2.1 Chemicals, Endophytic Bacteria and Microbial strains
Analytical grade Silver nitrate (AgNO3), tetracycline, kanamycin, nutrient agar, potato dextrose agar, Mueller-Hinton agar (Himedia Lab, Ltd., Mumbai, India) were used in the present study. The endophytic bacteria Pantoea anthophila (GenBank accession no. MN077163) identified earlier by 16S ribosomal RNA gene sequencing from W. indica were pure cultured and maintained in the laboratory. Bacterial pathogens such as Gram-positive Staphylococcus epidermidis (MTCC737), Bacillus subtilis (MTCC1133), Staphylococcus aureus (MTCC2940), Gram-negative Escherichia coli (MTCC40), Proteus mirabilis (MTCC425), Salmonella typhi (MTCC733), Klebsiella pneumoniae (MTCC2405) and fungal strains of Aspergillus niger (MTCC404), Candida albicans (MTCC183) and Penicillium chrysogenum (MTCC947) were obtained from Microbial Type Culture Collection (MTCC), Chandigarh.
2.2 Preparation of cell-free endophytic culture
P. anthophila (Pa) isolated from W. indica are subcultured and maintained in nutrient agar plates. A loopful of culture is inoculated in 100ml of Luria–Bertani broth at 37⁰C and placed in an orbital shaker at 200 rpm overnight. The cell-free extract was collected by centrifuging in a high-speed centrifuge (RM-03 Plus) (10,000rpm for 10min) and used immediately for AgNP synthesis.
2.3 Biosynthesis of Silver Nanoparticles
The 10 ml of bacterial cell free supernatant was mixed with 90ml of 1mM AgNO3 solution for the biosynthesis of silver nanoparticles. The reaction mixture was incubated at 30°C for 24 hours in dark to avoid any photochemical reactions. Simultaneously, the cell-free supernatant without silver nitrate was maintained as control. Formation of AgNPs was monitored at regular time intervals. Silver nanoparticles (Pa-AgNPs) formed were separated by centrifuging at 10,000rpm for 15 min. The water-soluble biological molecules, and other impurities are removed by washing the samples repeatedly (×3) with deionized water. The final mass of the AgNPs was collected and freeze-dried.
2.4 Biophysical characterization of Pa-AgNPs
The Pa-AgNPs are further characterized by UV-Visible spectroscopy, Dynamic Light scattering analysis (DLS), Zeta potential, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy Dispersive of X-Ray spectrum (EDX).
The formation of AgNPs was confirmed by measuring the absorption spectra in UV-Visible spectroscopy (SHIMADZU 1800). The reaction mixture was scanned at the speed of 300 nm min−1 in 200–600 nm range. DLS analysis determines the particle size and distribution pattern of Pa-AgNP. Zeta sizer (Malvern Instruments Ltd., U.K ZS90) was used to determine the surface charge potential, the magnitude of charge attraction or repulsion and electrophoretic stability of Pa-AgNP.
The FTIR analysis was carried out to identify the functional biomolecules in the endophytic extract that reduces Ag+ to AgNPs and the capping agents that stabilize the nanoparticles. The Pa-AgNPs is scanned at the transmission mode ranging from 500 to 4000 cm-1 in a solid phase, at a resolution of 1 cm−1 using FTIR spectrophotometer (SHIMADZU, IR PRESTIGE 21). XRD pattern provides the details of the structure and composition of Pa-AgNPs. The sample was analyzed by Shimadzu XRD 6000 diffractometer operated at 40kV voltage, 30mA current in a scanning mode range of θ-2θ between 10 to 80 degrees with sampling pitch of 0.1000 degree equipped with a Cu Kα radiation.
The crystallite structure of the Pa-AgNP was measured using TEM (JEOL JEM 2100). The sample was placed on copper grids coated carbon films, dried at room temperature and analysed at 200kV. SEM shows the size, shape and surface morphological properties of Pa-AgNP synthesized from the endophytic extract. The nanoparticle was observed in SEM (JEOL– JSM 6390) at a voltage of 15–20kV at different magnifications. The atomic composition of Pa-AgNP was confirmed by EDX analysis (Oxford instrument, INCA PentaFET x3) coupled with SEM.
2.5 Antimicrobial Screening of Pa-AgNPs
The antimicrobial activities of the synthesized Pa-AgNPs were analysed by Agar well diffusion method [22]. The microbial strains S. epidermidis, B. subtilis, S. aureus, E. coli, P. mirabilis, S. typhi, K. pneumonia at exponential growth phase were cultured in Muller Hinton agar plates. Six wells of about 5mm diameter were bored in each agar plates. Different concentrations of Pa-AgNPs (25µg/mL, 50µg/mL, 75µg/mL, 100µg/mL), 10µg/mL of dimethyl sulfoxide (DMSO) (negative control) and Tetracycline (positive control) were added to the wells.
The fungal strains A. niger, C. albicans and P. chrysogenum in potato dextrose agar are utilised for antifungal activity testing of Pa-AgNPs by agar well diffusion method, where Kanamycin (10µg/mL) is used as a positive control. The plates were incubated for 24 hours at 37°C to observe the clear zones of inhibition (ZOI). The diameter of clear zones was measured and recorded.
2.6 Antioxidant efficacy of Pa-AgNPs
2.6.1 1, 1-diphenyl-2- picrylhydrazyl (DPPH) radical scavenging assay
DPPH radical scavenging assay potential of Pa-AgNPs was assayed [23] at different concentrations (10, 20, 30, 40 & 50 μg/ml) of Pa-AgNPs and standard Ascorbic acid. In methanol solution, dissolved 50μl of 0.659 mM DPPH, added to the samples and the volume is made up to one with double distilled water. The tubes were incubated in dark at 25ºC for 20 minutes and the absorbance was recorded at 510 nm using Shimadzu UV 1800 spectrophotometer. The % inhibition (I%) was calculated as (I%) = 100 x (A0-A1)/A0, Where A0 is the absorbance of the control, A1 is the absorbance of the Pa-AgNPs and standard.
2.6.2 Nitric oxide radical scavenging assay
At physiological pH, in aqueous solution, sodium nitroprusside generates nitric oxide that interacts with oxygen to produce nitrite ions, which can be measured in the presence of Griess reagent [23]. To various concentrations (10-50μg/ml) of Pa-AgNPs and standard ascorbic acid, added 50μl of 10mM sodium nitroprusside dissolved in 0.5M phosphate buffer (pH 7.4) and incubated under fluorescent light at room temperature for 15 minutes. Added 125μl of Griess reagent, the tubes were incubated again at room temperature for 10 minutes and the absorbance was recorded at 546nm.
2.6.3 Hydrogen peroxide (H2O2) scavenging assay
The ability of the Pa-AgNPs to scavenge H2O2 was determined according to the method of Nabavi et al. [24]. 0.6ml of 40mM of H2O2 was prepared using 50mM phosphate buffer (pH 7.4) and added to varied concentrations (10-50μg/ml) of Pa-AgNPs and standard ascorbic acid. The tubes were incubated for 10 minutes and the absorbance was noted at 230 nm.
2.6.4 Total antioxidant capacity assay
The total antioxidant capacity assay was determined [25] by adding 1ml of reagent solution containing sulphuric acid (0.6M), sodium phosphate (28mM) and Ammonium molybdate (4mM) to Pa-AgNPs and standard ascorbic acid (10-50 μg/ml). The tubes were capped, incubated at 95ºC for 90 minutes in a thermal block and then cooled to room temperature, the absorbance was measured at 695 nm.
2.6.5 2,2’-Azino-bis-3-ethyl benzothiozoline-6-sulfonic acid (ABTS) radical scavenging activity
The assay is based on the scavenging of light by ABTS radicals. An antioxidant that donates a hydrogen atom will quench the stable free radical which can be quantified spectrometrically at 734nm [23]. 200μl of 70mM potassium persulphate and 50ml of 2mM ABTS were mixed before 2hours. To the 0.5ml of various concentrations (10-50μg/ml) of Pa-AgNPs and standard ascorbic acid, 0.3ml of ABTS radical cation and 1.7ml of phosphate buffer (pH 7.4) was added and the absorbance was measured.
2.6.6 Reducing Power Assay
Different concentrations (10-50 μg/ml) of Pa-AgNPs solution were mixed with 2.5ml of 200mM phosphate buffer (pH 6.6) and 1% potassium ferricyanide each. Incubated at 50ºC for 20 min, the mixture was cooled rapidly. Subsequently, added 2.5ml of 10% Trichloroacetic acid and centrifuged at 3000 rpm for 10 min. The supernatant (5ml) with an equal amount of distilled water were mixed and added to 1ml of 0.1% ferric chloride. Using ascorbic acid as a standard, the absorbance was measured at 700 nm [26].
2.6.7 Superoxide (O2-) radical Scavenging Assay
The superoxide scavenging activity of the Pa-AgNPs was assayed by the reduction of nitro blue tetrazolium (NBT) [27]. To Pa-AgNPs solution (10-50μg/ml), 3ml Tris–HCl buffer (16 mM, pH 8), 1ml NBT (50μM), 1ml Nicotinamide Adenine Dinucleotide (78μM) and 1ml phenazine methosulfate (PMS) solution (10μM) were mixed and kept for 5 min at 25°C. The absorbance was recorded at 560 nm. Ascorbic acid was used as standard.
Inhibition % versus concentration curve was plotted for each assay and the concentration of sample required for 50% inhibition was determined and expressed as IC50 value. The lower IC50 value indicates a high antioxidant capacity.