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It is essential to evaluate efficacy and reproducibility of sample pre-treatment as a first step to ensure the reliability of a metabolomic study. Among the reviewed methods, the comprehensive extraction method appears to provide a promising approach for extracting diverse types of metabolites.

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The metabolomic analysis of 11 Ilex species, I. argentina, I. brasiliensis, I. brevicuspis, I. dumosavar. dumosa, I. dumosa var. guaranina, I. integerrima, I. microdonta, I. paraguariensis var. paraguariensis, I. pseudobuxus, I. taubertiana, and I. theezans, was carried out by NMR spectroscopy and multivariate data analysis. The analysis using principal component analysis and classification of the (1)H NMR spectra showed a clear discrimination of those samples based on the metabolites present in the organic and aqueous fractions. The major metabolites that contribute to the discrimination are arbutin, caffeine, phenylpropanoids, and theobromine. Among those metabolites, arbutin, which has not been reported yet as a constituent of Ilex species, was found to be a biomarker for I. argentina,I. brasiliensis, I. brevicuspis, I. integerrima, I. microdonta, I. pseudobuxus, I. taubertiana, and I. theezans. This reliable method based on the determination of a large number of metabolites makes the chemotaxonomical analysis of Ilex species possible.

In recent years, a new type of solvents, natural deep eutectic solvents (NADES), have been developed and are gaining popularity as an alternative "green solvent" to conventional organic solvents. The traditional extraction of flavor or fragrance from plant materials such as vanillin is a tedious procedure that involves the use of organic solvents. In this case the solubility of vanillin and its extractability from vanilla pods in 14 NADES was evaluated. The solubility in some NADES such as lactic acid: propanediol (620 mg/mL) and lactic acid: fructose (320 mg/ mL) was found to be almost similar to that of organic solvents e.g. methanol (632.94 mg/mL) or ethanol (375.81 mg/mL). Interestingly, the extractability of vanillin from vanilla pods did not reflect the results observed for the solubility tests. Some NADES with constituents that are suitable as food ingredients such as citric acid-fructose-glucose (CAFG), fructose-glucose (FG), malic acid-glucose (MAG), and malic acid-fructose-glucose (MAFG), showed a higher extraction power (7.6, 10.1, 16.7, and 16.3 mg/g DW, respectively) than ethanol (4.4 mg/g DW). The discrepancy between solubility and extractability might be due to differences in the interaction with the matrix depending on the NADES. The effect of water content in some NADES on the extractability of vanillin was also studied. With the high extraction capacity and the absence of toxicity of their components, NADES are very suitable for extraction of flavours and fragrances used in food and cosmetics. plants. 4 However, in terms of quality, natural vanilla has a superior aroma and fragrance performance than pure vanillin. This is due to the presence of various minor compounds in the pods that give special notes to the vanilla flavor. The highest quality vanilla extract is obtained from vanilla pods using the percolation or oleoresin method. 5The percolation method consists of circulating aqueous ethanol (35-50%, v/v) over the pods, under vacuum. This process takes betweenThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.