2.1 Extraction of the target compounds

Fulfillment of present trends on green processes require to have in mind some key principles:

• • use of non-toxic, non-pollutant extractants;
• • reduction of the extractant volume if the extracted compounds are finally used in the solid state (thus decreasing energy consumption to remove the extractant).

A short extraction time is also desirable to increase production, particularly when dealing with stational crops that require treatment of huge amounts of the target raw material in short times to avoid drying and storage steps. Decrease of the extraction time and an increased efficiency is achieved by application of technologies based on the use of auxiliary energies. After surpassing the boom of supercritical fluid extraction (SFE), which elevated the use of supercritical CO₂ at the panacea category [2], this technology has not been forgotten, but it is almost devoted to the sole extraction of non-polar compounds (mainly fats and oils), as it corresponds to the non-polar nature of the extractant [3]. Other more versatile technologies, with lower costs and easy to scale to pilot plants and then to industrial plants, are gaining popularity. Among the auxiliary energies that apply these new technologies, are ultrasound (US) [4], microwaves (MW) [5] and high temperature + pressure, this last giving place to superheated liquid extraction (SHLE) [6]. Another name given to SHLE is pressurized liquid extraction (PLE), which does not express the effect of the two physical parameters involved in the technique: increased pressure is not the variable that provide to the technique their properties as overpressure is only required to maintain the liquid (extractant) in this state at temperature above its boiling point. High temperature of the extractant is the key for the success of the methods based on SHLE; while the increase of pressure has a negligible effect once the liquid is maintained as such. Fig. 2 shows laboratory extractors based on the use of the three types of energy.

The SHL extractor in Fig. 2A allows both static and dynamic steps. The former is achieved by switching off valves V1 and V2, then applying the pressure–temperature program. The dynamic step starts by switching on valve V1, and regulating V2 to the optimum flow rate and the optimum working pressure at the selected temperature [7].

US-assisted extraction (USAE) can also be developed in a static and/or a dynamic mode. Fig. 2B shows a dynamic extractor in which the extractant is recirculated to minimize dilution of the extract. With this aim, the flow direction is changed at preset intervals thus also avoiding increased compactness of the solid in the extraction chamber [8]. Easy automation of the extraction step is a common characteristic of these extractors that are endowed with reproducibility when the US device is a probe or a bath designed for analytical purposes [4].

MW has demonstrated to be an excellent energy to improve extraction, especially when dealing with polar compounds. Devices for MW irradiation to favor extraction range from domestic ovens to dedicated extractors with strict control of temperature, pressure, power and time for application. Also Soxhlet extraction has been accelerated by using MW with different degrees of similarity to the original technique and with also different success. One of these devices is the focused microwave-assisted Soxhlet extractor (FMASE) designed by the author's team, a dual version of which (with control of the extractant volume and the time for sample–extractant content together with continuous or discontinuous MW irradiation) is shown in Fig. 2C [9].

It could be said that these three technologies cover all the possible needs of matrix–extracted compounds: polar and non-polar compounds, thermostable and thermounstable compounds (US can work at very low temperatures), and compounds resistant to the action of free radicals, as those formed in polar media subjected to high-power US (frequencies in the range 20–40 kHz).

An example of the use of devices based on the three types of energy for the extraction of phenols from olive leaves by using ethanol–water mixtures (Table 1) shows that the superheated liquid extractor provides the most concentrated extract; US-assisted extraction requires the lowest ethanol percentage (probably due to the formation of free radicals which compensate for the lower ethanol content in the extractant); while the MW-assisted extraction is the fastest as it requires only 8 min for quantitative extraction [10]. Therefore, the most appropriate to the given application can be selected.

Formation of very fine emulsions between two immiscible extractants increases the surface contact between both and with the solid subjected to extraction (in this case a more correct name of the step is leaching). Emulsion formation is favored either by US (through the cavitation phenomenon) [11], or by boiling of the extractant with lower boiling point, in the case of MW [12], thus making possible simultaneous extraction of polar and non-polar compounds from plants, as shown in Fig. 3.

2.2 Identification and quantitation of valuable products in the extracts

Extraction is usually followed by one or several of the metabolomics strategies in Fig. 4 to obtain information on the vegetal metabolites extracted from each raw material or to know their effects on living organisms (particularly on humans).

The preferred metabolomics approach to classify the samples is fingerprinting, thus establishing the differences and similarities between cultivars, ripening state, crops, etc., while untargeted analysis is used to obtain a comprehensive profile of the metabolome of the target raw sample. Once the compounds of interest have been identified in the extract, targeted analysis allows quantitation of the given metabolites.

Examples of comprehensive identification of compounds in alperujo – more than 50 compounds – [13] and olive tree leaves – 26 compounds – [14], as well as in lees [15] or volatile fraction of vine shoots (more than 80 volatile compounds, and 18 studied cultivars) and oak ships (more than 50 volatile compounds, and 5 types of wood) [16] appears in recent literature on these subjects.

Once the compounds of interest have been identified, quantitation requires, most times, separation of the target compounds from the rest of the extract and sometimes also preconcentration. An excellent technique for both aims is solid-phase extraction (SPE), which is properly implemented on line with subsequent individual separation, generally by gas or liquid chromatography, and less commonly by capillary electrophoresis (CE). This coupling allows elution of the retained compounds by the chromatographic mobile phase. In this way the total amount of retained compounds is driven to the chromatographic column and maximum sensitivity is obtained as a result. Quantitation by MS is preferred because of its well-known characteristics. Precise quantitation is achieved only if standards of the target metabolites are available to run the corresponding calibration curve; otherwise, only semiquantitative determination is possible by interpolation of the signal provided by the given compound with no available standard within the calibration curve of the compounds with higher similarity.

3.1 Improvement of food quality

An example of how metabolites in the extracts from olive leaves or alperujo can improve the stability of oils is the research on refined oils [18]. These oils lose polar antioxidants in the extraction process that involves liquid–liquid extraction with hexane; therefore, the oils were enriched with extracts from olive leaves or alperujo at different concentrations, as shown in Fig. 5. Maize soy, high-oleic sunflower, sunflower, olive and rapeseed oils were enriched, and lowercase or capital letters were used to name the oils enriched with low (200 μg/mL) or high concentrations (400 μg/mL) of antioxidants in the extracts; “a” and “l” were used to express the type of extract (from alperujo or leaves). The stability of each oil, studied by the Rancimat method [19], is shown in Fig. 6. As can be seen in the figure, all oils improved their stability that, in some cases – as that of refined maize oil enriched with 400 μg/mL of alperujo extract (ROO-A) – became even higher than that of extra virgin olive oil (EVOO) with 400 μg/mL of original content of phenols.

3.2 Improvement of human welfare

A study which supports the effect of olive phenols on heated oils and their impact on the pathways of key, essential fatty acids is the following: virgin olive oil (VOO) containing 400 μg/mL of total phenols and sunflower oil enriched with either an artificial antioxidant (dimethylsiloxane, DMSO) or with an olive phenols extract (in both cases at 400 μg/mL) with the respective names of DSO and ASO were used, together with non-enriched sunflower oil (SO). All four oils were subjected to a simulated frying process consisting of twenty heating cycles of five min at 180 °C; then, the oils were used to prepare breakfasts to be randomly ingested by 26 obese individuals (men and women). Serum was sampled before and after breakfast intake. The effect of the intake through the long-chain alcohols, sterified and non-sterified fatty acids was clearly demonstrated [20], but the most crucial effect on human organisms was on the pathways catalyzed by lipo-oxygenases, cyclo-oxygenases and cytochrome P-450, which give place to prostanoids, including prostaglandins and thromboxanes [21]. These metabolites are involved in pro- and anti-inflammatory processes, and possess pro-aggregating, vasoconstriction and immunosuppressive properties [22], among others. Fig. 7 shows the results of the analyses of serum samples and the effect produced by the intake of the simulated fried oils containing different antioxidants on the changes in the pathways of essential oils (eicosanoids): Fig. 7A illustrates the scores graph by representing the three principal components with higher contribution to explain the observed variability (91%), thus demonstrating that the less affected by the fried oils were the individuals who ingested the fried VOO (closer to the profile of eicosanoids before intake). The most affected individuals were those who ingested the non-enriched oil. Closer to the behavior of VOO was ASO and then that of DSO. Fig. 7B confirms this behavior, showing the scores graph corresponding to each pair control/intervention breakfast, in which the situation more similar to that before intake corresponds to the intake of VOO, then to ASO and DSO and, finally, to the oil without any type of antioxidants.