Crocin and β-Carotene bleaching assays as analytical tools in the optimization of the extraction of hydrophilic and lipophilic antioxidants from tomato

Tomato (Lycopersicon esculentum Mill.) is the second most important vegetable crop worldwide and a rich source of hydrophilic (H) and lipophilic (L) antioxidants. The H fraction is constituted mainly by ascorbic acid and soluble phenolic compounds, while the L fraction contains carotenoids (mostly lycopene), tocopherols, sterols and lipophilic phenolics [1,2]. To obtain these antioxidants it is necessary to follow appropriate extraction methods and processing conditions. In this regard, this study aimed at determining the optimal extraction conditions for H and L antioxidants from a tomato surplus. A 5-level full factorial design with 4 factors (extraction time (I, 0-20 min), temperature (T, 60-180 •c), ethanol percentage (Et, 0-100%) and solid/liquid ratio (S/L, 5-45 g!L)) was implemented and the response surface methodology used for analysis. Extractions were carried out in a Biotage Initiator Microwave apparatus. The concentration-time response methods of crocin and P-carotene bleaching were applied (using 96-well microplates), since they are suitable in vitro assays to evaluate the antioxidant activity of H and L matrices, respectively [3]. Measurements were carried out at intervals of 3, 5 and 10 min (initiation, propagation and asymptotic phases), during a time frame of 200 min. The parameters Pm (maximum protected substrate) and V m (amount of protected substrate per g of extract) and the so called IC50 were used to quantify the response. The optimum extraction conditions were as follows: r~2.25 min, 7'=149.2 •c, Et=99.1 %and SIL=l5.0 giL for H antioxidants; and t=l5.4 min, 7'=60.0 •c, Et=33.0% and S/L~l5.0 g/L for L antioxidants. The proposed model was validated based on the high values of the adjusted coefficient of determination (R2.wi>0.91) and on the non-siguificant differences between predicted and experimental values. It was also found that the antioxidant capacity of the H fraction was much higher than the L one.

Ultrasound and microwave assisted extraction of ergosterol from Agaricus bisporus L.: optimization through response surface methodology

There is scientific evidence demonstrating the benefits of mushrooms ingestion due to their richness in bioactive compounds such as mycosterols, in particular ergosterol [I]. Agaricus bisporus L. is the most consumed mushroom worldwide presenting 90% of ergosterol in its sterol fraction [2]. Thus, it is an interesting matrix to obtain ergosterol, a molecule with a high commercial value. According to literature, ergosterol concentration can vary between 3 to 9 mg per g of dried mushroom. Nowadays, traditional methods such as maceration and Soxhlet extraction are being replaced by emerging methodologies such as ultrasound (UAE) and microwave assisted extraction (MAE) in order to decrease the used solvent amount, extraction time and, of course, increasing the extraction yield [2]. In the present work, A. bisporus was extracted varying several parameters relevant to UAE and MAE: UAE: solvent type (hexane and ethanol), ultrasound amplitude (50 - 100 %) and sonication time (5 min-15 min); MAE: solvent was fixed as ethanol, time (0-20 min), temperature (60-210 •c) and solid-liquid ratio (1-20 g!L). Moreover, in order to decrease the process complexity, the pertinence to apply a saponification step was evaluated. Response surface methodology was applied to generate mathematical models which allow maximizing and optimizing the response variables that influence the extraction of ergosterol. Concerning the UAE, ethanol proved to be the best solvent to achieve higher levels of ergosterol (671.5 ± 0.5 mg/100 g dw, at 75% amplitude for 15 min), once hexane was only able to extract 152.2 ± 0.2 mg/100 g dw, in the same conditions. Nevertheless, the hexane extract showed higher purity (11%) when compared with the ethanol counterpart ( 4% ). Furthermore, in the case of the ethanolic extract, the saponification step increased its purity to 21%, while for the hexane extract the purity was similar; in fact, hexane presents higher selectivity for the lipophilic compounds comparatively with ethanol. Regarding the MAE technique, the results showed that the optimal conditions (19 ± 3 min, 133 ± 12 •c and 1.6 ± 0.5 g!L) allowed higher ergosterol extraction levels (556 ± 26 mg/100 g dw). The values obtained with MAE are close to the ones obtained with conventional Soxhlet extraction (676 ± 3 mg/100 g dw) and UAE. Overall, UAE and MAE proved to he efficient technologies to maximize ergosterol extraction yields.

Flowers of Gomphrena globosa L. as an alternatve source of betacyanins: optimization of the extraction using response surface methodology

Betacyanins are betalain pigments that display a red-violet colour which have been reported to be three times stronger than the red-violet dye produced by anthocyanins [1]. The applications of betacyanins cover a wide range of matrices, mainly as additives or ingredients in the food industry, cosmetics, pharmaceuticals and livestock feed. Although, being less commonly used than anthocyanins and carotenoids, betacyanins are stable between pH 3 to 7 and suitable for colouring in low acid matrices. In addition, betacyanins have been reported to display interesting medicinal character as powerful antioxidant and chemopreventive compounds either in vitro or in vivo models [2]. Betacyanins are obtained mainly from the red beet of Beta vulgaris plant (between I 0 to 20 mg per I 00 g pulp) but alternative primary sources are needed [3]. In addition, independently of the source used, the effect of the variables that affect the extraction of betacyanins have not been properly described and quantified. Therefore, the aim of this study was to identifY and optimize the conditions that maximize betacyanins extraction using the tepals of Gomphrena globosa L. flowers as an alternative source. Assisted by the statistical technique of response surface methodology, an experimental design was developed for testing the significant explanatory variables of the extraction (time, temperature, solid-liquid ratio and ethanolwater ratio). The identification was performed using high-performance liquid chromatography coupled with a photodiode array detector and mass spectrometry with electron spray ionization (HPLC-PDAMS/ ESI) and the response was measured by the quantification of these compounds using HPLC-PDA. Afterwards, a response surface analysis was performed to evaluate the results. The major betacyanin compounds identified were gomphrenin 11 and Ill and isogomphrenin IJ and Ill. The highest total betacyanins content was obtained by using the following conditions: 45 min of extraction. time, 35•c, 35 g/L of solid-liquid ratio and 25% of ethanol. These values would not be found without optimizing the conditions of the betacyanins extraction, which moreover showed contrary trends to what it has been described in the scientific bibliography. More specifically, concerning the time and temperature variables, an increase of both values (from the common ones used in the bibliography) showed a considerable improvement on the betacyanins extraction yield without displaying any type of degradation patterns.

Optimization of microwave-assisted extraction of hydrophilic and lipophilic antioxidants from a surplus tomato crop by response surface methodology

Tomato is the second most important vegetable crop worldwide and a rich source of industrially interesting antioxidants. Hence, the microwave-assisted extraction of hydrophilic (H) and lipophilic (L) antioxidants from a surplus tomato crop was optimized using response surface methodology. The relevant independent variables were temperature (T), extraction time (t), ethanol concentration (Et) and solid/liquid ratio (S/L). The concentration-time response methods of crocin and β-carotene bleaching were applied, since they are suitable in vitro assays to evaluate the antioxidant activity of H and L matrices, respectively. The optimum operating conditions that maximized the extraction were as follows: t, 2.25 min; T, 149.2 ºC; Et, 99.1 %; and S/L, 45.0 g/L for H antioxidants; and t, 15.4 min; T, 60.0 ºC; Et, 33.0 %; and S/L, 15.0 g/L for L antioxidants. This industrial approach indicated that surplus tomatoes possess a high content of antioxidants, offering an alternative source for obtaining natural value-added compounds. Additionally, by testing the relationship between the polarity of the extraction solvent and the antioxidant activity of the extracts in H and L media (polarity-activity relationship), useful information for the study of complex natural extracts containing components with variable degrees of polarity was obtained.

Microwave-assisted extraction of phenolic acids and flavonoids and production of antioxidant ingredients from tomato: a nutraceutical-oriented optimization study

The production of natural extracts requires suitable processing conditions to maximize the preservation of the bioactive ingredients. Herein, a microwave-assisted extraction (MAE) process was optimized, by means of response surface methodology (RSM), to maximize the recovery of phenolic acids and flavonoids and obtain antioxidant ingredients from tomato. A 5-level full factorial Box-Behnken design was successfully implemented for MAE optimization, in which the processing time (t), temperature (T), ethanol concentration (Et) and solid/liquid ratio (S/L) were relevant independent variables. The proposed model was validated based on the high values of the adjusted coefficient of determination and on the non-significant differences between experimental and predicted values. The global optimum processing conditions (t=20 min; T=180 ºC; Et=0 %; and S/L=45 g/L) provided tomato extracts with high potential as nutraceuticals or as active ingredients in the design of functional foods. Additionally, the round tomato variety was highlighted as a source of added-value phenolic acids and flavonoids.

Solubility and solvation of monosaccharides in ionic liquids

Herein, solubility experimental data for six monosaccharides, viz. D-(+)-glucose, D-(+)-mannose, D-(-)-fructose, D-(+)-galactose, D-(+)-xylose and L-(+)-arabinose, in four ionic liquids (ILs), at temperatures ranging from 288.2 to 348.2 K, were obtained aimed at gathering a better understanding of their solvation ability and molecular-level mechanisms which rule the dissolution process. To ascertain the chemical features that enhance the solubility of monosaccharides, ILs composed of dialkylimidazolium or tetra-alkylphosphonium cations combined with the dicyanamide, dimethylphosphate or chloride anions were investigated. It was found that the ranking of the solubility of monosaccharides depends on the IL; yet, D-(+)-xylose is always the most soluble while D-(-)-fructose is the least soluble monosaccharide. The results obtained show that both the IL cation and the anion play a major role in the solubility of monosaccharides. Finally, from the determination of the respective thermodynamic properties of solution, it was found that enthalpic contributions are dominant in the solubilization process. However, the observed differences in the solubilities of monosaccharides in 1-butyl-3-methylimidazolium dicyanamide are ruled by a change in the entropy of solution.