Characterisation of the Phenolic Profile of Acacia retinodes and Acacia mearnsii Flowers’ Extracts

Acacia spp. is an invasive species that is widespread throughout the Portuguese territory. Thus, it is pertinent to better understand this species in order to find different applications that will value its use. To evaluate the phenolic profile in Acacia flowers, ethanolic extracts obtained through an energized guided dispersive extraction were analysed, focusing on two species, Acacia retinodes and Acacia mearnsii, at two flowering stages. The phytochemical profile of each extract was determined by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and high-performance liquid chromatography coupled with diode array detector. The FTIR-ATR technique was used to distinguish the different samples’ compositions. The results showed the presence of high concentrations of phenolic compounds (>300 mg GAE/g extract), among which are flavonoids (>136 mg QE/g extract), for all combinations of species/flowering stages. The phytochemical profile showed a complex composition with 21 compounds identified and quantified (the predominant ones being epicatechin, rutin, vanillin, and catechol). Both species and flowering stages presented significant variations regarding the presence and quantity of phenols and flavonoids, so much so that a principal component analysis performed with FTIR-ATR spectra data of the extracts was able to discriminate between species and flowering stages.


Introduction
Natural compounds obtained from plants offer research opportunities due to their significant pharmacological and toxicological properties [1]. They are often considered as potential new drugs against drug-resistant pathogens [2] and for treating diseases.
A variety of medicinal species belonging to the genus Acacia suggest the potential presence of bioactive metabolites. The most characteristic in this genus are flavonoids and tannins compounds [3]. The literature that is focused on plant extracts indicates that these species are rich in phytochemical compounds such as polyphenols, flavonoids, saponins, alkaloids, among others. The accumulations are made in different tissues/organs FS-flowering stages; EF-early flower; LF-late flower; TPC -total phenolic compounds; TFC-flavonoids content; n.s. for p > 0.05, *** p < 0.001; GAE-gallic acid equivalents; QE-quercetin equivalents. Means within the same column followed by different letters are significantly different (p < 0.05) according to the LSD Test.
The amount of TPC in flowers from the two flowering stages ranged, on average, between 300 and 350 mg GAE/mL for both A. mearnsii and A. retinodes.
The ANOVA results (Table 1) show a difference in total phenolic contents between the two species, where this difference accounted for 49.1% of the total variance for TPC. While, for A. retinodes, phenolic content increases as the flowering stage progresses, the opposite was observed for A. mearnsii, although with a less significant variation. In the species A. retinodes, the total phenolic content is higher in the late flowering stage (LF). However, in both species, no significant differences were observed between flowering stage. Flavonoids have beneficial biological activities, namely anti-inflammatory, antimicrobial, antioxidant, cytotoxic, and antitumour [31]. In this study, flavonoids were determined by the aluminium chloride colorimetric method (Table 1).
Concerning TFC, the flowers of the A. mearnsii species presented a significantly higher value in relation to the other species analysed, and this difference represented 46.8% of the total variation observed. In this case, the state of maturity was also highly significant (25.9% of the total variance), with the late flowers possessing a higher amount of TFC, independently of species. These results are similar to those obtained by A. dealbata [31], but in this case, the early flowering stage presents higher TPC and TFC than the late stage.
The TPC and TFC obtained in this study are in agreement with those observed for A. podalyriifolia flowers [32] but are lower than those observed for A. confusa [33].

Targeted and Untargeted Phytochemical Study
The samples were analysed by ultra-high performance liquid chromatography (UH-PLC) coupled to a QTOF-MS detector in order to identify the compounds and complement phytochemical characterization. The chromatographic analysis was performed using optimized conditions, providing a satisfactory separation in less than 25 min [34].
Analysis of phenolic compounds is reported in positive and negative ionization modes, but the latter mode was found more sensitive for the analysis of most compounds.
(i) Targeted analysis: Twenty-nine compounds were unambiguously identified and characterised by comparison of retention times and accurate mass spectra with those of authentic standards. A phytochemical library of 48 standard solutions (freshly prepared) was used to identify the metabolites in methanol extracts [35], and two groups of compounds were mainly present: hydroxybenzoic acids and flavonoids ( Table 2).  The samples were then analysed by a high-performance liquid chromatography-diode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table  S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5-methylfurfural, (+) catechin, quercetin, and 4 ,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification.
The results presented in Table 4 report the effects of species and flowering stages for all analysed compounds. The percentage of the variance of each factor analysed (species and flowering stages) was calculated based on the two-way ANOVA results.
Regarding Table 4, the concentration of gallic acid, syringaldehyde, and caffeic acid is not affected by species or flowering stages. Vanillin, p-coumaric acid, trans-cinnamic acid, 4 ,5,7-trihydroxyflavanone, 5-methyfurfural, quercetin, 4 ,5,7-trihydroxyflavanone, catechol, and (-)-epicatechin are highly influenced by the species. Comparing the compounds that appear in both species, it is possible to conclude that A. mearnsii is richer in the analysed compounds, with a significantly higher amount of vanillin, p-coumaric acid, trans-cinnamic acid, 4 ,5,7-trihydroxyflavanone, catechol, and (-)-epicatechin, where quercetin and 5-methyfurfural are present in a significantly higher amount in A. retinodes (Table 3). Gallic acid, syringaldehyde, and caffeic acid do not show significant differences between species. Table 3. The concentration of phenolic compounds (µg/g) in the ethanol extracts from A. retinodes and A. mearnsii flowers by HPLC-DAD using analytical standards and calibration curves (mean ± standard deviation). compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. diode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. diode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has s experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with t obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remai compounds were not detected or were below the limit of quantification. diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compound estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Tabl was in the species A. retinodes that it was possible to quantify most of the compoun of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic aci methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogra diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has s experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with t obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remai compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogr diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compound estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Tabl was in the species A. retinodes that it was possible to quantify most of the compoun of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic aci methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogra diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has s experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with t obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remai compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogr diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compound estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Tabl was in the species A. retinodes that it was possible to quantify most of the compoun of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic aci methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatograp diode array detector (HPLC/DAD) to identify the other compounds and fur complement the initial detection and phytochemical characterisation. Our group has s experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with th obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts w calibration curves constructed using the corresponding standard solut (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compounds of them, while in the species A. mearnsii, it was only possible to quantify gallic a vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remain compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogra diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogr diode array detector (HPLC/DAD) to identify the other compounds and f complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compound estimated by comparing their peak areas in the chromatograms of plant extract calibration curves constructed using the corresponding standard sol (Supplementary Material: Table S1. Data validation). The results are shown in Tab was in the species A. retinodes that it was possible to quantify most of the compoun of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic ac methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rem compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographyiode array detector (HPLC/DAD) to identify the other compounds and further omplement the initial detection and phytochemical characterisation. Our group has some xperience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, ompound identification was carried out by comparing their retention times with those btained from analytical standards. The concentration of the identified compounds was stimated by comparing their peak areas in the chromatograms of plant extracts with alibration curves constructed using the corresponding standard solutions Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It as in the species A. retinodes that it was possible to quantify most of the compounds, 20 f them, while in the species A. mearnsii, it was only possible to quantify gallic acid, anillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5ethylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining ompounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogra diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has s experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with t obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remai compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogr diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compound estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Tabl was in the species A. retinodes that it was possible to quantify most of the compoun of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic aci methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogra diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has s experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with t obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remai compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogr diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compound estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Tabl was in the species A. retinodes that it was possible to quantify most of the compoun of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic aci methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogra diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has s experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with t obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remai compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogr diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compound estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Tabl was in the species A. retinodes that it was possible to quantify most of the compoun of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic aci methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. Table 3. The concentration of phenolic compounds (µg/g) in the ethanol extracts from A. retinodes and A. mearnsii flowers by HPLC-DAD using analytical standards and calibration curves (mean ± standard deviation). The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. Table 3. The concentration of phenolic compounds (µg/g) in the ethanol extracts from A. retinodes and A. mearnsii flowers by HPLC-DAD using analytical standards and calibration curves (mean ± standard deviation). The samples were then analysed by a high-performance liquid chromatogra diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has s experience concerning the quantification of phenolic compounds [36][37][38][39]. T compound identification was carried out by comparing their retention times with t obtained from analytical standards. The concentration of the identified compounds estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Table  was in the species A. retinodes that it was possible to quantify most of the compound of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remai compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatogr diode array detector (HPLC/DAD) to identify the other compounds and fu complement the initial detection and phytochemical characterisation. Our group has experience concerning the quantification of phenolic compounds [36][37][38][39]. compound identification was carried out by comparing their retention times with obtained from analytical standards. The concentration of the identified compound estimated by comparing their peak areas in the chromatograms of plant extracts calibration curves constructed using the corresponding standard solu (Supplementary Material: Table S1. Data validation). The results are shown in Tabl was in the species A. retinodes that it was possible to quantify most of the compoun of them, while in the species A. mearnsii, it was only possible to quantify gallic vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic aci methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The rema compounds were not detected or were below the limit of quantification. then analysed by a high-performance liquid chromatography-(HPLC/DAD) to identify the other compounds and further etection and phytochemical characterisation. Our group has some the quantification of phenolic compounds [36][37][38][39]. Thus, n was carried out by comparing their retention times with those l standards. The concentration of the identified compounds was g their peak areas in the chromatograms of plant extracts with onstructed using the corresponding standard solutions al: Table S1. Data validation). The results are shown in Table 3. It tinodes that it was possible to quantify most of the compounds, 20 species A. mearnsii, it was only possible to quantify gallic acid, de, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5chin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining etected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. The samples were then analysed by a high-performance liquid chromatographydiode array detector (HPLC/DAD) to identify the other compounds and further complement the initial detection and phytochemical characterisation. Our group has some experience concerning the quantification of phenolic compounds [36][37][38][39]. Thus, compound identification was carried out by comparing their retention times with those obtained from analytical standards. The concentration of the identified compounds was estimated by comparing their peak areas in the chromatograms of plant extracts with calibration curves constructed using the corresponding standard solutions (Supplementary Material: Table S1. Data validation). The results are shown in Table 3. It was in the species A. retinodes that it was possible to quantify most of the compounds, 20 of them, while in the species A. mearnsii, it was only possible to quantify gallic acid, vanillin, syringaldehyde, caffeic acid, p-coumaric acid, trans-cinnamic acid, 5methylfurfural, (+) catechin, quercetin, and 4′,5,7-trihydroxyflavanone. The remaining compounds were not detected or were below the limit of quantification. Table 3. The concentration of phenolic compounds (µg/g) in the ethanol extracts from A. retinodes and A. mearnsii flowers by HPLC-DAD using analytical standards and calibration curves (mean ± standard deviation). Only chlorogenic acid and kaempferol are present in a significantly higher amount in EF.

λmax (nm)
The compounds (+)-catechin and coniferaldehyde appear only in A. mearnsii but do not show significant differences between LF and EF (Tables 3 and 4).
For the remaining compounds whose quantification was not performed by HPLC-DAD due to the absence of analytical standards, the concentration of the identified compounds by UHPLC/ESI-QTOF-MS was estimated by comparing their peak areas in the chromatograms from the plant extracts with those of the corresponding standard solutions freshly prepared and analysed by duplicate in the same batch as the samples (Supplementary Material Table S2). These results are presented in Table 5. (ii) Untargeted study: Finally, UHPLC-ESI-QTOF-MS/MS facilitates the characterization of known and unknown compounds on the basis of their molecular formula, exact mass measurements, and MS/MS fragmentations. Thus, the most abundant signal was analysed in both species (Supplementary Material: Figures S1 and S2). The compounds were tentatively identified by matching the accurate masses (±5 ppm error) of the detected molecular ions and their MS/MS patterns (Table 6). These patterns were obtained from those compounds previously reported in the literature and after comparison against openacess databases such as FOODB (http://foodb.ca, accessed on 2 February 2022) [40] and by using the on-line tool CEU Mass Mediator (http://ceumass.eps.uspceu.es/mediator, accessed on 2 February 2022) [41].
A global analysis was performed using a heat map to visualise hierarchical clustering that ordered similar groups to understand the behaviour of the different analysed compounds as a whole concerning each species and in the different flowering states to understand coherent patterns among them.
A heat map is a graphical representation of data where the individual values contained in a matrix are represented as colours [42].
The heat map (Figure 1) was generated from the content of compounds identified in each species and flowering stage. The blue colours represent a positive correlation between analyte levels and species, while the pink colours depict a negative correlation. Heat maps clustered the two Acacia species into two groups according to the abundance and presence of the different compounds. Total phenolic compounds, gallic acid, caffeic acid, (+)-catechin, p-coumaric acid, 4 ,5,7-trihydroxyflavanone, trans-cinnamic acid, vanillin, and syringaldehyde are more abundant in A. mearnsii, with some differences between EL and LF. A. retinodes presents a higher amount of the other compounds. Some of them appear only in this species, as aforementioned.
Regarding the cluster where A. retinodes have a higher amount, it was also possible to identify two sub-clusters related to the differences between early flowers and late ones. stand coherent patterns among them.
A heat map is a graphical representation of data where the individual values contained in a matrix are represented as colours [42].
The heat map (Figure 1) was generated from the content of compounds identified in each species and flowering stage. The blue colours represent a positive correlation between analyte levels and species, while the pink colours depict a negative correlation. Heat maps clustered the two Acacia species into two groups according to the abundance and presence of the different compounds. Total phenolic compounds, gallic acid, caffeic acid, (+)-catechin, p-coumaric acid, 4′,5,7-trihydroxyflavanone, trans-cinnamic acid,

FTIR-ATR Spectral Analysis
The spectra obtained with the ethanolic flowers' extract ( Figure 2) display the strong influence of the compounds in this matrix. These results are similar to those obtained for rosemary [43], Cucurbitaceae [44], and medicinal plant extracts [45]. vanillin, and syringaldehyde are more abundant in A. mearnsii, with some differences between EL and LF. A. retinodes presents a higher amount of the other compounds. Some of them appear only in this species, as aforementioned.
Regarding the cluster where A. retinodes have a higher amount, it was also possible to identify two sub-clusters related to the differences between early flowers and late ones.

FTIR-ATR Spectral Analysis
The spectra obtained with the ethanolic flowers' extract ( Figure 2) display the strong influence of the compounds in this matrix. These results are similar to those obtained for rosemary [43], Cucurbitaceae [44], and medicinal plant extracts [45].
The intense band at 3328 cm −1 was assigned by the stretching vibrations of -OH groups more highly influenced by water content in the samples. Still, a lower strength could also affect some alcohols, phenols, or peroxides.
The bands at 2973 cm −1 , 2927 cm −1 , and 2880 cm −1 are characteristics of C-H stretching vibrations mainly to -CH3 and -CH2 as well as aromatic groups present in some plant extracts and the ethanol present in the matrix. These peaks can also be related to the diterpene three-ring structure [44].
The band at 1454 cm −1 , corresponding to the combination of bending vibration of -CH2 and the vibration of the COO-group in the flavanol and organic acids [46] and also related to -CH3 in acetyl groups and at 1418 cm −1 , can be assigned by -CH2 and -OCH3 groups. The peak found at 1379 cm −1, and 1329 cm −1 can be related to C-H's symmetric deformation vibrations in methoxy groups and phenolic hydroxyls [47].
The peak at 1274 cm −1 could be related to asymmetric stretching vibrations of the C-O-C linkages in phenolic ethers (C-O stretch) and esters of phenolic hydroxyls [43].
The strong bands at 1087 cm −1 and 1045 cm −1 are related to the primary alcohol, C-O stretch, and the secondary alcohol [48].
At 880 and 802 cm −1 , it is possible to find the peaks associated with the C-C skeletal vibration and out-of-plane bending vibrations associated with some aromatic ring (aryl) group frequencies [48].  Figure 3 shows the PCA plot of the FTIR-ATR spectra obtained with all the samples. This non-supervised multivariate statistical analysis reveals that this technique is able to discriminate between flower raw materials based on their differences, among which their The intense band at 3328 cm −1 was assigned by the stretching vibrations of -OH groups more highly influenced by water content in the samples. Still, a lower strength could also affect some alcohols, phenols, or peroxides.
The bands at 2973 cm −1 , 2927 cm −1 , and 2880 cm −1 are characteristics of C-H stretching vibrations mainly to -CH 3 and -CH 2 as well as aromatic groups present in some plant extracts and the ethanol present in the matrix. These peaks can also be related to the diterpene three-ring structure [44].
The band at 1454 cm −1 , corresponding to the combination of bending vibration of -CH 2 and the vibration of the COO-group in the flavanol and organic acids [46] and also related to -CH 3 in acetyl groups and at 1418 cm −1 , can be assigned by -CH 2 and -OCH 3 groups. The peak found at 1379 cm −1, and 1329 cm −1 can be related to C-H's symmetric deformation vibrations in methoxy groups and phenolic hydroxyls [47].
The peak at 1274 cm −1 could be related to asymmetric stretching vibrations of the C-O-C linkages in phenolic ethers (C-O stretch) and esters of phenolic hydroxyls [43].
The strong bands at 1087 cm −1 and 1045 cm −1 are related to the primary alcohol, C-O stretch, and the secondary alcohol [48].
At 880 and 802 cm −1 , it is possible to find the peaks associated with the C-C skeletal vibration and out-of-plane bending vibrations associated with some aromatic ring (aryl) group frequencies [48]. Figure 3 shows the PCA plot of the FTIR-ATR spectra obtained with all the samples. This non-supervised multivariate statistical analysis reveals that this technique is able to discriminate between flower raw materials based on their differences, among which their phenolic and flavonoid profile of the Acacia's species and flowering stages have their impact. Similar results were found, but with FT-RAMAN, in a previous work [49]. The two first components can justify 84% of the variation being the first component capable of distinguishing between the two species. phenolic and flavonoid profile of the Acacia's species and flowering stages have their impact. Similar results were found, but with FT-RAMAN, in a previous work [49]. The two first components can justify 84% of the variation being the first component capable of distinguishing between the two species.

Plant Material
The flowers of A. retinodes and A. mearnsii were harvested during two flowering stages (early flower-EF; and late flower-LF). They were collected in March 2021 in the Lisbon region (A. retinodes at 38.747067; -−9.274577 and A. mearnsii at 38.713910, −9.192827). The flowers were freeze-dried and kept at −80 °C until extraction. Figure 4 shows an example of the different flowering stages.

Plant Material
The flowers of A. retinodes and A. mearnsii were harvested during two flowering stages (early flower-EF; and late flower-LF). They were collected in March 2021 in the Lisbon region (A. retinodes at 38.747067; −9.274577 and A. mearnsii at 38.713910, −9.192827). The flowers were freeze-dried and kept at −80 • C until extraction. Figure 4 shows an example of the different flowering stages.

Plant Material
The flowers of A. retinodes and A. mearnsii were harvested during two flowering stages (early flower-EF; and late flower-LF). They were collected in March 2021 in the Lisbon region (A. retinodes at 38.747067; -−9.274577 and A. mearnsii at 38.713910, −9.192827). The flowers were freeze-dried and kept at −80 °C until extraction. Figure 4 shows an example of the different flowering stages.

Early Flower
Late flower

Extraction Conditions
According to the procedure previously described, the energized dispersive guided extraction (EDGE) equipment was used for extraction [25].
The flower samples were ground in a hammer mill with a 1 mm mesh in the first step. After that, 1 g of powder sample underwent three successive extraction cycles using 20 mL of ethanol for 10 min at 40 • C in each cycle. The crude material of each sample was weighed directly into a Q-Cup containing a sandwich of tree Q-Discs composed of one fibreglass filter surrounded by two cellulose filters.
All extractions were performed in duplicate, and all subsequent measurements and analyses were performed in triplicate.

Determination of Total Phenolic and Flavonoid Contents
Total phenolic content (TPC) was determined by the Folin-Ciocalteu colorimetric method [50], using gallic acid as a standard. The ethanolic solution of each extract or standard (50 µL) was mixed with 450 µL of distilled water. Then, 2.5 mL of Folin-Ciocalteu reagent (0.2 N) was added, leaving the mixtures for 5 min before adding 2 mL of aqueous Na 2 CO 3 (75 g/L). The reaction mixtures were incubated for 90 min at 30 • C. Total phenols were determined by colourimetry at 765 nm.
The calibration curve was prepared using gallic acid standard solutions with concentrations between 0.016 and 3.2 mg/L (y = 0.2249 x; R 2 = 0.9973). TPC was expressed as gallic acid equivalents (mg GAE/g extract).
The total flavonoids content (TFC) was determined by the aluminium chloride colorimetric method according to Luís et al. [51].
Each ethanolic solution of the extracts (500 µL) was mixed with methanol (1.5 mL), 10% aluminium chloride (0.1 mL), 1 M potassium acetate (0.1 mL), and distilled water (2.8 mL). This solution was left in the dark at room temperature for 30 min, and the absorbance of the reaction mixture was measured at 415 nm using a spectrophotometer. The calibration curve was performed by preparing eight quercetin solutions at concentrations between 2.5