Some cruciferous plants are classed as “superfood”, because they may have beneficial effects in the treatment and prevention of several health disorders. This property is ascribed to their complex nutritional and phytochemical profile, which includes high concentrations of carotenoids, flavonoids, phenolic acids, and isothiocyanates [1
]. Isothiocyanates are one of the hydrolysis products of glucosinolates (a class of sulphured secondary metabolites from the botanical order Brassicales
) by the enzyme myrosinase [2
]. In the intact plant, myrosinase is stored separately from glucosinolates; when the plant tissue is damaged (by chopping or chewing), the enzyme contacts with glucosinolates and catalyses the lysis [3
]: the reaction produces indoles, nitriles, thiocyanates, or isothiocyanates. Glucosinolates are water-soluble and stable compounds, but isothiocyanates are hydrophobic and very reactive compounds.
Among the aforementioned superfoods, watercress (Nasturtium officinale
—WC) is the richest known source of the gluconasturtiin (phenylethyl glucosinolate) derived phenylethyl isothiocyanate (PEITC) [5
]. PEITC—one of the most interesting isothiocyanates—is a useful antioxidant, antimicrobial, and cancer chemo-preventive agent [3
]. Unfortunately, direct consumption of WC does not ensure the amount and bioaccessibility of PEITC, since, from a biological point of view, the formation of PEITC is easily affected by various factors such as temperature, pH, and presence of additives [3
]. Besides that, from a food-technological point of view, myrosinase is susceptible to heat and can be inactivated during the cooking process, inhibiting the hydrolysis of glucosinolates [7
Therefore, the production and extraction of PEITC from WC discards may represent an opportunity to extract it in a stable and bioactive form taking advantage of an important by-product, generating great value. In that sense, the reported work is scarce and, even more, the existent studies are not using a sustainable approach [9
]. The applied methodologies, in general, are analytical and use polluting organic solvents that are unfeasible at the industrial level. The few works with sustainable methodologies apply more complex techniques and expensive, and not easily scalable [9
]: microwave-assisted ethanol extraction, supercritical fluids.
In recent decades, a wide range of new non-toxic, non-flammable, and biodegradable solvents have been evaluated to develop environmentally friendly and sustainable extraction methods [14
]. Among them, certain surfactants have the mentioned properties, which represent an economic alternative to expensive and dangerous organic solvents. Non-ionic surfactants represent an important class of amphiphiles, which find wide applications in pharmaceutical and industrial formulations, and are widely used in the extraction process and purification of compounds of biological origin [15
]. As indicated by Katsoyannos et al. [18
], micellar systems using non-toxic surfactants (non-ionic, without branched aliphatic chains or aromatic moieties) are suitable for the isolation of natural antioxidants, which can then be used in dietary applications. Among non-ionic surfactants, polyethoxylates are the most numerous and technically interesting [15
], standing out for their widespread use Triton (X-100 and X-114), Brij (−30, −56 and −97), Genapol (X-080), and to a lesser extent, the Tergitols (15-S-X), among others [19
]. Genapol X-080 is a structured non-ionic surfactant of the ethoxylated primary aliphatic alcohols type, with a hydrophobic chain of 12 alkyls and 8 oxyethylene groups (OEs) in its hydrophilic region. For their part, the non-ionic surfactants Tergitol 15-S-7 are a mixture of ethoxylated secondary aliphatic alcohols with 11–15 carbons in the hydrophobic alkyl chain and with an average number of OEs of 7.3 as hydrophilic head. Both Genapol X-080 and Tergitol 15-S-7 are biodegradable and non-toxic. Furthermore, they have the quality of being transparent in the UV 240–280 nm region, facilitating the monitoring of purification processes of aromatic molecules or with conjugated double bonds that adsorb in that spectral zone. The micelles present in colloidal dispersions (aqueous micellar systems—AMSs) of these surfactants can interact with hydrophilic or hydrophobic molecules through different types of interactions, creating conditions for separate purposes [24
]. Previously, we have developed and optimised extractive processes with AMSs to recover low polar compounds, obtaining high yields [19
]. Currently, there is no study on AMSs for the extraction of PEITC, or any isothiocyanate, which opens the doors for an interesting analysis.
In this framework, we carried out a study to apply AMSs to produce extracts enriched with PEITC, developing a profitable process with low environmental impact. As a road map, we pose the following questions: Are Genapol X-080 and Tergitol 15-S-7 AMSs suitable for PEITC extractants, comparable to currently used solvents? What are the variables that most affect the extractive process of PEITC with AMSs? What are the optimal conditions that maximize PEITC extraction?
The use of conventional extraction with n-hexane has led to obtaining an amount of PEITC (Table 2
) almost half of that reported by Rodrigues et al. (3346 µg PEITC/g WC DB) under similar conditions [12
]. Farhana et al., reported higher levels, but not distant values (ca. 2300 µg/g WC DB), using dichloromethane as an extractant under similar conditions [7
]. The production of isothiocyanates can vary, depending on the conditions to which the plant is subjected, more specifically, the temperature and pH value to which the reaction occurs. Besides that, the production also depends on the plant species and age, as well as other factors such as place of cultivation, climatic conditions, storage, and processing [4
]. Considering this, more than in extraction, the difference lies in the production of PEITC itself. The use of the ACN/CF mixture, as an extractant in dispersive liquid-liquid microextraction (DLLME) technique, was developed and validated by Fusari et al. [10
] to determine the content of isothiocyanates in Brassicaceae vegetables. Taking these two methodologies as references, we can assert the efficacy of the analysed AMSs as PEITC extractants.
Nevertheless, we asked the question: Why are these AMSs so effective as organic solvents in extracting a hydrophobic molecule like PEITC? Genapol X-080 and Tergitol 15-S-7 have high effectiveness in interacting with low polar molecules in plant matrices. This appears to be closely related to the amphiphilic character of these surfactants and its ability to form micelles that can interact with hydrophobic groups of PEITC molecule. As previously discussed, it should be noticed that Genapol X-080 and Tergitol 15-S-7 have critical micellar concentrations (CMCs) of 4.6 10−3
and 3.9 10−3
% m/m, respectively [25
]. The concentrations applied in our study were higher than those CMCs. Then, by working on a concentration above the CMC, we provide a favourable environment (micelle) for the dispersion of PEITC in the aqueous solution.
Concerning the factors that affect the extractive process, Farhana, et al., with a one-factor approach, also observed similar effects of pH and temperature on PEITC production [7
]. In that study, they reported that myrosinase activity did not vary between 25 °C and 45 °C, decreasing slightly towards 65 °C. Regarding the pH, the enzymatic activity remained stable between pH 7.0 and 9.0, while, below 7.0, it decreased slightly. However, to produce PEITC, they found more marked behaviours than on enzymatic activity. They established that, at acidic pH, nitrile production is more favoured and therefore PEITC production decreases. However, from their univariate approach, they failed to reliably explain the variability that was not explained by the enzymatic activity of myrosinase. The authors concluded that the higher the activity of the enzyme, the greater the production of PEITC, this being optimal at mild temperatures (ca. 25 °C) and a pH value between neutral to slightly alkaline. Furthermore, they obtained a maximum PEITC production of 5611 µg PEITC/g WC DB in conditions like those predicted as optimal by our polynomial models.
At this point, it is important to mention the appropriateness of using solid statistical tools in the optimisation process. Biological matrices are complex systems strongly affected by different variables and their interactions, making it practically unfeasible to predict accurate behaviours based on the classic one-factor method (one variable at a time). Both multifactorial models indicate a maximisation towards one of the vertices of the design, leaving us the mystery of what could happen in conditions beyond that vertex, that is, at factor levels beyond those analysed. However, within the tested conditions, we can talk about an interesting scenario: these predictions of a unified condition, with similar results, provide the desired scenario of flexible technology, applicable more generally and not restricted to a particular surfactant.
Regarding antioxidant activity, the differences between the pure PEITC and the extracts may be due to the other phytochemicals that the AMSs were able to extract, such as phenolic compounds [19
]. Rodrigues et al. carried out pressurized fluid extraction in watercress and reported a relationship between the extractive increase of phenolic compounds and the antioxidant activity (ORAC) independent of the PEITC content [12
]. Now, this does not mean that PEITC does not have antioxidant capacity. PEITC has been reported as an antioxidant compound, but not by direct action on radicals, but by reducing the reactive oxygen species (ROS) load, being an important inducer/enhancer of antioxidant/detoxification enzymes [33
]. Besides, PEITC may act simultaneously as an oxidant, since it has a high capacity to generate ROS and induce oxidative damage in tumour cells, which makes PEITC “selective” for this cell type [33
]. Thus, the extracts produced have the advantage of providing an antioxidant action through different routes, synergistically combining the properties of PEITC and phenolic compounds.
Although there has been a growing interest in isothiocyanates in recent years, the information available is still scarce. There is practically no solid base of work with a technological focus and still less for the PEITC. Therefore, considering the potential of this compound and the technological relevance it may have soon, an exploratory study of scalable green methodologies for its production and extraction is of high impact.
In the present study, we report the development of a methodology to extract PEITC by applying cost-effective and low environmental impact systems, reaching the extractive capacity of classical organic solvents. Thus, our exploratory and optimisation work—with robust statistical tools—allows us to contribute with an innovative precedent in the state of the art.
The flexibility of selection of different surfactants (biodegradable, non-flammable, and non-toxic) for PEITC extraction with similar results, as well as the possibility of substituting organic solvents in large-scale extraction procedures, make these AMSs suitable for industrial production of PEITC from WC.
As a final consideration, the extracts obtained resulted in an interesting antioxidant product, which may synergistically combine different mechanisms of direct and cellular action against harmful oxidative states. However, this must be studied in greater depth and validated by other methodologies.