e., P. aeruginosa, S. enterica, Shigella spp. as per standard methods. Wells of 7 mm size are made in the Agar plates containing the bacterial lawn. The various concentration (25-100 mg/ml) of samples were lled in the wells made in the bacterial culture plates. Then the petri dishes consequently prepared were left at room temperature for ten minutes for allowing the diffusion of the samples into the agar bacterial lawn. After incubation for 24 h at 37°C, the plates had been observed. The zone of inhibition was observed and expressed in millimetres [38].

Bio lm Inhibition Assay
The ability of AgNPs with different concentrations (25-100μg/ml) to inhibit the formation of bacterial bio lms was recognized using 24 h old broth inoculums of E. coli and P. aeruginosa, S. enterica, Shigella spp using tissue culture plate method. The inoculums have been prepared using 10 ml of trypticase soy broth (TSB) with1%glucose and seeded into 1 cm 2 cover slides placed in culture plates. After 24 h, planktonic cells had been removed by way of washing using sterilized distilled water and the glass slides were stained with 0.2% crystal violet stain. Bio lms formation have been visualized by Trinocular Phase Contrast microscope (Kozo Optics) at X40magni cation. After visualization, the stain was solubilized with 1ml of 70% ethanol and the stained adherent bio lm was quanti ed using a micro-ELISA auto reader (model R, Epoch, USA) at wavelength 570 nm [39].

Photocatalytic degradation of dye
Photocatalytic things to do of the green synthesized AgNPs estimated from the degradation of methylene blue (MB) under the sun light irradiation for distinctive time interval. 1mg of methylene blue dye used to be dissolved in 100mL of double distilled water used as inventory solution. About 20 mg of green synthesized AgNPs was dispensed to 50mL of methylene blue dye solution. A control was also maintained without addition of AgNPs. Before exposing to irradiation, the reaction suspension was nicely blended by means of being magnetically stirred for 30min to certainly make the equilibrium of the working solution. Then, the dispersion used to be put under the sunlight and it monitored from morning to sunset. At particular time intervals, aliquots of 5mL suspension had been taken and ltered to evaluate the photocatalytic degradation of dye via UV spec two at 200-700nm [43] 2.9. Statistical analysis Green synthesized AgNPs were assayed for antibio lm, antimicrobial, anticancer and photo-catalytic activity. All the experiment were run in triplicates and data were reported as mean ± standard deviation and the data were analysed by one-way ANOVA Tukey's HSD analysis of variance, with a P-value of 0.05 being signi cant, using the (IBM SPSS statistics 20) statistical software package.

Results And Discussion
3.1. Ultra-sonic intensi ed synthesis and characterization of AgNPs using B. buxifolia leaf extract: The present study integrates ultra-sound intensi ed green approach to produce silver nanoparticles using B. buxifolia leaf extract as both reducing and stabilizing agent. In a typical greener procedure, an appropriate amount (10ml) of leaf extract was dissolved in 50 ml of silver nitrate solution. Then, an ultrasonic probe was immersed into the mixture solution for various time interval (0-240 min), whereas exposing to ultrasound waves on Ag + ions containing leaf extract changed from light yellow to darkish brown colour, which suggest the formation of AgNPs via bioreduction process (Ag + converted to Ag 0 , during the addition of polyphenols). The Ag + ions without leaf extract did not show any colour changes even exposing to ultra-sonication for 240min. From Fig. 1a. the UV-Vis results, it is found that green synthesised AgNPs are mono dispersed in nature and broad surface plasmon resonance(SPR) peak was also observed at 435 nm with high intensity for the increasing time, which indicates the formation of AgNPs and their stability. Earlier reports recommended that an SPR shift situated from 410 to 450 nm has been detected for AgNPs, which also attributed to spherical in nature [1,44] and their size range in 2nm-100nm [45]. Also our results consist with [2,46,47] that synthesized silver nanoparticles using plant extract; they obtained the maximum absorption shift at 440, 430, 450 nm and they cited the band came about due to the surface plasmon resonance of AgNPs. According to these results we suggest, that the process of ultra-sonication makes the silver nanoparticles intrinsically capping with plant molecules and it produced size controlled spherical shape like non-aggregated mono dispersed particles, as well as these techniques gives to a treasured contribution in nano-biotechnology.
The phase purity and crystalline nature of synthesised AgNPs is studied with the aid of X-ray diffraction analysis as given in Fig. 1b [48,49]. In this manner, the normal grain size of the AgNPs was computed from the solid re ection peak by (111) arrangement utilizing Scherer formula.
From this equation, the common size of AgNPs became approximately in 80 nm. In addition, the X-ray diffraction results clearly shows that the silver nanoparticles formed by the reduction of Ag+ ions by the B. buxifolia leaf extract are crystalline in nature. Further, unassigned peaks at 2θ = 32.35°, 46.31°, 54.56°, and 57.58° denoted by (*) that's indicating the presence of plant extract (capping agent) with the AgNPs as summarized in Figure 2b, the similar results were reported by Awwad et al. 2013 [50].
The nano structures of the green synthesized AgNPs by B. buxifolia leaf extract have been studied through scanning electron microscopy (SEM) and transmission electron microscopy analysis which illustrated that AgNPs are mostly spherical in shape without aggregation of nanoparticles. A typical SEM and TEM image showing the size and morphology of the nanoparticles is given in Fig. 1c and 1d. The presence of chemical elements was analysed using an EDS study of 0 to 10. keV and showed the characteristic peaks for pure metal silver and the presence of 52.40 % silver was a clear indicator of the AgNP synthesis, which is shown in Fig. 1g. The average size of the colloidal AgNPs was measured by dynamic light scattering (DLS) detector. Fig. 1e shows that the average particles size was found to be 80 nm in diameter with poly-dispersed (pdi-0.243) in nature.
FTIR measurements were carried out in direction to pick out the presence of various functional group in green synthesized AgNPs using B. buxifolia. Fig. 1f

ABTS radical scavenging activity
Antioxidants are molecules that prevent cell damage by reacting with the free radicals and have proved critical in infection management of bacterial, fungal and viral disease as well as Cancer, HIV and in ammatory diseases in human. ABTS radical inhibition of leaf extract biomass and AgNPs using different concentration compared to standard (ascorbic acid) which was shown in (Fig 2) respectively. Although ascorbic acid used as a positive control in this manner, it has been showed the highest antiradical action. Thus, AgNPs show tremendous free radical activity in signi cantly with expanding concentration in the range of 25-150 µg/ml when compared with standard and leaf extract biomass. The greatest free radical-scavenger activity was represented the green synthesised AgNPs, standard and leaf extract biomass estimations of 95.65 ±1.6%, 89.57±3.1 % and 70.85±1.5 % separately. Our study strongly agreement with earlier studies, that the ability of AgNPs synthesized using leaf extracts prepared from E. scaber and P. granatum as good scavengers of free radicals [53,54] 3.3. Analysis of antibacterial activity of green synthesized Ag-NPs The antibacterial property of green synthesized AgNPs, leaf extract biomass and amikacin was tested against MDR strains such as E. coli and P. aeruginosa, S. enterica, Shigella spp. This was quanti ed by the agar well diffusion assay, wherein the zone of inhibition acquired by plating the organisms on a Muller-Hinton agar plate and performing the well diffusion test for various concentrations (25, 50and 100 µg/ml) for 24hr. The AgNPs, leaf extract biomass and amikacin are exhibited signi cant (ANOVA, P<0.05) antibacterial activity against tested pathogens at dose dependant manner and the results were summarized in Table 1. Overall, our results indicate that Silver nanoparticles have better antibacterial property than the commercial antibiotic and leaf extracts biomass. In addition, here we observed highest zone of inhibition present at maximum inhibitory concentration of AgNPs (100µg/ml) on S.enterica (25.40±0.83) followed by E. coli (21.6±1.10), Shigella spp (19.4±0.97) and P. aeruginosa (18.6±1.25). Furthermore, the minimum inhibitory concentration (25µg/ml) also shows better activity which are displayed in Table 1. Other than that, silver nanoparticles synthesised with aqueous extract of Rivina humilis leaves and Pistacia atlantica leaf extract were also strongly inhibited at low concentrations in the growth of all tested bacteria [55,56]. Also our results strongly committed with old reports, that the AgNPs exhibited more antibacterial activity than other nanoparticles because of their physical and chemical properties [57,58]. As well as some studies reports, that green synthesized AgNPs initiate continuous oxidative stress on bacterial cell wall, it may leads to bacterial cell death [59,60]. Therefore, our ndings suggest, that the green synthesized AgNPs from B. buxifolia and its antibacterial properties may be useful to food preservation technique, biomedical, cosmetic and agriculture eld.

Anti-bio lm and EPS inhibition activity of Ag-NPs
Bio lms inhibition of E. coli and P. aeruginosa, S. enterica, Shigella spp with different concentration of AgNPs were imaged by light microscopy. The samples were stained with crystal violet to differentiate between control (without AgNPs) and treated bio lm (with AgNPs). Violet colour indicates the presence of bacterial cells with compromised membranes, which is shown in Fig. 3. From this results here we observed, that bio lm inhibition and bio lm disruption or cell dispersion occurred at dose dependant manner. The AgNPs showed effective anti-bio lm activity towards the tested bio lm producers. From Fig.   4 (a, b, c, d), it was observed that all the concentrations of AgNPs showed good anti-bio lm activity, even at minimal concentration of 25μg/ml. Current results revealed the anti-bio lm activity of AgNPs on Salmonella was signi cantly higher than other tested bacteria (p < 0.05). In this case, the amount of bio lm formation was sharply decreased by increasing concentration (BIC) of AgNPs at 100μg/ml (96.1 ±1.37% inhibition) and (90.3 ± 1.1% inhibition) (86.7 ± 1.5% inhibition) (76.3 ± 1.2% inhibition) for E. coli, S. enterica, P. aeruginosa and Shigella spp. The toxicity of green synthesized silver nanoparticles against bacterial pathogen, may be due to the small size of nanoparticles, which penetrate into the cell wall, where they interfere with moulting and change the physiological processes [39].
The synthesized AgNPs were able to reduce the exo poly substances (EPS) of E. coli and P. aeruginosa, S. enterica, Shigella spp. The concentration of the AgNPs used to assess the EPS inhibition ranged from 25 μg -100 μg /ml. From Fig. 4 [61] reported, that silver nanoparticles had more toxic against bacterial cells and their extracellular substance.

Cytotoxicity/cell viability of AgNPs in MCF-7 cells
Breast cancer cell line (MCF-7) were exposed to AgNPs and Doxorubicin (DOX) at the concentrations of 0 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL and 100 μg/mL for 48 hours, and cytotoxicity was determined by MTT assay which are summarized in Fig 5 a. This results revealed, that % of cell viability was sharply decreased by increase in AgNPs and Dox concentration. While in case of AgNPs, the 50 % inhibition in breast cancer cell line was seen at 48 μg/mL for MCF-7 cell line. In addition, the IC 50 values for Dox were 37 μg/mL for MCF-7 cell line respectively. From these results, it was understood that the green synthesized AgNPs were less toxic, as well as similar anticancer activity compared with conventional drug. This results are strictly dealing with the earlier investigations [62]. In cancer cells, AgNPs cause reactive oxygen species to damage the cellular components that lead to cell death [63].
In comparison with untreated cells, morphological changes were observed in AgNPs (IC 50 and 100 g/mL) and DOX (IC 50 and 100 g/mL) treated MCF-7 cells. Cytoplasmic condensation, cell shrinkage, which is summarised in this study Fig 5b, was the most identi able morphological changes of AgNPs and DOXtreated cells seen in this study. In agreement with our ndings, recent research pointed out that AgNPs may attached to the membrane of cancer cells due to their electrostatic interaction and cause a process of pore formation on cell surface, cell shrinkage, membrane blabbing and deactivation of DNA and Mitochondria, that may ultimately lead to the cell death [64,65].

Photocatalytic activity of AgNPs
Methylene Blue is a highly toxic, synthetic dye that can essentially be used in the pharmaceutical, textile and dyeing industries. The effect of this poisonous compound can result in the destruction of equilibrium in water bodies and living systems. [66,67]. In this direction, current study examined photocataytic activity of AgNPs against MB. The experiment resulted in the execution of the following conditions: 50 ml of MB with 20 mg of AgNPs nanoparticle and this reaction mixture was kept in sunlight for various time intervals. During this reaction, the MB was photo degraded in the stepwise manner with the colour of the solution changing from a preliminary deep blue to almost transparent. The absorbance intensity decreases gradually with increasing of the contact times, which shows the photocatalytic degradation that was proven in Fig 6a. Methylene blue does not degrade under the irradiation of sun light without the use of AgNPs. Fig. 6b shows the amounts of MB degraded for various concentrations of AgNPs (2.5mg, 5mg, 10mg, 15mg, 20mg) and their % removals are 25, 56, 77, 82 and 83% respectively. In line with our results, it could be concluded that the green synthesized AgNPs had a dose dependent and time dependent manner degradation of MB. Our results strongly agree with previous reports that the green synthesized AgNPs have effective photocatalytic properties [15].

Conclusion
In conclusion, we have developed a simple and ultra-sonication intensi ed green approach closer the enhancement of a new technique for the synthesis of silver nanoparticles (Ag NPs). The formation of AgNPs starts quickly after the addition of AgNO 3 with B. buxifolia leaf extract with the aide of ultrasonic vibration. According to our knowledge, it is very rst time for synthesis of AgNPs using B. buxifolia via ultra-sonication process. The AgNPs have been accurately characterized by UV, XRD, SEM, TEM, DLS and FTIR analysis. Characterization fact showed that the AgNPs had been crystalline in nature and physical identi cation expose the spherical shape with size range at 80 nm. The green synthesized AgNPs exhibit strong antioxidant properties and enhanced antibacterial, anti-bio lm and EPS inhibition activity against MDR strains such as E. coli, S. enterica, P. aeruginosa and Shigella spp. Additionally good anticancer impact towards breast cancer cell lines (MCF-7) and less toxic compared to commercial drug (Doxorubicin). As well as the MB is successfully degraded by AgNPs act as photocatalyst under the sun light condition. From our studies, we stated that a simple, unhazardous, cost-less, eco-friendliness is some promising points of the ultrasonic aided green process, and this method reduces the time to make the nano crystals without agglomeration. Therefore, we believe that our ndings make a valuable contribution to different elds such as food preservation, pharmaceutical, agricultural and textile industries. Declarations