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www.nature.com/scientificreports/ Peak Rt (min) Identified compounds UV–Vis (λmax) [M−H]− (m/z) Fragment ions (m/z) Percentage (%) Refs 1 3.4 2 4.2 3 4.8 4 4.82 5 5.3 6 5.6 7 5.9 8 6.6 9 6.9 10 7.3 11 7.67 12 9.7 13 10.5 14 15.3 15 25.7 16 26.2 17 27.3 18 39.2 19 39.3 20 39.8 21 40.58 22 41.32 23 43.32 Galloyl-HHDP-hexoside Galloyl-HHDP-DHHDP- hexoside (Granatin B) Gallic acid Castalagin derivative Bis-HHDP-hexoside Digalloyl-HHDP-hexoside Cyanidin pentoside HHDP-hexoside Ellagic acid Procyanidin dimer Galloyl hexoside Dimethyl ellagic acid glucu- ronide Ellagic acid deoxyhexoside Ellagic acid dihexoside Ellagic acid hexoside Feruloyl coniferin Galloyl-HHDP-gluconate Coumaroyl hexose Caffeoyl quinic acid Dicaffeoyl quinic acid Pelargonidin Delphindin dihexoside Phloretin-hexoside (Phlo- rizin) 333, 260 365, 274 274 275 320, 256 375, 268 515, 278 267 367, 275 377, 285 375, 265 365, 270 366 319, 286 361, 252 225 375, 265 315 325, 210 325, 220 369, 229 519, 277 287 633.2 301, 249 0.54 951.2 933, 301 0.27 169.2 125 1.25 965.2 301 0.89 783 765, 481, 301 0.21 801.1 649, 301 0.12 417.1 287 0.12 481.2 301, 275, 229 0.16 301 229, 185 2.89 577.2 441, 289 0.35 331.1 271, 269, 125 0.28 505.1 329, 314 0.08 447.3 301 0.19 625 425, 301 0.08 463.2 301, 271 0.32 517.1 337, 271 0.62 649.2 497, 301 1.83 325.1 163 0.13 353.1 191 0.12 515.4 353, 191 0.09 271.1 181 0.05 627 303 0.31 435.4 167 0.02 15,52,53 15,53 15,52–54 15 13,47,48 13 49 13,47,48 13,47,48 49 13,47 48 13,47 13 13,47 47 13,47 49 13 49 49 13,49 47,49 Table 1. LC–MS–MS data of the tentatively identified compounds in the ethyl acetate fraction. Scientific RepoRtS | y = 0.6701x + 2.1231 and R2 = 1 (Fig. 1d). It was found that each mg of the fraction contains 43.14 ± 0.57 μg ellagic acid. LC–MS–MS analysis of ethyl acetate fraction of PL. It is the first time to report the polyphenols pro- file of the ethyl acetate fraction (the polyphenols rich fraction) using a UPLC–PDA–ESI–MS method (Table 1). Twenty-three compounds were tentatively identified; most of them (11 compounds) were found to be phenolic acids and their derivatives. Furthermore, 8 compounds were found to be tannins; 7 of them were hydrolysable tannins while only one compound was a condensed tannin. Moreover, 3 anthocyanins and 1 flavonoid derivative were detected. Green synthesis of AgNPs and its optimization. Firstly, crude extract as well as the ethyl acetate and n-butanol fractions at different concentrations were preliminary tested in the green synthesis process. It was found that the AgNPs that were synthesized by the aid of the ethyl acetate fraction was the best, in terms of its SPR bands (λmax = 417 nm) and particle size (34 nm). Systematic optimization was carried out to find the best conditions for synthesis of AgNPs by PL for the first time. Fourteen experimental trials were carried out and responses were recorded (Table 2), the obtained data were subjected to the chosen models followed by performing fitting analysis using various statistical parameters. After data modelling, two polynomial equations were generated (Eqs. 1 and 2). Those equations proved that there is an interaction and curvature effect for the two variables analysed (particle size and PDI). Furthermore, good fitting of data within the selected models was illustrated in the 3D plots of responses (Fig. 2) Particle size = −36.265 + 9.289A + 23.031B + 0.805C + 0.450AB − 0.084AC − 0.308BC (1) PDI = 0.166+0.0022A+0.0146B−0.0334C+0.0028AB−0.0568AC++0.0135BC+0.0398A2++0.0215B2+0.0505C2 (2) where A is the fraction concentration (mg/mL), B is the silver nitrate concentration (mMol) and C is the tem- perature (°C). Regarding the particle size response, ANOVA results showed that the 2FI model is significant with P-value < 0.05, where A, B, and C are significant model terms. R2 = 0.9733, with lack of fit F-value of 1.92 that implies non-significant lack of fit relative to the pure error. The quadratic model for the PDI response was found to be non-significant, with a significant lack of fit F-value of 455.14, this could be attributed to closeness of the PDI results to each other within the tested ranges, as they are all in the acceptable range below 0.400 (Table 3). (2020) 10:14851 | https://doi.org/10.1038/s41598-020-71847-5 3 Vol.:(0123456789)PDF Image | pomegranate leaves and their role in green silver nanoparticles
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