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www.nature.com/scientificreports/ Organism Inhibition zone diameter (mm) Silver nanoparticles Ethyl acetate fraction 0.45 mg/100 μL Silver nitrate solution 0.45 mg/100 μL 0.05 mg/100 μL 0.15 mg/100 μL 0.25 mg/100 μL 0.35 mg/100 μL 0.45 mg/100 μL Bacillus subtilis Staphylococcus aureus Sarcina lutea Salmonella paratyphi Escherichia coli Pseudomonas aeruginosa Candida albicans 12 16 18 11 13 14 13 16 17 10 13 15 12 13 14 11 11 12 0 11 14 FTIR analysis. FTIR analysis was carried out to determine the important functional groups on the surface of the nanoparticles (Fig. 3a). IR spectrum showed strong peaks at 1635 cm−1 and 3,300 cm−1 corresponding to presence of C=C and phenolic –OH respectively. Hence, the obtained results indicate involvement of pome- granate phenolic compounds in the reduction of silver ions and stabilization of the size and shape of the nano- particles. Particle size analysis. The average diameter of the synthesized nanoparticles was determined by light scattering technique and found to be 26.22 ± 0.63 nm with PDI 0.189 (Fig. 3b). XRD analysis. The crystalline nature of the AgNPs was confirmed by X-ray crystallography. The XRD pattern of the synthesized AgNPs is shown in (Fig. 3c). The diffracted intensities were recorded from 2θ of 4° to 2θ of 80°. The XRD pattern showed four strong Bragg reflections at 37.84°, 44.00°, 64.20° and 77.17°, with relative intensities of 100%, 32.26%, 27.28% and 26.63% respectively. Those four peaks correspond to the planes of (1 1 1), (2 0 0), (2 2 0) and (3 1 1) respectively, which could be indexed according to the facets of face centred cubic crystal structure of silver23. Moreover, the nature of crystals was confirmed to be silver by the interplanar spacing (dcalculated) values of 2.378, 2.056, 1.451 and 1.236 Å for (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes respectively that are matched with standard silver values. The average crystalline size is calculated using Debye–Scherrer formula, k β cos θ where D is the average crystalline size of the nanoparticles, k is the geometric factor (= 0.9), λis the wavelength of the X-ray radiation source in Å and β is the angular full-width at half maximum (FWHM) in Rad of the XRD peak at the diffraction angle θ. The calculated average crystalline size of the AgNPs is ~ 32 nm. Scanning electron microscopy. The SEM analysis was performed to determine the exact shape and size of the synthesized nanoparticles (Fig. 3). The figures show that the average diameter of the particles was from 20 to 40 nm, and the shape was nearly polygonal. Size range and shape distribution was nearly equal all over the sam- ple (Fig. 3e). The high magnification photo (Fig. 3f) illustrated the capping of bioactive compounds from the ethyl acetate fraction on the surface of the nanoparticles. Antimicrobial activity. The antimicrobial activity of AgNPs, polyphenols rich fraction and silver nitrate were investigated. The results shown in table (3) revealed that the fraction exhibited antimicrobial activity against Bacillus subtilis, Staphylococcus aureus and Sarcina lutea, while other microorganisms were resistant to the frac- tion, the highest activity was against Sarcina lutea with inhibition zone diameter 34 mm. The highest antimicro- bial activity of AgNPs was found to be against Bacillus subtilis, the rest of tested microorganisms have more or less equal sensitivity to the synthesized AgNPs with a concentration dependent manner. Silver nitrate solution showed the highest antimicrobial activity against all the tested microorganisms except Sarcina lutea. Discussion Currently, green synthesis is gaining a great interest in the production of metal nanoparticles, due to their ease of synthesis, eco and environmentally friendly, and utilization of plants waste products24. Aqueous extract of PL was found to have a good potential in the production of AgNPs with anticancer activity on human cervical cancer cells, liver cancer cells (HepG2) and antidiabetic activity18,20,25. Based on the reported data, there was a need for preparation of a standardized phytochemically analysed fraction and correlate that action with its components. To achieve that goal, the aqueous alcohol extract of PL was fractionated using n-hexane, methylene chloride, ethyl acetate and n-butanol. The crude extract as well as the different fractions at different concentrations were preliminary tested for their ability to synthesize AgNPs with good characteristics. The comparison between the outcome of each experiment was based on its SPR band as the best nanoparticles would have the lowest λmax with the highest intensity22. Moreover, spectrophotometric determination of total (2020) 10:14851 | https://doi.org/10.1038/s41598-020-71847-5 5 Vol.:(0123456789) 20 23 19 31 15 17 22 23 19 19 34 27 15 18 0 20 16 18 0 22 15 16 0 22 16 17 0 27 Antimicrobial activity of the synthesized silver nanoparticles at different concentrations, ethyl acetate fraction and silver nitrate solution, tested by well diffusion method. Table 3. D= Scientific RepoRtS |PDF Image | pomegranate leaves and their role in green silver nanoparticles
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