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www.nature.com/scientificreports/ Scientific RepoRtS | the capability of reducing silver ions, resulting in the formation of AgNPs, further, the AgNPs in the presence of ellagic acid were highly effective as antibacterial agents, this finding support our results30. From the optimization 3D plot diagrams shown in (Fig. 2), it was deduced that there is an increase in the particle size in correlation to the increase in concentration of the ethyl acetate fraction and/or silver nitrate, while there is an inverse correlation with temperature. It was reported that increasing in the silver nitrate concentra- tion increases the particle size, as they were easy to aggregate into larger particles, due to the rise of the collision frequency with an increasing of silver salt content31. Moreover, the temperature was found to have a significant effect on the formation, shape, size and size distribution of particles32. Liu et al. (2017) found that the size of AgNPs was slightly increased from 70 to 80 °C, while it decreased sharply from 80 to 90 °C, they concluded that the decrease of size at high temperature is a result from the sharply increased nucleation rate constant instead of the decreased growth rate constant33. Although, the statistical model of the PDI was found to be not significant, it is obvious from the diagrams (Fig. 2d–f) that PDI has an optimum range of temperature to be minimized, less or more than that range, PDI will increase. A characterization profile was carried out for the AgNPs obtained from the optimized experiment. Particle size was analysed by light scattering technique and SEM, the results were relatively in the same range and in consistence with the theoretically calculated particle size from the polynomial equation obtained after data mod- elling and the Debye–Scherrer formula obtained from the XRD analysis. In the current study, the particle size of the synthesized AgNPs by the aid of the polyphenols rich fraction was found to be relatively smaller than those reported before in literature which obtained from crude aqueous extracts of different organs of pomegranate34, the range of particle sizes of AgNPs prepared by crude aqueous extract of pomegranate leaves was found to be 35–70 nm18,20. The UV–Visible spectral analysis showed λmax in the range that is similar to those reported before for AgNPs synthesized by pomegranate leaves18,20. Similar type of analysis with nearly the same range of results was carried out before in recent studies of synthesis of AgNPs using extracts of different plants, such as Allium cepa (onion), Parkia speciose, and Salvia hispanica35–37. FTIR analysis confirmed the role of pomegranate phytochemicals as reducing and stabilizing agents for the AgNPs. The spectrum revealed presence of C=C stretching vibrations of the phenolic compounds and phenolic hydroxyl groups. Similar findings were obtained from the AgNPs prepared by Diospyros lotus and Thunbergia grandiflora extracts38,39. As mentioned before, ellagic acid is the major compound in the PL, it was reported that ellagic acid which is the major antioxidant component in the combined peels extract of pomegranate, orange, banana and apple is responsible for the synthesis of AgNPs, as it has an easy electron loosing capacity which results in formation of H+ radical, that reduces the size of silver to nano size40. XRD analysis confirmed the cubic nature of the AgNPs (Joint Committee on Powder Diffraction Standards; JCPDS no. 04-0783), the results are consistent with previous studies, reporting similar diffraction peaks20,23. Size and nature of AgNPs was confirmed by the high magnification using SEM technique, which also confirmed presence of an organic shell around the nanoparticles. Antimicrobial activities of the synthesized AgNPs, polyphenols rich fraction and silver nitrate solution were tested against different Gram positive, Gram negative bacteria, and fungi. The majority of the tested microor- ganisms had resistance to the fraction. On the other hand, AgNPs showed antimicrobial activity against all the tested microorganisms. As silver nitrate solution exhibited strong antimicrobial activity in contrast to the frac- tion, that could rationalize the antimicrobial characters of the AgNPs by presence of silver metal rather than the coating organic material. The antimicrobial activity of silver metal is reported for a wide range of over 650 microorganisms from different classes such as viruses, gram positive and gram negative bacterial, and fungi41. The bactericidal effect of silver nitrate was reported before42,43, it was found that at lower concentrations it induced synthesis of AgNPs, whereas at higher concentrations it induced cell death44. The higher antimicrobial activity of silver nitrate solution than AgNPs against all tested bacteria is highly supported with those reported by Li et al.45 and Kedziora et al.46. It was reported that different extracts of PL had moderate antimicrobial activity against various human pathogens47. Pomegranate peels extract was also found to have in-vitro and in-vivo antibacterial activity against different strains of Salmonella48. In conclusion, in this study twenty three polyphenolic compounds were tentatively identified by LC–MS–MS techniques in ethyl acetate fraction. This fraction was standardized by UPLC–PDA–UV method with reference to its major compound ellagic acid. AgNPs were biosynthesized using polyphenol rich fraction of PL in a sim- ple one step process. The production of AgNPs was correlated to the phenolic content of PL and FTIR results confirmed this finding. Systematic optimization of AgNPs using PL proved the direct correlation between the particle size with silver nitrate and fraction concentrations, and inverse correlation with the temperature. AgNPs were found to have an average size of 26.22 nm size, polygonal structure, absorbance maxima at 425 nm and the presence of biocompatible capping over it. The antibacterial activity of the synthesized AgNPs on tested organ- isms further confirms the activity to the silver metal rather than the polyphenols adsorbed on the surface of the AgNPs. Finally, pomegranate polyphenols could provide promising opportunities in silver nanoparticles industry. Material and methods chemicals. 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). plant material. Punica granatum leaves were collected from the botanical garden of Cairo University. Iden- tity was verified by Prof. Mohammed El-Sayed, Horticultural Research Institute, Agriculture Research Centre, (2020) 10:14851 | https://doi.org/10.1038/s41598-020-71847-5 7 Vol.:(0123456789)PDF Image | pomegranate leaves and their role in green silver nanoparticles
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