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www.nature.com/scientificreports/ Scientific RepoRtS | Vol:.(1234567890) 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, meth- ylene 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 fla- vonoids 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 yellow- ish 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 chromatog- raphy (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 tan- dem 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 con- tinuous 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., Min- neapolis, 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 (2020) 10:14851 | https://doi.org/10.1038/s41598-020-71847-5 8PDF Image | pomegranate leaves and their role in green silver nanoparticles
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