1H NMR Mixture Design-Fingerprints and ASCA Analysis in Ilex paraguariensis: Model Stability in Search of a Global Metabolome.

The effects of experimental repetitions and solvent extractors on the 1H NMR fingerprinting of yerba mate extracts, obtained from two genders and two light environments, were analyzed in-depth by ANOVA-simultaneous component analysis (ASCA). Different solvents were used according to a mixture design based on ethanol, dichloromethane, and hexane and their combinations. The number of experimental repetitions significantly affected the ASCA results. Increasing repetitions led to decreases in the percentage effect variance values and an increase in the percentage residual variance. However, secondary sexual dimorphism, light availability, and their interaction effects became more significant with decreasing p-values at or above the 95% confidence level. The choice of a solvent extractor significantly affects the chemical profile and can lead to distinct conclusions regarding the significance of effect values. Pure solvents yielded different conclusions about the significance of factorial design effects, with each solvent extracting unique metabolites and maximizing information for specific effects. However, the use of binary solvent mixtures, such as ethanol-dichloromethane, proved more efficient in extracting sets of compounds that simultaneously differentiate between different experimental conditions. The mixture design-fingerprint strategy provided satisfactory results expanding the range of extracted metabolites with high percentage of residual variances and low explained percentage effect variances in the ASCA models. Ternary and even higher-ordered mixtures could be good alternative extracting media for work-intensive procedures. Our study underscores the significance of experimental design and solvent selection in metabolomic analysis, improving the accuracy, robustness, and interpretability of metabolomic models, leading to a better understanding of the chemical composition and biological implications of plant extracts.

Leaf gas exchange and bean quality fluctuations over the whole canopy vertical profile of Arabic coffee cultivated under elevated CO2.

Leaves in different positions respond differently to dynamic fluctuations in light availability, temperature and to multiple environmental stresses. The current hypothesis states that elevated atmospheric CO2 (e[CO2]) can compensate for the negative effects of water scarcity regarding leaf gas exchanges and coffee bean quality traits over the canopy vertical profile, in interactions with light and temperature microclimate during the two final stages of berry development. Responses of Coffea arabica L. were observed in the 5th year of a free air CO2 enrichment experiment (FACE) under water-limited rainfed conditions. The light dependent leaf photosynthesis curves (A/PAR) were modelled for leaves sampled from vertical profile divided into four 50-cm thick layers. e[CO2] significantly increased gross photosynthesis (AmaxGross), the apparent quantum yield efficiency, light compensation point, light saturation point (LSP) and dark respiration rate (Rd). As a specific stage response, considering berry ripening, all parameters calculated from A/PAR were insensitive to leaf position over the vertical profile. Lack of a progressive increase in AmaxGross and LSP was observed over the whole canopy profile in both stages, especially in the two lowest layers, indicating leaf plasticity to light. Negative correlation of Rd to leaf temperature (TL) was observed under e[CO2] in both stages. Under e[CO2], stomatal conductance was also negatively correlated with TL, reducing leaf transpiration and Rd even with increasing TL. This indicated coffee leaf acclimation to elevated temperatures under e[CO2] and water restriction. The e[CO2] attenuation occurred under water restriction, especially in A and water use efficiency, in both stages, with the exception of the lowest two layers. Under e[CO2], coffee produced berries in moderate- and high light level layers, with homogeneous distribution among them, contrasted to the heterogeneous distribution under actual CO2. e[CO2] led to increased caffeine content in the highest layer, with reduction of chlorogenic acid and lipids under moderate light and to raised levels of sugar in the shaded low layer. The ability of coffee to respond to e[CO2] under limited soil water was expressed through the integrated individual leaf capacities to use the available light and water, resulting in final plant investments in new reproductive structures in moderate and high light level layers.

PARAFAC HPLC-DAD metabolomic fingerprint investigation of reference and crossed coffees.

In this study two cultivars of Coffea arabica L., Bourbon (reference) and IPR101 (crossing) were analyzed. The extracts were prepared according to a simplex centroid design with four components, ethanol, ethyl acetate, dichloromethane, and hexane. Multiway data were obtained by HPLC-DAD analysis of the fifteen different mixtures for each cultivar. The PARAFAC methodology was used to investigate the chromatographic fingerprint. For both cultivars, Factor 1 was able to discriminate mixtures containing ethyl acetate as solvent. Factor 2 indicated that mixtures in pure ethanol and binary mixtures containing ethanol were the most efficient in extracting chlorogenic acids and factor 3 identified methylxanthines through spectrophotometric profile in all mixtures. Higher concentrations were obtained by the ethanol, dichloromethane and hexane ternary mixture for the Bourbon cultivar and by the quaternary mixture of these solvents with ethyl acetate for the IPR101 cultivar. Trigonelline and cafestol were extracted in both cultivars. The reference coffee showed higher relative abundances of cafestol ester, chlorogenic acids and trigonelline whereas the crossed coffee showed higher levels of caffeine. To confirm these results, UPLC-MS was used as a complementary method to confirm the presence of the metabolites in these extracts. The three way PARAFAC strategy determines correlations of HPLC-DAD chromatographic and spectral data simultaneously with samples permitting a more unambiguous assignment of metabolic groups than can be obtained treating chromatographic and spectral data separately by two way methods. This can provide higher quality chromatographic fingerprints for food chemistry.