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www.nature.com/scientificreports/ Scientific RepoRtS | Vol:.(1234567890) Figure 1. Standardization of the ethyl acetate fraction, (a) UPLC Chromatogram of the ethyl acetate fraction, (b) UV spectrum of the peak at Rt = 19.4 min, (c) UPLC Chromatogram of the isolated ellagic acid at different concentrations, (d) Calibration curve of the isolated ellagic acid. Punica granatum (Pomegranate) is a plant whose fruits are largely consumed all over the world. It is well known for its richness in polyphenols15. Apart from the fruits, other plant parts such as peels, leaves, flowers are included in different food preparations like beverage tea, jams, jellies, sauces and salad dressings16. Different plant parts like seeds, peels and leaves were reported before in the green synthesis of AgNPs17–20. The present study aimed at profiling the polyphenols of pomegranate leaves (PL) grown in Egypt and proving their role in the green synthesis process of AgNPs. This incorporates extraction, fractionation, standardization, and polyphenols profiling of PL extract adopting UPLC-PDA-UV and LC–MS–MS methods. Besides, isolation and structural elucidation of the main polyphenol component. Moreover, facile green synthesis of AgNPs with the aid of pome- granate polyphenols, characterization, and investigation of its antimicrobial activity. Results Extraction and standardization of the extract. One of the main goals of this study is to prepare a standardized polyphenols rich fraction from the aqueous alcohol extract of pomegranate leaves (PL). The spec- trophotometric assay of the total polyphenols and flavonoids contents showed that ethyl acetate fraction was the richest fraction in polyphenols and flavonoids with 586.25 mg gallic acid equivalent (GAE)/g fraction and 194.67 mg rutin equivalent (RE)/g fraction, followed by the crude extract with 396.75 mg GAE/g extract and 125.60 mg RE/g extract, while the least amount was found in n-butanol fraction with 353.00 mg GAE/g fraction and 91.30 mg RE/g fraction. The ethyl acetate fraction was subjected to automated flash chromatography to isolate its major phenolic compound, that was isolated as buff colored powder (342 mg) with 1H-NMR (DMSO-d6, 400 MHz, δ = ppm), 7.47 (2H, s, H-5 and H-5′), and 13C-NMR (DMSO-d6, 100 MHz, δ = ppm), 112.80 (C1), 136.84 (C2), 140.17 (C3), 148.60 (C4), 110.65 (C5), 108.03 (C6), and 159.64 (C7). The UV–Visible spectrum of that compound exhibited λmax at 253.7 nm and 367.5 nm. The received spectral data were consistent with those reported for ellagic acid21. The ethyl acetate faction was standardized by UPLC technique, the total ion current chromatogram of the fraction was recorded at 370 nm (Fig. 1a), that showed a major peak (44.54%) at Rt = 19.4 min, with UV spectrum, that exhibited λmax at 253.5 nm and 367.3 nm (Fig. 1b) that was identified as ellagic acid by co-chromatography with the previously isolated compound. A calibration curve of the isolated ellagic acid was established on the same UPLC device with the same conditions and parameters (Fig. 1c). The curve equation was found to be (2020) 10:14851 | https://doi.org/10.1038/s41598-020-71847-5 2PDF Image | pomegranate leaves and their role in green silver nanoparticles
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