Untargeted metabolomic profiling of fresh and dried leaf extracts of young and mature Eucalyptus globulus trees indicates differences in the presence of specialized metabolites

Abstract

Aqueous extracts from Eucalyptus globulus leaves contain a wide variety of specialized metabolites, mainly polyphenols and appreciable amounts of volatile compounds, which are responsible for their diverse biological activities, such as antioxidant, antimicrobial, and allelopathic features. For this reason, several studies have been conducted to explore the composition of E. globulus leaf extracts for multiple therapeutic and commercial applications. However, so far, the available bibliographic reports only refer to the chemical composition of extracts prepared with leaves from mature trees, leaving much to clarify about the composition of juvenile eucalyptus leaf extracts. Furthermore, there is no consensus regarding the type of leaves, fresh or dried ones, to be used in the extraction procedure, considering the highest recovery of biologically active compounds. In this sense, this study aimed to characterize the chemical composition of aqueous extracts prepared with fresh and dried leaves from young and mature E. globulus trees. For this, leaf biomass from young and mature E. globulus trees was collected in three distinct places from a forest area, and after oven-drying a portion of the leaves, an extraction in hot water was carried out, followed by GC-MS and HPLC-MS/MS analyses. The results revealed that the maturity of eucalyptus trees and biomass drying significantly influenced the volatile and non-volatile composition of the aqueous extracts. Accordingly, while fresh leaf extracts of young trees had great levels of hydrolysable tannins, extracts prepared with fresh leaves from mature trees presented a wide range of terpenes. When dried leaf material was used, extracts had notorious contents of amino acids derivatives, C13 norisoprenoids, fatty and other organic acids. Overall, this study showed, for the first time, that plant maturity (young vs mature) and pre-processing (fresh vs dried) of foliar biomass of E. globulus trees need to be considered in the preparation of leaf aqueous extracts depending on the desired purposes, since major changes in what regards biologically active compounds were found.

Eucalyptus globulus Leaf Aqueous Extract Differentially Inhibits the Growth of Three Bacterial Tomato Pathogens

“…In comparison with other approaches, like AMP or other single-molecule therapies, the acquisition of bacterial resistance to plant extracts, as in the case of DLE, is less likely to occur, as they comprise a complex mixture of different compounds with distinct modes of action. In fact, as mentioned earlier, DLE is composed of many phenolic acids like gallic acid, galloylglucose digalloylglucose and the benzoic acid protocatechuic acid, which induce membrane disruption, organic acids such as arabinaric and tartaric acids, and flavonoids like quercetin [26]. Organic acids have the ability to cross membranes and can disrupt ATP production and induce cytoplasmic acidification [29], whereas flavonoids display multiple modes of action in bacterial cells, as they can inhibit biofilm formation, bacterial virulence, and nucleic acid, cell envelope, and peptidoglycan synthesis, besides disrupting membrane permeability [28].…”

“…The growth of Pst, Xeu, and Cmm was differentially inhibited by DLE, resulting in distinct susceptibilities to the plant extract. A recent report from our group, which analysed the metabolomic profile of aqueous extracts of eucalyptus leaves, revealed that DLE-the one prepared with dried leaves of young E. globulus trees -, in comparison with those prepared with fresh leaves of juvenile and mature trees and dried leaf extracts of mature trees, presented appreciable amounts of phenolic acids, more specifically, gallic acid, digalloylglucose I, IV, and V, and galloylglucose I and II [26]. Due to their great affinity for lipids, phenolic acids, a class of specialized metabolites with notorious antibacterial properties, can easily cross membranes, causing cytoplasmic acidification and consequently inducing membrane disruption [32].…”

“…To prepare the aqueous extract, dried leaves of young E. globulus trees were incubated for 30 min in deionised water warmed to 70 • C, according to the procedure described by Pinto, Soares, Pereira, Rodrigues, Fidalgo, and Valente [26]. The metabolomic profile of DLE (250 g L −1 ) has been previously detailed in Pinto, Soares, Pereira, Rodrigues, Fidalgo and Valente [26].…”

“…To prepare the aqueous extract, dried leaves of young E. globulus trees were incubated for 30 min in deionised water warmed to 70 • C, according to the procedure described by Pinto, Soares, Pereira, Rodrigues, Fidalgo, and Valente [26]. The metabolomic profile of DLE (250 g L −1 ) has been previously detailed in Pinto, Soares, Pereira, Rodrigues, Fidalgo and Valente [26]. In brief, this extract presented a great richness in gallic acids and derivatives (namely, gallic acid, galloylglucose I and II, and digalloylglucose I, IV, V), fatty acids [tetrahydroxyoctadecenoic acid, galactosylgycerol or glucosylglycerol, phloionolic acid, 6,8-dihydroxy-octanoic acid, and FA hydroxy (11:2/11:2) I], benzoic acids like protocatechuic acid, organic acids such as arabinaric acid, tartaric acid or meso-tartaric acid II, 2-hydroxypropanedioic acid, 2-methylcitric acid or homocitric acid I, and erythronic acid or threonic acid I, and amino acids like methylserine hexoside, serine glucoside, and n-1-deoxy-1-fructosylalanine, and flavonoids like quercetin [26].…”

“…In this sense, E. globulus leaf extracts could represent a more practical, affordable, and feasible approach to harnessing the bioactivity of eucalyptus specialized metabolites. A previous work [26] extensively characterized the phytochemical profile of aqueous extracts prepared with fresh and dried leaves of young and mature E. globulus trees, suggesting potential applications for these plant extracts. This study revealed that aqueous extracts prepared with dried leaves of young trees were rich in hydroxybenzoic acids, flavonoids, and organic acids, three chemical classes whose ability to disrupt bacterial membranes has already been described, thus having strong antibacterial properties [27][28][29].…”