All solvents were of HPLC grade, while those used in LC–MS–MS analysis were of MS grade. Rutin, gallic acid, and silver nitrate were purchased from Sigma-Aldrich (Schnelldorf, Germany).
Punica granatum leaves were collected from the botanical garden of Cairo University. Identity was verified by Prof. Mohammed El-Sayed, Horticultural Research Institute, Agriculture Research Centre, Ministry of Agriculture, Giza. A voucher specimen (No. PG002) has been deposited in the herbarium of the Faculty of Pharmacy, BUE, Cairo, Egypt.
Preparation of extract and fractions
Five hundred gram of PL were extracted by maceration in 1 L of 70% ethanol for 48 h at room temperature, followed by filtration. The extraction process was repeated 3 times. The extracts were collected and dried under reduced pressure at 45 °C. The dried hydroalcoholic extract was suspended in distilled water and fractionated using solvents of increasing polarities, which are n-hexane, methylene chloride, ethyl acetate and n-butanol. Fractions were dried to yield 0.45 g, 9 g, 12 g and 10.5 g respectively.
Spectrophotometric analysis of total polyphenols and flavonoids contents
Total polyphenols and flavonoids contents of ethyl acetate and n-butanol fractions in comparison to the crude extract were assayed using Folin-Ciocalteu method and aluminium chloride method respectively, that were reported by Attard49 and Herald et al.50, utilizing the microplate reader Fluostar Omega (BMG Labtech, Germany). Gallic acid and rutin were used as standards to calculate the total polyphenols content (mg GAE/g extract or fraction) and total flavonoids content (mg RE/g extract or fraction). The range of gallic acid concentrations to establish a calibration curve was from 7.8 to 500 μg/mL, while that of rutin was from 50 to 1,000 μg/mL.
Isolation and identification of the major compound
The major phenolic compound in the PL extract was isolated by automated flash chromatography technique (Puriflash 4100 system—Interchim; Montlucon, France) with PDA–UV–Visible detector 190–840 nm and equipped with Puriflash column 30 C18 HP (20 bar). For system controlling and process monitoring, Interchim Software 5.0 was used. 10 g of the polyphenols rich fraction was dissolved in 50 mL of ethanol, then introduced into the column via dry loading technique using 10 g celite gel. Mobile phase was composed of 0.1% formic acid in water (Solvent A), and acetonitrile (Solvent B). The total run was for 210 min, and the gradient program was; 0–15 min (5–10% B), 15–135 min (10% B), 135–165 min (25% B), 165–180 min (45% B) and 180–210 min (100% B). The flow rate was 30 mL/min, and totally, 300 fractions were collected, each was with volume of 20 mL. The compound was precipitated as yellowish buff crystals from the fractions 20–80. The structure of the compound was elucidated by 1H and 13C NMR analyses that were recorded by Bruker Avance III HD FT-high resolution, Geramany- 1H-NMR (400 MHz), 13C-NMR (100 MHz) at Faculty of Pharmacy-Mansoura University.
Standardization of the polyphenols rich fraction using Ultra performance liquid chromatography (UPLC) analysis
The polyphenols rich fraction was standardized. The experiment was carried out on Thermo Fisher UPLC Model Ultimate 3,000 (USA), equipped with PDA–UV–Visible light detector, on a column Hypersil GOLD (250 mm × 4.6 mm i.d.) and particle size 5 µm. Mobile phase was composed of 0.1% phosphoric acid in water as solvent A, and acetonitrile as solvent B, with constant flow rate at 0.7 mL/min. The gradient program was, 0–7 min (5–15% B), 7–10 min (15% B), 10–22 min (15–35% B), 22–35 min (35–100% B) and 35–40 min (100–5% B). Injection volume was 20 μL and column oven temperature was 30 °C. A calibration curve of the isolated major phenolic compound was established with concentrations range (31.25–1,000 μg/mL) and ethyl acetate fraction concentration was 5 mg/mL. All analyses were carried out in triplicate.
LC–MS–MS analysis of the polyphenols rich fraction
The parameters of the LC–MS analysis were adjusted according to the method developed by Alfaifi et al.51. In details, the chromatographic separation was carried on Waters Acquity Xevo TQD system, on column Acquity BEH C18 100 mm × 2.1 mm column (p.s., 1.7 µm) (Waters, Ireland). Absolute ethanol was used to solubilize the sample at concentration of 1 mg/mL and filtered through a micropore filter of size 0.2 µm, the injection volume was 10 μL. The mobile phase system composed of Solvent A (0.1% formic acid in water) and Solvent B (0.1% formic acid in acetonitrile) at flow rate 200 μL/min with gradient elution: 0–4 min (15% B), 4–8 min (20% B), 8–30 min (55% B), 30–35 min (90% B) and 35–40 min (15% B). The high resolution mass spectra were recorded by Xevo TM triple-quadrupole tandem mass spectrometer with electrospray ionization (ESI) interface (Waters Corp., Milford, MA, USA) at mass ranges from 100 to 1,000 m/z, capillary voltage, 3.5 kV; detection at cone voltages, 20 V—95 V; radio frequency (RF) lens voltage, 2.5 V; source temperature,150 °C and desolvation gas temperature, 500 °C. Nitrogen was used as desolvation and cone gas at a flow rate of 1,000 and 20 L/h, respectively. System operation and data acquisition were controlled using Mass Lynx 4.1 software (Waters) .
Green synthesis of AgNPs
AgNPs were synthesized by reduction of silver ions (Ag+) of silver nitrate solution to silver metal (Ago). Briefly, 250 mL of silver nitrate solution at varying concentrations (1–5 mM) were heated to a temperature ranging from 60 to 80 °C, followed by addition of 50 mL of the plant material solution (hydroalcoholic extract, ethyl acetate fraction, or n-butanol fraction) at concentrations 0.5–5 mg/mL, with continuous stirring at 1,500 rpm for 1 hAfter that, AgNPs were pelleted by centrifugation at 10,000×g for 90 min at 4 °C, then washed thrice by deionized water, lyophilized and stored at − 18 °C for further analysis.
Systematic optimization of the green synthesis process
Box-Behnken design was used to optimize the green synthesis process for production of AgNPs with the aid of Design Expert ver. 11.0 (Stat-Ease Inc., Minneapolis, USA). The independent variables for the optimization process were silver nitrate concertation, plant material concentration and temperature. Each variable was tested at three different levels; low (− 1), medium (0), and high (+ 1). A total of 14 trials were suggested by the selected design. Particle size and Polydispersity index (PDI) were analyzed as responses. After feeding the data in the design, mathematical modelling was carried out for analysis of results. Two factor interaction (2FI) process order was the best fitting model for the particle size response, while quadratic second order model was the suggested fitting model for the PDI response. After that the results were analyzed by ANOVA. The optimum conditions were identified by graphical optimization techniques and numerical desirability function.
Characterization of AgNPs
UV–Visible spectral analysis
Surface plasmon resonance (SPR) bands of AgNPs were recorded on UV–Visible spectrophotometer device (Jasco-V630, Jasco Inc., MD, USA) at different time intervals during the green synthesis process. The wavelengths range of the spectral analysis was from 300 to 700 nm.
Fourier transform infrared spectroscopy (FTIR)
FTIR spectroscopy analysis for AgNPs was carried out on Vertex 70 FTIR, Brucker (USA). A thin film of the sample was allowed to form on KBr pellet and spectra were recorded.
Particle size analysis by light scattering technique
The mean particle size (diameter) and size distribution (PDI) of the synthesized nanoparticles were analysed with dynamic light scattering measurements at a scattering angle of 173°. Measurements were performed using a Zetasizer Nano ZS (Malvern Instruments, UK), where the dried nanoparticles were reconstituted in deionized water.
X-ray diffraction (XRD)
XRD measurement was carried out on X-ray diffractometer (Empyrean—Malvern Panalytical—Netherland), at X-ray power 40 kV and 30 mA and the spectrum was recorded by CuKα radiation with wavelength of 1.5406 Å in the 2θ range of 4°–80°, with a continuous scan type, step size (2θ) was 0.0200 and scan step time was 0.5 s.
Scanning electron microscopy (SEM)
Field emission SEM (Quattro S, Thermo scientific, USA) was used for the purpose of imaging of the synthesized AgNPs in order to study their shape and size. A small amount of AgNPs were placed on carbon coated copper grid. Then images were recorded at a magnifications × 160,000, × 480,000 and × 960,000.
Antimicrobial activity of the prepared AgNPs
Antimicrobial activity of the AgNPs were analysed by well diffusion method that was reported by Jyoti et al.23 as their AgNPs were synthesized using a plant extract and the particle sizes range was nearly the same as our findings. The analysis was carried out against G + ve bacteria (Bacillus subtilis, Staphylococcus aureus, and Sarcina lutea), G -ve bacteria (Salmonella paratyphi, Escherichia coli, and Pseudomonas aeruginosa) and fungi (Candida albicans). In each plate, five different concentrations of AgNPs were tested (0.05, 0.15, 0.25, 0.35 and 0.45 mg/100 µL), ethyl acetate fraction (0.45 mg/100 µL) and silver nitrate (0.45 mg/100 µL). The plates were incubated for 24 h at 37 °C, then the inhibition zone diameters were recorded.