Seasonal Changes in the Metabolic Profiles and Biological Activity in Leaves of Diospyros digyna and D. rekoi "Zapote" Trees

Abstract

Leaves of semi-domesticated Diospyros digyna and wild D. rekoi trees, sampled seasonally in Mexico in 2014, were analyzed. Metabolic fingerprints revealed higher metabolite diversity in D. rekoi leaves. The TLC bands characteristic of glycosylated flavonoids, predominant in this species, matched the detection of quercetin and quercetin 3-O-glucuronides by liquid chromatography (UPLC-MS) of spring leaf extracts (LEs). Further gas chromatography (GC-MS) analysis revealed abundant fatty acids, organic acids, and secondary metabolites including trigonelline, p-coumaric, and ferulic and nicotinic acids. Phenolic-like compounds prevailed in D. digyna LEs, while unidentified triterpenoids and dihydroxylated coumarins were detected by UPLC-MS and GC-MS. A paucity of leaf metabolites in leaves of this species, compared to D. rekoi, was evident. Higher antioxidant capacity (AOC) was detected in D. digyna LEs. The AOC was season-independent in D. digyna but not in D. rekoi. The AOC in both species was concentrated in distinct TLC single bands, although seasonal variation in band intensity was observed among trees sampled. The AOC in D. digyna LEs could be ascribed to the coumarin esculetin. The LEs moderately inhibited phytopathogenic bacteria but not fungi. Leaf chemistry differences in these Mesoamerican Diospyros species substantiated previous variability reported in tree physiology and fruit physical chemistry, postulated to result from domestication and seasonality.

Keywords: Antioxidant capacity; Diospyros; flavonoid glycosides; metabolic fingerprints; phenolics; secondary metabolites.

Figure 1

Seasonal variation in the content of phenolic secondary metabolites in Diospyros rekoi. High performance-thin layer chromatography (HP-TLC) traces of D. rekoi leaf extracts visualized under (a) UV light (254 nm) and (b) UV light (366 nm) after derivatization with the NP/PEG reagent. Lanes: 1–5 represent leaf extracts from five trees (1–5) that were systematically sampled in the spring, summer, autumn, and winter of 2014. Band Rf values are represented on the right-side end of the figures.

Figure 2

Seasonal variation in the content of phenolic secondary metabolites in Diospyros digyna. The HP-TLC traces of D. digyna leaf extracts visualized under (a) UV light (254 nm) and (b) UV light (366 nm) after derivatization with the NP/PEG reagent. Lanes: 1–5 represent leaf extracts from five trees (1–5) that were systematically sampled in the spring, summer, autumn, and winter of 2014. Band Rf values are represented on the right-side end of the figure.

Figure 3

Seasonal variation of total soluble phenols (TSPs) and flavonoids (TFs) in leaves of Diospyros digyna and D. rekoi. Average seasonal variation in (a) TSPs and (b) TFs, expressed as caffeic acid and catechin equivalents, respectively, was determined in vitro in 60% aqueous methanolic leaf extracts of D. digyna and D. rekoi trees. The bars represent the mean values obtained from leaf extracts produced from the pooled leaves of five trees sampled in the spring, summer, autumn, and winter of 2014, respectively. Intervals over the bars represent the standard error of the means, whereas different letters over the bars represent statistically different values at p ≤ 0.05 (Tukey–Kramer test). DW = dry weight.

Figure 4

Seasonal metabolic diversity in leaves of D. digyna (Dg) and D. rekoi (Dr). The metabolic heat map was obtained from acidified methanol extracts obtained from leaves collected from Dg and Dr trees at different seasons: SPR, spring 2014; SUM, summer 2014; AUT, autumn 2014, and WIN, winter, 2013 and 2014 (for (Dr) and 2014 (Dg)). The 100 most abundant ionizable metabolites were selected to obtain the metabolic heat map within an 80–1300 m/z range. All measurements were performed in triplicate. The color-coded matrix elements and adjacent dendrograms indicate the functional relationships among the following variables: 100 ionizable metabolites’ abundance, detected by untargeted direct-injection electrospray mass spectrometry (DIESI-MS) analysis; two Diospyros tree species; and five seasons. The blank column represents a solvent control used to calibrate the system.

Figure 5

Average seasonal variation in antioxidant activities in aqueous methanol leaf extracts sampled from Diospyros digyna and D. rekoi trees. Antioxidant activity was measured using the (a) DPPH (2, 2′-diphenyl-1-picrylhydrazyl) and (b) FRAP (ferric ion-reducing antioxidant power) assays. Both were expressed as ascorbic acid (AA) equivalents. The bars represent the mean values obtained from leaf extracts produced from the pooled leaves of five trees sampled in the spring, summer, and autumn, and winter of 2014, respectively. Intervals over the bars represent the standard error of the means, whereas different letters over the bars represent statistically different values at p ≤ 0.05 (Tukey–Kramer test). DW = dry weight.

Figure 6

Antioxidant activity in D. rekoi and D. digyna leaf extracts. Bands showing antioxidant activity using the DPPH reagent were detected in polar leaf extracts previously separated on HP-TLC plates. The aqueous methanol extracts were obtained from leaves of five (a) D. rekoi and (b) D. digyna trees (Trees: 1–5) that were systematically sampled in the spring, summer, autumn, and winter of 2014, respectively. Band Rf values are represented on the left-side end of the figures.