3.2. Chemical Analyses

According to the AOAC methods [72], the proximate composition was determined in the lyophilized plant material and expressed in g/100g of fresh weight (fw). The incineration at 550 ± 15 °C was used to determine the ash content. Crude protein was estimated by the macro-Kjeldahl method (N × 6.25) using an automatic distillation and titration unit (model Pro-Nitro-A, JP Selecta, Barcelona). Soxhlet extraction was used to determine the crude fat, with petroleum ether during 7 h. Total carbohydrates content was calculated by difference using the formula: Total carbohydrates (g/100 g fw) = 100 − (g moisture + g fat + g ash + g proteins). The energetic value was calculated according to the Atwater system using the formula: Energy (kcal/100 g fw) = 4 × (g proteins + g carbohydrates) + 9 × (g fat).

The extraction of tocopherols from the lyophilized plant material was carried out following the procedure described by Silva et al. [73].The analysis was made using a high performance liquid chromatography coupled to a fluorescence detector (HPLC-FL; Knauer, Smartline system 1000, Berlin, Germany) as described by the authors. For the identification of the tocopherol compounds, chromatographic comparisons with authentic standards were made, and the quantification was performed using tocol as internal standard (IS) and calibration curves obtained from commercial standards (Sigma, St. Louis, MO, USA). The results were expressed in mg per 100 g of fw.

The extraction of free sugars from the lyophilized plant material was carried out according to Silva et al. [73]. The compounds were identified using a HPLC with a refraction index detector (HPLC-RI) operating as previously described for the authors cited. Peaks identification was performed by comparisons of their relative retention time (Rt) with authentic standards. Quantification was completed using melezitose as IS, (Sigma-Aldrich, St. Louis, MO, USA). Results were processed in a Clarity Software (Data Apex, Prague, Czech Republic) and expressed in g per 100 g of fw.

The extraction of organic acids from the lyophilized plant material was done as previously described and optimized by Barros et al. [74] and subsequent determination by ultra-fast liquid chromatography coupled to diode-array detection (UFLC-DAD); (Shimadzu Corporation, Kyoto, Japan). Compounds were identified by comparison of spectra and retention time with the authentic standards (Sigma-Aldrich, St. Louis, MO, USA). The quantification was performed based on calibration curves (245 nm for ascorbic acid and 215 nm for remaining acids). The results were recorded and processed using LabSolutions Multi LC-PDA software (Shimadzu Corporation, Kyoto, Japan), and expressed in mg/100 g of fw.

The fatty acids in the lyophilized plant material were determined after the trans-esterification process, as previously described by Silva et al. [73]. The analysis was made using a gas chromatographer DANI model GC 1000 instrument equipped with a split/splitless injector and a flame ionization detector (GC-FID, 260 °C). The identification and quantification of the compound were performed by comparing the relative retention times of fatty acid methyl ester (FAME) from commercial standards. Fatty acids were processed using Clarity Software (DataApex 4.0, Prague, Czech Republic) and the results expressed in percentage.

Powder from freeze-dried plants was extracted by stirring with 30 mL of ethanol/water (80:20, v/v) for 1 h and subsequently filtering through Whatman No. 4 paper. The residue was then extracted with an additional 30 mL of ethanol/water for 1 h. The combined hydroethanolic extracts were evaporated to dryness and redissolved in ethanol/water for the evaluation of the phenolic compounds and bioactive assays [75].

For phenolic compounds was weighted 20 mg of prepared lyophilized extracts, re-dissolved in 2 mL of ethanol:water (80:20, v/v). The compounds were evaluated using a Dionex Ultimate 3000 UPLC system and a diode array coupled in-series to an electrospray ionization mass spectrometry detector (LC-DAD-ESI/MSn) [76]. The identification of the individual phenolic compounds was performed by comparing the retention times, UV-visible spectra, and MS fragmentation patterns of the detected compounds with those of authentic standards; data available from the literature were also used. The quantification was based on the calibration curves of authentic standards. The results were presented as mg/g of plant fw.