3.1. Ultra-sonic intensified synthesis and characterization of AgNPs using B. buxifolia leaf extract:
The present study integrates ultra-sound intensified 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 Ag0, 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. Herein, we observed the crystalline peaks at 2Ө= 38.21°, 44.38, 64.56° and 77.39°, these peaks are attributed to the (111), (200), (220) and (311) crystallographic planes. Those peaks are consistent with the JCPDS card No 65-2871of the AgNPs. Also our results strongly agreement with previous reports, that the sharp diffraction peaks with negligible noise verify the high quality of crystallinity that attributed to face-centered cubic (fcc) spinel structure of AgNPs [48, 49]. In this manner, the normal grain size of the AgNPs was computed from the solid reflection 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 shows absorption bands at 1348.20, 1382.96, 1593.20, 2360.67, 2725.42, 2810.28, 2926.06 and 3410.15 cm-1 specifying the presence of plant extract in AgNPs. The broad shift present at 3410.15 cm-1in the spectra agrees to O-H stretching vibration showing the presence of phenols and alcohol. In addition, the band at 1593.20 cm-1 corresponds to amide C=O stretching and some other peaks located at 2926.06, 1348.20, and 1382.96cm-1respectively, which are agree to C-H stretch (alkanes); C=C stretch (aromatic ring); C-H (aromatics) suggesting, that presence of phytochemicals. FT-IR results revealed that alcoholic, phenols, aromatic and amine groups may act as a capping and stabilizing agent of Ag+ ions to AgNPs [29, 51, 52].
3.2. 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 inflammatory 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 significantly 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 quantified 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 significant (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 findings suggest, that the green synthesized AgNPs from B. buxifolia and its antibacterial properties may be useful to food preservation technique, biomedical, cosmetic and agriculture field.
3.4. Anti-biofilm and EPS inhibition activity of Ag-NPs
Biofilms 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 biofilm (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 biofilm inhibition and biofilm disruption or cell dispersion occurred at dose dependant manner. The AgNPs showed effective anti-biofilm activity towards the tested biofilm producers. From Fig. 4 (a, b, c, d), it was observed that all the concentrations of AgNPs showed good anti-biofilm activity, even at minimal concentration of 25μg/ml. Current results revealed the anti-biofilm activity of AgNPs on Salmonella was significantly higher than other tested bacteria (p < 0.05). In this case, the amount of biofilm 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 (a1, b1, c1, d1), it was observed that all the concentrations of AgNPs showed significant reduction of EPS production. The test AgNPs at 100 µg/ml exhibited 98.4±1.4%, 95.3±1.0%, 91.7±1.4 and 84.6% decrease in EPS production of E. coli, S. enterica, P. aeruginosa and Shigella spp. This result also indicates the disruption of biofilm architecture. Our results correlated with Park et al. 2013 [61] reported, that silver nanoparticles had more toxic against bacterial cells and their extracellular substance.
3.5. 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 IC50 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 (IC50 and 100 g/mL) and DOX (IC50 and 100 g/mL) treated MCF-7 cells. Cytoplasmic condensation, cell shrinkage, which is summarised in this study Fig 5b, was the most identifiable morphological changes of AgNPs and DOX-treated cells seen in this study. In agreement with our findings, 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].
3.6. 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].