Supercritical CO2 Extraction As a Tool to Isolate Anti-Inflammatory

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Article Supercritical CO2 Extraction as a Tool to Isolate Anti-Inﬂammatory Sesquiterpene Lactones from Cichorium intybus L. Roots

João P. Baixinho 1,† , José D. Anastácio 1,2,† , Viktoriya Ivasiv 1 , Katarina Cankar 3 , Dirk Bosch 3, Regina Menezes 1,2 , Matthew de Roode 4, Cláudia Nunes dos Santos 1,2 , Ana A. Matias 1 and Naiara Fernández 1,*

1 iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; [email protected] (J.P.B.); [email protected] (J.D.A.); [email protected] (V.I.); [email protected] (R.M.); [email protected] (C.N.d.S.); [email protected] (A.A.M.) 2 CEDOC, Chronic Diseases Research Centre, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Campo dos Mártires da Pátria, 130, 1169-056 Lisboa, Portugal 3 Wageningen University and Research, Wageningen Plant Research, BU Bioscience, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; [email protected] (K.C.); [email protected] (D.B.) 4 Sensus B.V., Oostelijke Havendijk 15, 4704 RA Roosendaal, The Netherlands; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work.

Abstract: Cichorium intybus L. or chicory plants are a natural source of health-promoting compounds in the form of supplements such as inulin, as well as other bioactive compounds such as sesquiterpene Citation: Baixinho, J.P.; Anastácio, lactones (SLs). After inulin extraction, chicory roots are considered waste, with most SLs not being J.D.; Ivasiv, V.; Cankar, K.; Bosch, D.; harnessed. We developed and optimized a new strategy for SL extraction that can contribute to Menezes, R.; de Roode, M.; dos the conversion of chicory root waste into valuable products to be used in human health-promoting Santos, C.N.; Matias, A.A.; Fernández, applications. In our work, rich fractions of SLs were recovered from chicory roots using supercritical N. Supercritical CO2 Extraction as a CO2. A response surface methodology was used to optimize the process parameters (pressure, Tool to Isolate Anti-Inflammatory temperature, flow rate, and co-solvent percentage) for the extraction performance. The best operating Sesquiterpene Lactones from conditions were achieved at 350 bar, 40 ◦C, and 10% EtOH as a co-solvent in a 15 g/min flow Cichorium intybus L. Roots. Molecules 2021, 26, 2583. https://doi.org/ rate for 120 min. The extraction with supercritical CO2 revealed to be more selective for the SLs 10.3390/molecules26092583 than the conventional solid–liquid extraction with ethyl acetate. In our work, 1.68% mass and a 0.09% sesquiterpenes yield extraction were obtained, including the recovery of two sesquiterpene Academic Editor: Mara G. Freire lactones (8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin), which, to the best of our knowledge, are not commercially available. A mixture of the abovementioned compounds were tested at Received: 7 April 2021 different concentrations for their toxic profile and anti-inflammatory potential towards a human Accepted: 26 April 2021 calcineurin/NFAT orthologue pathway in a yeast model, the calcineurin/Crz1 pathway. The SFE Published: 28 April 2021 extract obtained, rich in SLs, yielded results of inhibition of 61.74 ± 6.87% with 50 µg/mL, and the purified fraction containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin inhibited the Publisher’s Note: MDPI stays neutral activation of the reporter gene up to 53.38 ± 3.9% at 10 µg/mL. The potential activity of the purified with regard to jurisdictional claims in fraction was also validated by the ability to inhibit Crz1 nuclear translocation and accumulation. published maps and institutional affil- These results reveal a possible exploitable green technology to recover potential anti-inflammatory iations. compounds from chicory roots waste after inulin extraction.

Keywords: supercritical CO2 extraction; sesquiterpene lactones; anti-inﬂammatory potential

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons At present, there is a growing interest in the potential beneﬁts of nutritional com- Attribution (CC BY) license (https:// pounds for disease prevention, which in turn also means an increasing demand for natural creativecommons.org/licenses/by/ and nutritional food [1]. Biologically active compounds and functional ingredients (vi- 4.0/). tamins, antimicrobials, antioxidants, colorants, etc.) from natural sources are of great

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commercial interest, especially in the form of dietary supplements. The taproot of chicory (Cichorium intybus L., Asteraceae family) contains several health-related compounds such as inulin, flavonoids, vitamins, and sesquiterpene lactones (SLs) [2–4]. Chicory leaves have been consumed in Europe for many centuries, and the roots are currently used for the production of inulin, a fructose-based polymer that is added to numerous food products as a dietary fiber and sweetener [5]. After inulin extraction, the remaining biomass is considered waste, even though the roots are rich in other health-promoting compounds such as SLs. Several bioactivities have been previously associated with SLs, for instance, those that are antimicrobial, anti-inflammatory, and antiparasitic [3,5–12]. The most rel- evant SLs previously identified in chicory roots [13–15] include lactucin, lactucopicrin, 11β-13-dihydrolactucin, 11β-13-dihydrolactucopicrin, and 8-deoxylactucin [16]. SL extraction from several Asteraceae species has been carried out using different organic solvents or mixtures such as methanol, ethyl acetate, chloroform, n-hexane, and ethanol (EtOH) [17–22]. The need to minimize toxic solvent utilization has motivated the development of new environment-friendly processing technologies that can be carried out at the industrial scale. In this field, high pressure technologies are revealed to be remarkably efficient in enabling a more selective and effective recovery of high-value compounds from natural sources [23–25]. One such technology, supercritical fluid extraction (SFE), is generally performed using carbon dioxide (CO2). CO2 has a moderately low critical pressure and temperature (74 bar, 32 ◦C) and is nontoxic; it is available in high purity at relatively low costs and can be easily removed from the extract [26–28]. Supercritical CO2 is an excellent solvent for the extraction of nonpolar and low molecular weight compounds [25,29]. Solvent extraction power and selectivity have been widely studied and depend especially on SFE operating conditions, which includes the possibility of adding a co-solvent (usually water or EtOH) to modify supercritical CO2 polarity, enhancing target molecules solubility [23,25,26]. The combination of CO2 properties turns SFE into a sustainable and safe extraction technique for the extractions of ingredients for supplements and health care products [24,26,27]. The purpose of this study is to contribute to the valorization of chicory root by developing for the first time a high-pressure strategy to extract SLs for human health- promoting applications. Chicorium intybus L. SFE extract is highly rich in different SLs such as lactucin, lactucopicrin, 11β-13-dihydrolactucin, 11β-13-dihydrolactucopicrin, 8- deoxylactucin, and 11β,13-dihydro-8-deoxylactucin. After a chromatographic process, a purified fraction of a mixture of 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin could be isolated. The SFE extract and the purified fraction are tested in vitro for its toxicity at different concentrations as well its anti-inflammatory potential. In our study, the SFE extract was able to inhibit 61.74 ± 6.87% of the transcription factor activity with a concentration of 50 µg/mL an inhibition rate in the same range of those observed for the immunosuppressant pharmaceutical drug FK506 also known as tacrolimus. Furthermore, the purified fraction was also tested for the potential anti-inflammatory capacity, revealing a capacity to inhibit Crz1 activation by 53.38 ± 3.9% at 10 µg/mL.

2. Results and Discussion 2.1. Supercritical CO2 Extraction of SLs Freeze-dried and milled (particle size < 1 mm) Cichorium intybus L. roots were subjected to SFE. Based on the preliminary extractions (data not shown) with supercritical CO2 as a solvent, the procedure resulted in a low mass yield extraction (0.25 mg extract/g raw material) while the mass yield increased when an organic co-solvent (10% EtOH) was used (10 mg extract/g raw material). An extraction kinetic was made (data not shown) at 300 bar and at 50 ◦C with a ﬂow rate of 10 g/min, including 10% EtOH as a co-solvent, and 120 min was considered the best extraction time for the SFE processing of chicory roots. Molecules 2021, 26, 2583 3 of 17 Molecules 2021, 26, x FOR PEER REVIEW 3 of 17

A design of experiments (DOE)(DOE) waswas plannedplanned in in order order to to optimize optimize the the extraction extraction proce- pro- ceduredure and and to to study study the the effect effect of of process process variables variables on on the the mass mass yieldyield andand SLSL concentrationconcentration in the the extract extract.. SFE SFE extracts extracts were were characterized characterized by byHPLC HPLC-DAD-DAD and and were were based based on the on SLs the thatSLs thatare most are most abundant abundant in chicory in chicory roots roots[8,14,18,30] [8,14,18. Four,30]. Fourdifferent different compounds compounds were iden- were tifiedidentified by comparing by comparing the the retention retention times times of of the the commercially commercially available available SL SL standards: 1111β,13β,13-dihydrolactucin,-dihydrolactucin, lactucin lactucin,, 1111β,13β,13-dihydrolactucopicrin,-dihydrolactucopicrin, and lactucopicrin. The SL concentration in the extract was calculated based on the sum of the abovementioned SLs (Figure1 1).). InIn aa firstfirst approachapproach flowflow rate rate (10 (10 g/min), g/min), thethe extractionextraction timetime (120(120 min),min), andand thethe cco-solvento-solvent percentagepercentage (10%(10% EtOH)EtOH) were were kept kept constant constant to to evaluate evaluate the the effect effect of temperatureof tempera- ◦ ture(40–80 (40–C)80 and°C) and pressure pressure (100–550 (100–550 bar) bar) (see (see Table Table1, DOE1, DOE 1, 1, two two factors factors with with three three level level faceface-centered-centered composite design).

Figure 1. Major sesquiterpene lactones ident identiﬁedified in Cichorium intybus L. root extracts.

Table 1. Variables used in the two different design of experiments (DOE) and actual values of var- Table 1. Variables used in the two different design of experiments (DOE) and actual values of iables for the coded values. First process optimization (DOE 1) with 2 process variables, i.e., tem- variables for the coded values. First process optimization (DOE 1) with 2 process variables, i.e., perature and pressure, and after the final process parameters optimization (DOE 2) with 3 process variables,temperature i.e., and flow pressure, rate, co and-solvent after percentage the final process, and temperature. parameters optimization (DOE 2) with 3 process variables, i.e., flow rate, co-solvent percentage, and temperature. Levels Levels Variable, unit −1 0 1 Variable,Temperature, unit −1 0 1 Temperature, ◦C40 60 80 DOEDOE 1 1 °C Pressure, bar 100 325 550 Pressure, bar 100 325 550 ◦ Temperature,Temperature,C 25 33 40 DOE 2 Co-solvent, % EtOH25 10 33 25 40 Flow rate,°C g/min 10 20 30 Co-solvent, % DOE 2 10 25 40 EtOH The obtained results regarding mass and SL concentration in the extract are shown in Flow rate, Figure2. The regression analysis of the data shows10 that for a 90%20 confidence level30 ( p < 0.10), the SL concentration ing/min the extract was negatively affected by extraction temperature (p = 0.060). While the effect of CO2 pressure was not significant for the SL concentration, this parameterThe obtained had aresults significantly regarding quadratic mass and effect SL onconcentration mass yield (inp = the 0.040), extract which are shown means thatin Figure the optimal 2. The levels regression of pressure analysis are of found the data in the show middles that of for the a experimental90% confidence range. level (p < 0.10), the SL concentration in the extract was negatively affected by extraction temperature (p = 0.060). While the effect of CO2 pressure was not significant for the SL concentration, this parameter had a significantly quadratic effect on mass yield (p = 0.040), which means that the optimal levels of pressure are found in the middle of the experimental

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range.

FigureFigure 2. 2.The The extraction extraction of of SL SL was was carried carried out out following following a a Central Central Composite Composite Face-Centered Face-Centered Design Design (DOE (DOE #1) #1) as as a a function function ofof pressure pressure and and temperature. temperature.Experiment Experiment numbersnumbers (1–10)(1–10) correspondcorrespond toto thetheextraction extraction numbernumber assay,assay, white white diamonds diamonds (values(values linked linked to to right right axis) axis) represent represent extraction extraction mass mass yield yield and and coloured coloured bars bars (values (values linked linked to to left left axis) axis) represent represent the the

concentrationconcentration of of the the major major SLs SLs extracted extracted from fromCichorium Cichorium intybus intybusL. L roots. roots from from supercritical supercritical CO CO2 2extraction extraction process. process.

AsAs graphically graphically described described in Figurein Figure2, the 2, maximum the maximum mass mas yields wasyield obtained was obtained (5.73 mg/g (5.73 rawmg/g material) raw material) when the when conditions the conditions depicted depicted in assay in 9 assay were applied9 were applied (80 ◦C, ( 32580 ° bar,C, 325 and bar a , continuousand a continuous flow rate flow of 10 rate g/min of 10 CO g/min2 with CO 10%2 with of 10% EtOH of as EtOH the co-solvent), as the co-solvent corroborating), corrob- withorating the predictedwith the predicted results by results the model. by the model On the. On other the hand, other thehand, minimum the minimum yield yield was achievedwas achieved when thewhen extraction the extraction was conducted was conducted at lower at pressures lower pressures (100 bar) (100 as represented bar) as repre- in assayssented 1, in 4, andassays 8, with 1, 4, theand lowest 8, with value the lowest (0.43 mg/g value raw (0.43 material) mg/g raw achieved material) at high achieved temper- at atureshigh (assaytemperatures 8). Regarding (assay the 8). SLRegarding concentration the SL in concentration the extract, the in effect the extract of the, temperature the effect of isthe visible temperature when comparing is visible extraction when comparing assays 1, 4, extraction and 8. At assays the same 1, pressure4, and 8. (100 At bar),the same the ◦ SFEpressure performed (100 atbar), a lower the SFE temperature performed (40 atC) a lowe resultedr temperature in an extraction (40 °C) four resulted times higher in an ex- in SLstraction (42.7; 8.2four and times 11.6 higherµg/mg in extract, SLs (42.7 respectively); 8.2 and 11. compared6 µg/mg extract to the, onesrespectively performed) compared at high ◦ temperatureto the ones (80performedC). The at most high selective temperature extraction (80 °C). is representedThe most selective in assay extraction 1 (42.7 µ g/mgis rep- extract)resented and in the assay lowest 1 (42.7 in assay µg/mg 4 (8.2extract)µg/mg and extract). the lowest Since in noassay optimal 4 (8.2 extraction µg/mg extract). point wasSince obtained no optimal with extraction DOE 1, a point second was DOE obtained (Table with1, DOE DOE 2) 1, wasa second created DOE to (Table narrow 1, theDOE optimal2) was extractioncreated to regionnarrow based the optimal on previously extraction obtained region resultsbased on with previously a fixed temperature obtained re- ◦ atsults 40 C with and a pressure fixed temperature at 350 bar. Aatset 40 of°C experiments, and pressure including at 350 bar. three A set center of experiments, points, was carried out to optimize new variables, namely the co-solvent percentage (10–40% EtOH), including three center points, was carried out to optimize◦ new variables, namely the co- flowsolvent rate (10–30percentage g/min), (10– and40% lowEtOH), temperature flow rate (25–40(10–30 g/min)C) in the, and extraction low temperature process. (25–40 °C)Figure in the3 extraction shows mass process. and SL concentration in the extract obtained in DOE 2. They were used toFigure estimate 3 shows the linear mass andand quadraticSL concentration effects of in the the variables extract obtained and their in synergies. DOE 2. They The response surfaces (Figure3) fitted to the SL concentration in the extract can be described were used to estimate the linear and quadratic effects of the variables and their synergies. by second-order polynomial models as a function of co-solvent percentage, temperature, The response surfaces (Figure 3) fitted to the SL concentration in the extract can be de- and flow rate (Table2). In these response surface models, for a 95% confidence level, scribed by second-order polynomial models as a function of co-solvent percentage, tem- the significant effects of p < 0.05 were included in the model equations. The high values perature, and flow rate (Table 2). In these response surface models, for a 95% confidence for both R2 and R 2 of these models (Table2) suggest a close agreement between the level, the significantadj effects of p < 0.05 were included in the model equations. The high experimental data and the theoretical values predicted by the model. values for both R2 and Radj2 of these models (Table 2) suggest a close agreement between the experimental data and the theoretical values predicted by the model.

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FigureFigure 3. 3.The The extractionextraction ofof SLSL waswas carriedcarried outout followingfollowing aa CentralCentral CompositeComposite Face-CenteredFace-Centered Design Design (DOE (DOE #2) #2) as as a a function function ofof pressure, pressure, temperature, temperature, flow flow rate rate and and co-solvent co-solvent percentage. percentage. Experiment Experiment numbersnumbers (1–17)(1–17) correspondcorrespond toto the the extraction extraction numbernumber assay, assay, white white diamonds diamonds (values (values linked linked to right to right axis) axis) represent represent extraction extraction mass yieldmass andyield coloured and coloured bars (values bars (values linked linked to left axis) represent the concentration of the major SLs extracted from Cichorium intybus L. roots from supercritical to left axis) represent the concentration of the major SLs extracted from Cichorium intybus L. roots from supercritical CO2 CO2 extraction process. extraction process. The analysis of the second response surface model for SL extraction (Figure 4) shows Table 2. Model equations for the response surfaces, after final SFE optimization (DOE 2), fitted to the values of mass yield that there is a positive effect of temperature and a negative effect of co-solvent percentage (MY) and the four major SLs extracted as a function of temperature (A), co-solvent percentage (B), and flow rate (C), and 2 2and flow rate in the SL concentration in the extract. their respective R and Radj .

Table 2. Model equations for the response surfaces, after final SFE optimization (DOE2 2), fitted to the values of mass2 yield Polynomial Model Equations R Radj (MY) and the four major SLs extracted as a function of temperature (A), co-solvent percentage (B), and flow rate (C), and MY = 1.232 + 0.936A + 0.403B − 0.124A2 + 0.285BC 0.99 0.98 their respective R2 and Radj2. 11β,13-dihydrolactucin = 4.2 + 0.748A − 6.096B − 1.519C + 4.01B2 0.98 0.97 − − Lactucin = 6.262 +Polynomial 1.046A 9.261B Model2.155C Equations + 6.088B2 0.98R2 0.98Radj2 11β,13-dihydrolactucopicrin = 1.688 − 0.876B 0.90 0.87 LactucopicrinMY = 1.232 = 5.564 + 0.936A− 4.293B + 0.403B− 1.271C − 0.124A2 + 1.236B2 + 0.285BC 0.960.99 0.940.98 11β,13-dihydrolactucin = 4.2 + 0.748A − 6.096B − 1.519C + 4.01B2 0.98 0.97 Lactucin = 6.262 + 1.046A − 9.261B − 2.155C + 6.088B2 0.98 0.98 The analysis of the second response surface model for SL extraction (Figure4) shows 11β,13-dihydrolactucopicrinthat there is a positive = 1.688 effect − 0.876B of temperature and a negative0.90 effect of co-solvent0.87 percentage Lactucopicrin =and 5.564 ﬂow − 4.293B rate in − the 1.271C SL concentration + 1.236B2 in the extract. 0.96 0.94 As already observed in the previous design of experiments, temperature is the most importantAs already parameter observed for the in selectivity the previous of the design process. of experiments, By varying the temperature temperature is again,the most it wasimportant possible parameter to obtain thefor the value selectivity that best of favors the process the extraction. By varying of the the SLs temperature from Cichorium again, intybusit was L.possible roots. to This obtain positive the value effect that means best favors that the the increase extraction in of temperature the SLs from favors Cichorium the extractionintybus L up. roots. to 40 This◦C, andpositive beyond effect this means value, that this the parameter increase has in atemperature negative effect favors in thethe process.extraction As shownup to 40 in °C Figure, and4 beyond, the best this temperature value, this toparameter extract all has the a fournegative SLs is effect 40 ◦ C.in the process. As shown in Figure 4, the best temperature to extract all the four SLs is 40 °C.

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FigureFigure 4.4. ResponseResponse surfacessurfaces (DOE(DOE 2)2) fittedfitted toto thethe SLSL concentrationconcentration inin thethe extractextract asas aa functionfunction ofof temperature,temperature, co-solventco-solvent percentage (% EtOH), and flow rate. The white squares show the concentration (µg/mg extract) of each compound de- percentage (% EtOH), and flow rate. The white squares show the concentration (µg/mg extract) of each compound pending on the conditions of the three axes. In blue, the minimum extraction; in red, the maximum extraction achieved depending on the conditions of the three axes. In blue, the minimum extraction; in red, the maximum extraction achieved for for each SL. To maximize SL extraction, the best SFE is conducted at high temperature◦ (40 °C) with a low flow rate (10 eachg/min) SL. and To maximizelow co-solvent SL extraction, percentage the (10% best EtOH). SFE is conducted at high temperature (40 C) with a low flow rate (10 g/min) and low co-solvent percentage (10% EtOH). When comparing all extraction attempts (Figure 3), we can see that the best selectivity When comparing all extraction attempts (Figure3), we can see that the best selectivity results are obtained with low co-solvent percentages (assays 1, 2, 6, 13, and 14). Therefore, results are obtained with low co-solvent percentages (assays 1, 2, 6, 13, and 14). Therefore, the best selectivity result was obtained when the extraction was conducted at 40 °C with the best selectivity result was obtained when the extraction was conducted at 40 ◦C with a low flow rate (10 g/min) and low co-solvent percentage (10% EtOH). This extraction is a low flow rate (10 g/min) and low co-solvent percentage (10% EtOH). This extraction is characterized by presenting a 5.91% SL content on the final extract, and it is represented characterized by presenting a 5.91% SL content on the final extract, and it is represented in in assay 13 (Figure 3). assay 13 (Figure3). In contrast, the mass yield was higher when the extraction process was performed at In contrast, the mass yield was higher when the extraction process was performed at a a high flow rate (30 g/min) with high co-solvent percentages (40% EtOH) (assays 5, 12, high flow rate (30 g/min) with high co-solvent percentages (40% EtOH) (assays 5, 12, and 17;and Figure 17; Figure3). This 3). resultThis result was expected was expected because because a reduced a reduced selective selecti extractionve extraction is obtained is ob- whentained high when flow high rates flow and rates co-solvents and co-solvents are applied are applied in SFE. in However, SFE. However, an increase an increase in extract in massextract does mass not does mean not an mean increase an increase in SL content, in SL co asntent shown, as shown in Figure in3 F.igure As it can3. As be it noted,can be whennoted,the when polarity the polarity of the of extractant the extractant solvent solvent is changed, is changed, the finalthe final mass mass characterization characterization can also be altered. Moreover, as the solvent (CO2 + EtOH) flow rate was increased,

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Molecules 2021, 26, 2583 7 of 17 independently from the temperature applied, the extraction capacity of the described system increased (red area, Figure 5). Here, the best conditions to increase the mass yield were 40 °C, 30 g/min flow rate with 40% EtOH as the co-solvent, resulting in a 260 mg/g

canraw alsomaterial be altered. with 0.91% Moreover, of SLs in as its the composition solvent (CO (assay2 + EtOH) 17; Figure ﬂow 3) rate. was increased, independentlyAn optimal from condition the temperature to maximize applied, both responses the extraction (mass yield capacity and ofSL theyield) described was not systemobtained, increased but a region (red area, where Figure all criteria5). Here, we there met best was conditions found at to 350 increase bar, 40 the °C mass with yielda con- weretinuous 40 ◦flowC, 30 rate g/min of 13 ﬂow–19 rateg/min with flow 40% with EtOH 10% as EtOH the co-solvent, as a co-solvent resulting; this inregion a 260 is mg/g com- rawmonly material called with the sweet 0.91% spot. of SLs in its composition (assay 17; Figure3).

FigureFigure 5. 5.Response Response surfacessurfaces (DOE 2) fitted fitted to to the the extraction extraction mass mass yield yield (g/g (g/g raw raw material) material) as asa function a function of temperature, of temperature, co- co-solventsolvent percentage percentage (%EtOH) (%EtOH),, and and flow flow rate. rate. The The white white squares squares show show the the mass mass (mg) extracted depending onon thethe conditionsconditions ofof the the three three axes. axes. In In blue, blue, the the minimum minimum extraction; extraction in; in red, red, the the maximum maximum mass mass extracted extracted in SFE. in SFE. To maximizeTo maximize mass mass yield, yield, the bestthe best SFE isSFE conducted is conducted at a highat a high flow flow rate (30rate g/min) (30 g/min) with with a high a high co-solvent co-solvent percentage percentage (40% (40% EtOH). EtOH).

AnFrom optimal the sweet condition spot, the to maximizebest operating both conditions responses were (mass set yield, corresponding and SL yield) to a was 120 ◦ notmin obtained,, at 350 bar, but 40 a region°C, and where 15 g/min all criteriaflow with were 10% met EtOH was foundas the atco- 350solvent bar,.40 ThisC optimi- with a continuouszation resulte flowd in rate a 16 of.8 13–19 mg/g g/minraw material flow with extraction 10% EtOH with as53a µg/mg co-solvent; extract this of regionSLs in isits commonlycomposition, called accounting the sweet for spot. an acceptable balance between mass yield and SL concentration Fromin the theextract sweet. spot, the best operating conditions were set, corresponding to a 120 min, at 350 bar, 40 ◦C, and 15 g/min flow with 10% EtOH as the co-solvent. This optimization2.2. Conventional resulted Solid– inLiquid a 16.8 Extraction mg/g raw of SL material extraction with 53 µg/mg extract of SLs in its composition, accounting for an acceptable balance between mass yield and SL SFE performance was compared to the conventional solvent extraction. Water and concentration in the extract. ethyl acetate were used as two different solvents to extract SLs from freeze-dried and 2.2.milled Conventional Cichorium Solid–Liquid intybus L. roots. Extraction Overall, ofSL the two extractions resulted in a low SL concentration, i.e., 7.5 and 6.6 µg/mg extract, respectively. Although the mass yield was higher SFE performance was compared to the conventional solvent extraction. Water and in the water extract (31%) compared to SFE extraction (1.68%), the ethyl acetate extract ethyl acetate were used as two different solvents to extract SLs from freeze-dried and milled resulted in a similar mass yield (1.54%). This fact is explained by the solvent extraction Cichorium intybus L. roots. Overall, the two extractions resulted in a low SL concentration, power as the capacity of water to extract compounds with a variety of polarities [4,31,32]. i.e., 7.5 and 6.6 µg/mg extract, respectively. Although the mass yield was higher in the water extract (31%) compared to SFE extraction (1.68%), the ethyl acetate extract resulted 2.3. Fractionation of Cichorium Intybus L. Extract in a similar mass yield (1.54%). This fact is explained by the solvent extraction power as the capacityThe best of waterSFE chicory to extract extract compounds was fractionated with a variety by flash of polarities column [ 4 chromatography.,31,32]. Three different fractions were obtained: fraction 1 (retention factor = 0.79; 6.99% purifica- 2.3.tion Fractionation yield), fraction of Cichorium 2 (retention intybus factor L. = Extract 0.62; 7.01% purification yield), and fraction 3 (re- tentionThe factor best SFE = 0.53, chicory 2.82% extract purification was fractionated yield) The by fractions flash column were chromatography.analyzed by HPLC Three and different11β,13-dihydrolactucin fractions were and obtained: lactucin fraction were identified 1 (retention in fraction factor = 2 0.79; and 6.99%11β,13 purification-dihydrolac- yield),tucopicrin fraction and 2 lactucopicrin (retention factor in fraction = 0.62; 7.01%3 (Figure purification 6). yield), and fraction 3 (retention factor = 0.53, 2.82% purification yield) The fractions were analyzed by HPLC and 11β,13- dihydrolactucin and lactucin were identified in fraction 2 and 11β,13-dihydrolactucopicrin and lactucopicrin in fraction 3 (Figure6).

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FigureFigure 6. 6. HPHPLC-DADLC-DAD chromatograms chromatograms (λ = (λ 480= 480nm) nm) of standards of standards (numbers (numbers represent represent the different the different SL obtained SL obtained in the incor- the respondingcorrespondingFigure 6. fraction: HP fraction:LC-DAD 1-11β,13 1-11chromatograms-βdihydrolactucin;,13-dihydrolactucin; (λ = 480 2- Lactucin;nm) 2-Lactucin; of standards 3-8-deoxylactucin 3-8-deoxylactucin (numbers represent and and 11β,13 11theβ-dihydro ,13-dihydro-8-deoxylactucin;different-8 SL-deoxylactucin; obtained in the 4- corresponding fraction: 1-11β,13-dihydrolactucin; 2-Lactucin; 3-8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin; 4- 11β,134-11β-,13-dihydrolactucopicrin;dihydrolactucopicrin; 5-Lactucopicrin), 5-Lactucopicrin), the the best best SFE SFE extract extract (350 (350 bar, bar, 40 40 °C,◦C, 10% 10% EtOH EtOH as as co co-solvent-solvent and and 15 15 g/min g/min flow11β,13 rate during-dihydrolactucopicrin; 120 min) and the 5 three-Lactucopicrin), different fractions the best obtained SFE extract by flash(350 bar,chromatography 40 °C, 10% EtOH using as Silica co-solvent gel 60 and mesh 15 asg/min flow rate during 120 min) and the three different fractions obtained by flash chromatography using Silica gel 60 mesh as stationaryflow rate phase during and 120Dichloromethane:Methanol min) and the three different (95:5 fractions v/v) as mobile obtained phase. by flash chromatography using Silica gel 60 mesh as stationarystationary phase phase and and Dichloromethane:Methanol Dichloromethane:Methanol (95:5 (95:5v/v v)/ asv) as mobile mobile phase. phase. FractionFraction 1 1could could not not be be identified identified with with the the available available SL SL standards standards,, and and so so it it was was then then Fraction 1 could not be identified with the available SL standards, and so it was then analyzedanalyzed using using LC LC-MS-MS (Figure (Figure 7)7).. The The analysis analysis show showeded that that two two compounds compounds were were present present analyzed using LC-MS (Figure 7). The analysis showed that two compounds were present inin th thatat fraction. fraction. Compound Compound 1 1eluting eluting at at the the retention retention time time of of 17.5 17.5 min min,, with with the the accurate accurate massin [M+H]that fraction.+ of+ 261.11252 Compound, correspond 1 elutings to at thethe elementalretention tformulaime of 17.5 C15 Hmin16O, 4with typical the ofaccurate 8- mass [M+H] of 261.11252, corresponds to the elemental formula C15H16O4 typical of mass [M+H]+ of 261.11252, corresponds to the elemental formula C15H16O4 typical of 8- deoxylactucin8-deoxylactucin (at (at1.47 1.47 ppm ppm mass mass accuracy). accuracy). Compound Compound 2 eluting 2 eluting at atthe the retention retention time time of of deoxylactucin (at 1.47 ppm mass +accuracy). Compound 2 eluting at the retention time of 18.0618.06 min min,, with with accurate accurate mass mass [M+H] [M+H] +ofof 263.12811 263.12811,, corresponds corresponds to to the the elemental elemental formula formula + C15H18.0618O4, mincharacteristic, with accurate of 11β,13 mass- dihydro[M+H] -of8- 263.12811deoxylactucin, corresponds (at 1.23 ppm to the mass elemental accuracy) formula. C15H18O4, characteristic of 11β,13-dihydro-8-deoxylactucin (at 1.23 ppm mass accuracy). BothBothC compounds15 compoundsH18O4, characteristic were were previously previously of 11β,13 described described-dihydro in in-8 chicory- chicorydeoxylactucin extracts extracts (at[17, [17 1.233,333].]. ppm mass accuracy). Both compounds were previously described in chicory extracts [17,33].

FigureFigure 7. 7. MassMass spectrum spectrum of of the the compounds compounds 1 1and and 2. 2. Compound Compound 1 1was was identified identified as as 8- 8-deoxylactucin,deoxylactucin, and and compound compound 2 2was was Figure 7. Mass spectrum of the compounds 1 and 2. Compound 1 was identified as 8-deoxylactucin, and compound 2 was identifiedidentified as as 11β,13 11β,13-dihydro-8-deoxylactucin.-dihydro-8-deoxylactucin. identified as 11β,13-dihydro-8-deoxylactucin. 2.4. SFE Extract Potential Anti-Inflammatory Activity 2.4. SFE Extract Potential Anti-Inflammatory Activity 2.4.The SFE SFE Extract extracts Potential were Anti-Inflammatory then tested for their Activity potential for anti-inflammatory activity. CytotoxicTheTheity SFE evaluationSFE extracts extracts wereon were yeast then then cells tested testedshowed for for their a theirtoxicity potential potential for concentration for for anti-inflammatory anti-inflammatorys higher than activity. activity 500 . µg/mLCytotoxicityCyto (Ftoxicigureity evaluation 8A evaluation). The potential on on yeast yeast of cells cellsan SFE showed showed extract aa toxicitytoxicity to attenuate for concentration concentrations inflammatorys higher higherresponses than than 500 500µg/mLµg/mL (F (Figureigure 8A8A).). The The potential ofof anan SFE SFE extract extract to to attenuate attenuate inflammatory inflammatory responses responses

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throughthrough the the modulation modulation of ofCrz1, Crz1, the the yeast yeast ortholog orthologueue of the of human the human nuclear nuclear factor factor of acti- of vatedactivated T-cells T-cells (NFAT) (NFAT) pathway pathway,, was also was evaluated also evaluated for the for nontoxic the nontoxic concentrations concentrations of the SFEof the extract SFE extract(Figure (Figure 8B). SFE8B). demonstrated SFE demonstrated a higher a higher anti-inflammatory anti-inflammatory potential potential at con- at centrationsconcentrations such such as 50 as and 50 and 75 75µg/mL,µg/mL, reducing reducing the the calcineurin calcineurin/Crz1/Crz1 pathway pathway a activation,ctivation, upup to to 61.74 61.74 ± ±6.876.87%% and and 59.5 59.5 ± 0±.040.04%,%, respectively respectively,, at rates at rates statistically statistically comparable comparable to the to positivethe positive control control FK506 FK506 (62.48 (62.48 ± 2.10±%2.10%)) with a with calculated a calculated IC50 of IC38.8150 of ± 38.810.32 µg/mL± 0.32 (µFg/mLigure 8B(Figure). Both8 B).in human Both in and human in yeast, andin i.e., yeast, NFAT i.e., and NFAT Crz1, and respectively, Crz1, respectively, are under are the under control the ofcontrol calcineurin. of calcineurin. Human Humancalcineurin calcineurin and yeast and calcineurin yeast calcineurin share a sharehigh aevolutionary high evolutionary simi- laritysimilarity,, making making this thismodel model ideal ideal for forthe the screening screening of ofcompounds compounds with with anti anti-inflammatory-inflammatory potential.potential. In In the the presence presence of of a a stim stimulusulus (MnCl (MnCl22),), Crz1 Crz1 dephosphorylates dephosphorylates from from calcineurin calcineurin andand migrates migrates towards towards the nucleus nucleus,, thusthus promotingpromoting thethe expression expression of of calcineurin calcineurin dependent depend- entresponse response element element (CDRE) (CDRE) regulated regulated genes genes similarly similarly to humanto human NFAT. NFAT. SFE SFE extract extract is rich is richin SLs, in SL whichs, which have have been been already already demonstrated demonstrated to to possesspossess anti-inflammatoryanti-inflammatory potential potential inin a a previous previous study study [ [3344].]. However, However, this this extract extract contains contains unexplored unexplored SLs SLs such such as as the the 8 8-- deoxylactucindeoxylactucin and and 11 11ββ,13,13-dihydro-8-deoxylactuin,-dihydro-8-deoxylactuin, whose whose anti anti-inflammatory-inflammatory activity activity was was notnot previously previously described described towards towards the the c calcineurin/Crz1alcineurin/Crz1 pathway. pathway.

Figure 8. SFE extract potentialFigure for inhibiting 8. SFE extract calcineurin/Crz1 potential for activation. inhibiting calcineurin/Crz1(A) Toxicity of SFE activation. extract was (A) evaluated Toxicity of at SFE the extract indicated concentrations of SFEwas in evaluated µg/mL. ( atB) theAnti indicated-inflammatory concentrations potential of SFE extract in µg/mL. as demonstrated (B) Anti-inflammatory by the inhibi- potential tion of the calcineurin/Crz1 ofpathway SFE extract in S. ascerevisiae demonstrated. Statistical by differences the inhibition are ofnoted the calcineurin/Crz1as * p < 0.1, ** p < pathway0.01, *** p in < S.0.001 cerevisiae . relative to the activated controlStatistical (cells treated differences with areMnCl noted2). as * p < 0.1, ** p < 0.01, *** p < 0.001 relative to the activated control (cells treated with MnCl2). 2.5. Purified Fraction Anti-Inflammatory Potential 2.5. Purified Fraction Anti-Inflammatory Potential As mentioned above, other pure SLs present in the SFE extract, such as lactucin, lac- As mentioned above, other pure SLs present in the SFE extract, such as lactucin, tucopicrin, 11β,13-dihydrolactucin and 11β,13-dihydrolactucopicrin, all of which are com- lactucopicrin, 11β,13-dihydrolactucin and 11β,13-dihydrolactucopicrin, all of which are mercially available, were already investigated for their anti-inflammatory potential in our commercially available, were already investigated for their anti-inflammatory potential in previous study [34]. Therefore, we decided to explore the potential of this novel purified our previous study [34]. Therefore, we decided to explore the potential of this novel puri- fraction containing a mixture of 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin. In fied fraction containing a mixture of 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin. order to measure its associated toxicity, yeast cells were exposed to different concentra- In order to measure its associated toxicity, yeast cells were exposed to different concentrations of SFE purified fraction. Here, 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin tions of SFE purified fraction. Here, 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin

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demonstrated no toxicity towards yeast cells at concentrations ranging from 12.5 µg/mL demonstrated no toxicity towards yeast cells at concentrations ranging from 12.5 µg/mL toto 100 100 µg/mLµg/mL (Figure (Figure 9A).9A).

FigureFigure 9. 9. PurifiedPurified fraction fraction potential potential for for inhibiting inhibiting calcineurin/Crz1 calcineurin/Crz1 activation. activation. (A) Toxicity (A) Toxicity was was evaluatedevaluated at the indicatedindicated concentrationsconcentrations of of the the SFE SFE purified purified fraction. fraction. (B ()B Anti-inflammatory) Anti-inflammatory potential po- tentiaof SFEl of purified SFE purified fraction fraction demonstrated demonstrated by the by inhibitionthe inhibition of theof the calcineurin/Crz1 calcineurin/Crz1 pathway pathwayin in S. S.cerevisiae cerevisiae. Statistical. Statistical differences differences are are noted noted as *asp *< p 0.1, < 0.1 **, p**< p 0.01, < 0.01, *** ***p < p 0.001 < 0.001 relative relative to theto the activated acti- 2 vatedcontrol control (cells treated(cells treated with MnCl with 2MnCl). ).

TheThe SFE purified purified fraction’sfraction’s ability to reducereduce thethe activationactivation ofof calcineurin/Crz1calcineurin/Crz1 was testedtested as anan indicatorindicator of of an an anti-inflammatory anti-inflammatory potential. potential. The The fraction fraction presents present itss maximum its maxi- mumactivity activity at 10 µ atg/mL, 10 µg/mL promoting, promoting a reduction a reduction in Crz1 in activation Crz1 activation of 53.38% of 53.38%± 3.9 ( Figure± 3.9 (Fig-9B ). ureThis 9B result). This demonstrated result demonstrated that this that compound this compound is able tois reduceable to thereduce activation the activation of the cal- of thecineurin/Crz1 calcineurin/Crz1 pathway pathway at levels at similarlevels similar to those to of those theimmunosuppressant of the immunosuppressant drug used drug as useda positive as a positive control FK506control with FK506 a calculated with a calculated IC50 of 7.2IC50± of3.15 7.2 µ±g/mL. 3.15 µg/mL. TheThe SL SL 8 8-deoxylactucin-deoxylactucin contains an α--methylene-methylene-γ--lactonelactone ring ring in its structure. This moietymoiety was was described described to to be be the the most most bioactive bioactive group group present present in in SLs SLs with with respect respect to to anti anti-- inflammatoryinflammatory activity activity by by interacting interacting with with protein proteinss that that contain contain exposed exposed sulfhydryl sulfhydryl groups groups viavia Michael Michael-type-type additionaddition [[3535].]. ThisThis compoundcompound may may be be able able to to interact interact with with calcineurin calcineurin or orthrough through its its exposed exposed sulfhydryl sulfhydryl groups groups present present in in its its catalytic catalytic subunit subunit [36 [,3637,].37 Although]. Althoughα- αmethylene--methyleneγ--lactonesγ-lactones rings rings are are the the most most bioactive bioactive group, group they, they may may also also present present the the ability abil- ityto createto create conjugates conjugates via via Michael-type Michael-type addition addition with with glutathione glutathione intracellularly—a intracellularly— processa pro- cessalready already described described for otherfor other chicory chicory SLs, SLs lactucin, lactucin and and lactucopicrinand lactucopicrinand other other SLs SLs such such as asnoblin—that noblin —that become become unavailable unavailable for for further further reaction reaction with with the the target target proteins proteins [38 ,[38,3939]. ]. β α InIn a a previous previous study, study, chicory chicory SL SL 11β,13 11 ,13-dihydrolactucin,-dihydrolactucin, lacking lacking the the α-methylene-methylene--γ- γ lactone-lactone ring ring,, was was able able to modulate to modulate the activation the activation of calcineurin/Crz1 of calcineurin/Crz1 pathway pathway due to duethe α β β presenceto the presence of α,β- ofunsaturated, -unsaturated carbonyl carbonyl groups groups[34]. In [ 34addition,]. In addition, 11β,13- 11dihydro,13-dihydro-8--8-deoxy- α γ lactucindeoxylactucin also does also does not contain not contain the the α-methylene-methylene--γ-lactones-lactones group, group, but but it itcontains contains other other functional groups such as α,β-unsaturated carbonyl, similarly to 11β,13-dihydrolactucin, functional groups such as α,β-unsaturated carbonyl, similarly to 11β,13-dihydrolactucin, possibly still being able to modulate the calcineurin/Crz1 pathway acting as a potential possibly still being able to modulate the calcineurin/Crz1 pathway acting as a potential anti-inflammatory compound [34]. anti-inflammatory compound [34]. To confirm the effect of purified fraction containing 8-deoxylactucin and its dihydro variant on Crz1 activity, fluorescence microscopy was used as a means to assess their

Molecules 2021, 26, x FOR PEER REVIEW 11 of 17

Molecules 2021, 26, 2583 11 of 17 To confirm the effect of purified fraction containing 8-deoxylactucin and its dihydro variant on Crz1 activity, fluorescence microscopy was used as a means to assess their interference with Crz1 nuclear translocation and the accumulation of the transcription factor interference with Crz1 nuclear translocation and the accumulation of the transcription Crz1 at the concentration of the previously calculated IC50 of the fraction (7.2 µg/mL). The factor Crz1 at the concentration of the previously calculated IC of the fraction (7.2 µg/mL). Crz1 signal is dispersed throughout the cells in the control condition50 , challenging cells The Crz1 signal is dispersed throughout the cells in the control condition, challenging cells with MnCl2 triggered Crz1 nuclear accumulation (48.7%), which was inhibited by SFE pu- with MnCl triggered Crz1 nuclear accumulation (48.7%), which was inhibited by SFE rified fraction2 treatment (14.6%) in a similar manner to that of the positive control FK506, puriﬁed fraction treatment (14.6%) in a similar manner to that of the positive control FK506, known to inhibit calcineurin activity and thereby preventing the translocation of Crz1 to known to inhibit calcineurin activity and thereby preventing the translocation of Crz1 to the nucleus (Figure 10). the nucleus (Figure 10).

Figure 10. Purified fraction inhibitsFigure 10.Crz1Purified–GFP nuclear fraction accumulation. inhibits Crz1–GFP Yeast cells nuclear were accumulation. first treated either Yeast cellswith wereor with firstout treated µ 7.2 µg/mL of purified fraction,either challenged with or with without 3 mM 7.2 MnClg/mL2, and of purifiedCrz1–GFP fraction, subcellular challenged distribution with 3 was mM monitored MnCl2, and using Crz1–GFP fluorescence microscopy. Thesubcellular immunosuppressant distribution FK506 was monitoredwas used as using a positive fluorescence control. microscopy.Approximately The 1800 immunosuppressant cells representing each condition were FK506counted. was Re usedpresentative as a positive imagens control. are shown, Approximately and the 1800values cells are representing the mean of eachpercentage condition of were nuclear Crz1–GFP ± SD of threecounted. biological Representative replicates. Statistical imagens aredifferences shown, andare denoted the values as *** are p the < 0.001 mean relative of percentage to activated of nuclear cells. Crz1–GFP ± SD of three biological replicates. Statistical differences are denoted as *** p < 0.001 relative to activated cells. 3. Materials and Methods 3. Materials and Methods 3.1. Chemicals 3.1. Chemicals CO2 pure grade (99.95%, Air Liquide, Lisbon, Portugal) and EtOH (96%) were used for highCO pressure2 pure grade extraction (99.95%, experiments. Air Liquide, Solvents Lisbon, used Portugal) for conventional and EtOH (96%)extractions were usedpu- rificationfor high pressuresteps and extraction chromatographic experiments. analysis Solvents included used forethyl conventional acetate (99.98%, extractions Fisher purifi- sci- cation steps and chromatographic analysis included ethyl acetate (99.98%, Fisher scientific entific U.K. Limited, Loughborough, UK), methanol (99.8%, Fisher scientific U.K. Limited, U.K. Limited, Loughborough, UK), methanol (99.8%, Fisher scientific U.K. Limited, Lough- Loughborough, UK), dichloromethane (Honeywell Riedel-de Haën, Germany), distilled borough, UK), dichloromethane (Honeywell Riedel-de Haën, Germany), distilled water, water, and ultrapure water purified with a Milli-Q water purification system (Merck Mil- and ultrapure water purified with a Milli-Q water purification system (Merck Millipore, lipore, Billerica, MA, USA). For the inflammation assays, the inflammatory stimulus was Billerica, MA, USA). For the inflammation assays, the inflammatory stimulus was carried out by the usage of MnCl2 (Merck, Darmstadt, Germany), and the anti-inflammatory positive control was carried by FK506 (Cayman Chemicals, Ann Arbor, MI, USA). Cellular lysis was preformed using Yeast Protein Extraction Reagent (Y-PER; ThermoFisher Scientific, Sci-

Molecules 2021, 26, 2583 12 of 17

entiﬁc, Rockford, IL, USA). To quantify the expression of reporter gene lacZ, we employed a solution buffer composed of Na2HPO4 (ROTH, Karlsruhe, Germany), NaH2PO4·H2O (Merck, Buchs, Switzerland), KCl (Panreac, Barcelona, Spain), and MgSO4·7H2O (Merck, Buchs, Switzerland), containing o-nitrophenyl β-D-galactopyranoside (ONPG; Sigma– Aldrich®–Poole, Dorset, UK). Nuclear staining was carried out using nuclear dye Hoechst 33342 (Sigma, Buchs, Switzerland).

3.2. Raw Material Chicory roots were obtained from a processing industry (Sensus, The Netherlands), which were harvested in November 2018. For sample preparation, roots were received, cleaned, and cut into pieces (1 cm) for freeze-drying (Coolsafe Superior Touch 55-80, Scanvac). The samples were further milled, and the particle size was determined using an AS 200 basic vertical vibratory sieve shaker (Retsch, Haan, Germany), with a measuring range between 250 and 1000 µm. The milled roots were stored and protected from light while kept at room temperature in a desiccator until the day of the experiments.

3.3. Extraction and Fractionation Procedures 3.3.1. Supercritical CO2 Extractions High-pressure extractions were carried out in an SFE system (Thar Technology, Pittsburgh, PA, USA, model SFE-500F-2-C50) [40]. Liquid CO2 was fed into the extraction vessel using a Thar SFC P-50 high pressure pump (Thar Technology, Pittsburgh, PA, USA). The extraction vessel was filled with 10 g of freeze-dried, milled chicory roots and glass beads in order to decrease solvent consumption. EtOH used as a co-solvent was pumped to the extraction vessel using a Thar SFC P-50 high pressure pump (Thar Technology, Pittsburgh, PA, USA). The vessel was pressurized with CO2 until reaching the required pressure (100–550 bar), which was maintained by an automated back pressure regulator (Thar SFC ABPR, Thar Technology, Pittsburgh, PA, USA). CO2 was expanded into the first fraction collector, and the extracts were recovered in a flask placed in an ice bath.

3.3.2. Experimental Design Analysis/Statistical Analysis Response surface methodology, a collection of statistical and mathematical techniques for improvement and optimize processes, is proposed to model the effect of different parameters such as pressure, temperature, co-solvent percentage, and flow rate on the extraction performance by SFE [41–43]. In this study, response surface methodology was used to model the recovery of SLs from chicory roots and to optimize the SFE process conditions. The extraction of SL was carried out following two different central composite face-centered (CCFC) designs as a function of pressure (100–550 bar), temperature (25–80 ◦C), flow rate (10–30 g/min), and co-solvent percentage (10–40%) to optimize the mass and SL extraction yield. After the selection of independent variables and their ranges, experiments were established based on a CCFC design, and each one was coded at three levels, i.e., −1, 0, and +1 (Table1). The results of the CCFC, particularly the total mass and the SL concentration in the extract value, were analyzed using the software MODDE (MODDE Go 12.1 32-bit, Umetrics, Sweden). Both linear and quadratic effects of each factor under study, as well as their interactions, were calculated based on different extractions that were performed in a random order, as defined by the software. A surface, as described by a second-order polynomial equation, was fitted to each set of experimental data points. First- and second-order coefficients of the polynomial equations were generated by regression analysis. The fit of the models was 2 2 2 evaluated using the determination coefficients (R ) and adjusted R (Radj ).

3.3.3. Conventional Solid–Liquid Extraction The solid–liquid extraction, a process implemented by an industrial project partner (Sensus, The Netherlands), was performed for comparison with the SFE results. Brieﬂy, the freeze-dried and milled Cichorium intybus L. roots were extracted with either ethyl acetate Molecules 2021, 26, 2583 13 of 17

or water (raw material:solvent 1:10 (w/v)) for 1 h at 60 ◦C with mechanic stirring (RW20.n IKA, Labortechnik, Germany) at 900 rpm. Then, the extract was ﬁltered (qualitative ﬁlter paper, 125 mm, from Filter-Lab) and centrifuged (Eppendorf Centrifuge 5810 R) to remove any remaining solid part of the unsolved raw material. The extract was dried in a rotary evaporator under reduced pressure at 40 ◦C. The experiments were performed in triplicate.

3.3.4. Flash Column Chromatography Purification Purification of the crude extract obtained from SFE consisted of dry loading approximately 1.5 g of the sample into a chromatographic column (30 × 400 mm) packed with silica gel 60 (0.04–0.06 mm, 230–400 mesh ASTM). The eluent for the purification was dichloromethane:methanol (9.5:0.5), which was used with an isocratic flow. Thin-layer chromatography (TLC) was conducted with TLC silica gel 60 F 254 and visualized under UV light to follow the purification, using the same eluent system. The pure fractions were collected, and the solvent was evaporated in a rotary evaporator under reduced pressure at 40 ◦C, for further analysis.

3.4. SL Analysis 3.4.1. Quantification of SL by HPLC with Diode-Array Detector (HPLC-DAD) HPLC analyses were carried out on a Thermo HPLC Dionex Ultimate 3000, equipped with a quaternary pump, solvent degasser, autosampler, and column oven, coupled to a Photodiode Array Detector Thermo Dionex DAD-3000. The separation was performed on a reversed-phase column (LiCrospher® 100 RP-18, 250 × 4 mm; 5 mm; Merck®) at 35 ◦C. Elution was carried out in gradient mode employing the following solvent system: mobile phase A, methanol/water 14/86 (v/v); mobile phase B, methanol/water 64/36 (v/v). The gradient program used was 0 to 20 min, 100–58% A; 20 to 30 min, 58% A; 30 to 45 min, 58–0% A; 45 to 50 min, 0% A; 50 to 52 min, 0–100% A; 52 to 62 min, 100% A. The flow rate used was 0.5 mL/min, and the injection volume was 20 µL. A photodiode array detector was used to scan the wavelength absorption from 210 to 600 nm. The software used was Thermo Scientific™ Dionex™ Chromeleon™ (version 7.2 SR4) processed data.

3.4.2. Identification of SL by Liquid Chromatography–Mass Spectrometry (LC-MS) For LC-MS analysis purified fraction 1 was dissolved in 70% methanol containing formic acid (0.1%) at the concentration of 10 µg/mL. LC-MS analysis was performed using the LC-PDA-LTQ-Orbitrap FTMS system (Thermo Scientific), which consists of an Acquity UPLC (H-Class) with Acquity eLambda photodiode array detector (220–600 nm) connected to a LTQ/Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionizator (ESI). The injection volume was 5 µL. Chromatographic separation was conducted on a reversed phase column Luna C18 column (2.0 × 150 mm, 3 µm; Phenomenex, USA) at 40 ◦C. Degassed eluent A (ultra-pure water: formic acid (1000:1, v/v)) and eluent B (acetonitrile:formic acid (1000:1, v/v)) were used at a flow rate of 0.19 mL min−1. A linear gradient from 5 to 75% acetonitrile (v/v) in 45 min was applied, which was followed by 15 min of washing and equilibration. FTMS full scans (m/z 90.00–1350.00) were recorded with a resolution of 60,000.

3.5. Anti-Inflammatory Evaluation 3.5.1. Saccharomyces Cerevisiae Strains and Growth Conditions The S. cerevisiae strains used in this study were YAA3 (his3::CRZ1-GFP-HIS3) and YAA5 (aur1::AUR1-C-4xCDRE-lacZ) [39]. The growth conditions were similar to those previously described [34]. Briefly, a preinoculum was prepared in an SC (synthetic com- plete) medium (0.79% (w/v) CSM (MP Biomedicals, Inc.—Fisher Scientific, Irvine, CA, USA), 0.67% (w/v) YNB (DifcoTM Thermo Scientific Inc., Waltham, MA, USA), and 2% (w/v) glucose), and cultures were incubated overnight at 30 ◦C under orbital shaking. Cultures were diluted in a fresh medium and incubated under the same conditions until the optical density at 600 nm (OD600) reached 0.5 ± 0.05. Readings were performed in a Molecules 2021, 26, 2583 14 of 17

96-well microtiter plate using a Biotek Power Wave XS plate spectrophotometer (Biotek® Instruments, Winooski, VT, USA).

3.5.2. Cell Viability Assays Cell viability was monitored as previously described [34]. Briefly, 100 µL of cultures diluted to a final OD600 = 0.025 ± 0.0025 were placed into 96-well microplates; 10 µL of cell titer blue reagent (Promega, WI, USA) were added, and the plates were incubated for 3 h at 30 ◦C. Fluorescence was measured in 30 min intervals at emission wavelength 580 nm using the Biotek Power Wave XS Microplate Spectrophotometer (Biotek® Instruments, Winooski, VT, USA). Doses of chicory SFE extracts ranging from 100 to 1000 µg/mL, and purified fractions containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin in the range of 12.5–100 µg/mL were tested in the viability assays. Readings were performed using a Biotek Power Wave XS plate spectrophotometer (Biotek® Instruments, Winooski, VT, USA).

3.5.3. Measurement of Reporter Gene Activity Measurement of β-galactosidase activity was performed as previously described [34]. Briefly, cells were challenged with an SFE chicory extract with concentrations ranging from 1 µg/mL to the highest nontoxic concentration. For the purified fraction of the SFE extract containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin, the concentrations tested ranged from 10 ng/mL up to 100 µg/mL. The immunosuppressant drug FK506 (Cayman Chemicals, Ann Arbor, MI, USA) was used as a positive control at a final concentration of 12.5 µM. The final results were expressed as Miller units (MU) according to the following equation, where V is the volume of culture assayed in mL, and t is the reaction time in minutes [44]:

Miller unit = 1000 × (OD420 − 1.75 × OD550)/(t × V × OD600)

3.5.4. Fluorescence Microscopy The monitoring of Crz1 subcellular localization was performed as previously described [34]. Briefly, cells were treated with 7.2 µg/mL of purified fraction of SFE extract containing the SLs 8-deoxylactucin and its dihydro variant, or12.5 µM of FK506. The preparations were monitored for GFP fluorescence as previously described using a Zeiss Imager Z2 (Zeiss, Germany) fluorescence microscope [45]. Photographs were taken with an Axiocam 506 mono (Zeiss). At least 600 individual cells were counted for each condition. Images were analyzed using Fiji-ImageJ1.53c (United States).

3.6. Statistical Assays Results for the toxicity and bioactivity of SFE extract and fraction are the averages of three independent experiments and are reported as mean ± SD. Differences between the controls and the experimental concentrations were assessed by an analysis of variance with Dunnett’s multiple comparison tests (α = 0.05), utilizing GraphPad Prims 8.4.2 software. The IC50 value for the SFE total extract and the SFE puriﬁed fraction was also calculated using the GraphPad Prims 8.4.2 software.

4. Conclusions

In this study, a green extraction approach using supercritical CO2 was explored for the first time to recover SLs from Cichorium intybus L. roots. The best operating conditions to obtain sesquiterpene-rich extracts were achieved at 350 bar and 40 ◦C in a 15 g/min flow rate (10% EtOH). SFE proved to be an efficient process for extracting SL (53 µg/mg extract) while reducing the quantity of organic solvents consumed. The SFE method produced an extract rich in SLs, compounds that had been previously described as anti- inflammatory in S. cerevisiae model. Moreover, a purified fraction containing a mixture of noncommercially available 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin was Molecules 2021, 26, 2583 15 of 17

obtained. The SFE extract was then tested for its anti-inflammatory potential, where it was able to decrease the activity of the calcineurin/NFAT orthologue Crz1 in a S. cerevisiae model with an IC50 of 38.81 ± 8.32 µg/mL, indicating the possibility of exploiting this extraction method to produce a possible anti-inflammatory extract using green technology with reduced costs. The purified fraction’s anti-inflammatory capacity was also measured, resulting in an inhibition of the pathway with an IC50 of 7.2 ± 3.15 µg/mL, which are results similar to those of the immunosuppressant pharmaceutical drug FK506. We also verified the ability of this purified fraction to inhibit the translocation of the transcription factor Crz1 to the nucleus by cellular localization, observed by fluorescence microscopy. Calcineurin is highly conserved among eukaryotes, and the docking of the NFAT sequence recognized by calcineurin is highly similar to Crz1. This makes the yeast model suitable for studying the interaction of compounds with the mammalian equivalent calcineurin-NFAT pathway. However, further studies are required in order to validate this anti-inflammatory potential achieved through the inhibition of NFAT using other advanced mammalian cellular models and to address the molecular targets of the purified fraction. NFAT nuclear translocation and target genes expression can confirm the potential previously observed. A final validation in animal models subjected to an inflammatory insult is ultimately required for a deeper understanding of the mechanisms, doses, and role of the compounds on inflammatory biomarkers. Based on these results, we can consider pressurized fluid extraction technology as a good alternative to traditional solvent extraction methods for the development of high- value products from Cichorium intybus L. with potential human health-promoting applications.

Author Contributions: J.P.B. performed SFE extractions and sample analysis, composed the first draft of the manuscript, performed review and editing; J.D.A. performed the bioactivity tests, review, and editing; V.I. performed the chromatographic purification and sample analysis; K.C. performed sample analysis, review, and editing; D.B. performed sample analysis, review, and editing; R.M. supervised the development of the work related with the bioactivity test, participated in the discussion and analysis of the results, and performed review and editing; M.d.R. performed review and editing; C.N.d.S. conceived the presented idea, acquired funding, supervised the development of the work, participated in the discussion and analysis of the results, and performed review and editing; A.A.M. conceived the presented idea, acquired funding, and performed review and editing; N.F. supervised the development of the work, participated in the discussion and analysis of the results, and performed review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This work was conducted under the “CHIC” project (H2020-NMBP-BIO-2017) with financial support received from the EU Horizon 2020 research and innovation programme under grant agreement N. 760891. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors acknowledge the financial support received from the EU Horizon 2020 research and innovation programme under grant agreement N. 760891. The iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior, through national funds and funding from INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC), is also gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: Samples of the compounds are available on request from the authors.

References 1. Espín, J.C.; García-Conesa, M.T.; Tomás-Barberán, F.A. Nutraceuticals: Facts and ﬁction. Phytochemistry 2007, 68, 2986–3008. [CrossRef] Molecules 2021, 26, 2583 16 of 17

2. Bais, H.P.; Ravishankar, G.A. Review Cichorium intybus L–cultivation, processing, utility, value addition and biotechnology, with an emphasis on current status and future prospects. J. Sci. Food Agric. 2001, 484, 467–484. [CrossRef] 3. Al-snafi, P.A.E.; Medicine, C. Medical importance of Cichorium intybus–A review. Iosr J. Pharm. 2016, 6, 41–56. 4. Siddhan, N.; Kumari, B.D.R. Phytochemical and Antibacterial Studies of Chicory (Cichorium intybus L.)-A Multipurpose Medicinal Plant Phytochemical and Antibacterial Studies of Chicory. Adv. Biol. Res. 2014, 1, 17–21. 5. Street, R.A.; Sidana, J.; Prinsloo, G. Cichorium intybus: Traditional uses, phytochemistry, pharmacology, and toxicology. Evidence- based Complement. Altern. Med. 2013, 2013.[CrossRef] 6. Mares, D.; Romagnoli, C.; Tosi, B.; Andreotti, E.; Chillemi, G.; Poli, F. Chicory extracts from Cichorium intybus L. as potential antifungals. Mycopathologia 2005, 160, 85–92. [CrossRef] 7. Mai, F.; Glomb, M.A. Structural and Sensory Characterization of Novel Sesquiterpene Lactones from Iceberg Lettuce. J. Agric. Food Chem. 2016, 64, 295–301. [CrossRef] 8. Bischoff, T.A.; Kelley, C.J.; Karchesy, Y.; Laurantos, M.; Nguyen-Dinh, P.; Arefi, A.G. Antimalarial activity of Lactucin and Lactucopicrin: Sesquiterpene lactones isolated from Cichorium intybus L. J. Ethnopharmacol. 2004, 95, 455–457. [CrossRef] 9. Wesołowska, A.; Nikiforuk, A.; Michalska, K.; Kisiel, W.; Chojnacka-Wójcik, E. Analgesic and sedative activities of lactucin and some lactucin-like guaianolides in mice. J. Ethnopharmacol. 2006, 107, 254–258. [CrossRef] 10. Peña-Espinoza, M.; Valente, A.H.; Thamsborg, S.M.; Simonsen, H.T.; Boas, U.; Enemark, H.L.; López-Muñoz, R.; Williams, A.R. Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: A review. Parasites Vectors 2018, 11, 1–14. [CrossRef][PubMed] 11. El-Sayed, Y.S.; Lebda, M.A.; Hassinin, M.; Neoman, S.A. Chicory (Cichorium intybus L.) root extract regulates the oxidative status and antioxidant gene transcripts in CCl4-induced hepatotoxicity. PLoS ONE 2015, 10, 1–12. [CrossRef] 12. Ripoll, C.; Schmidt, B.M.; Ilic, N.; Poulev, A.; Dey, M.; Kurmukov, A.; Raskin, I. Anti-inflammatory Effects of a Sesquiterpene Lactone Extract from Chicory (Cichorium intybus L.) Roots. Nat. Prod. Commun. 2007, 2, 717–722. [CrossRef] 13. Perović,J.; Tumbas Šaponjac, V.; Kojić,J.; Krulj, J.; Moreno, D.A.; García-Viguera, C.; Bodroža-Solarov, M.; Ilić,N. Chicory (Cichorium intybus L.) as a food ingredient–Nutritional composition, bioactivity, safety, and health claims: A review. Food Chem. 2021, 336, 127676. [CrossRef][PubMed] 14. van Beek, T.A.; Maas, P.; de Groot, A.; King, B.M.; Leclercq, E.; Voragen, A.G.L. Bitter Sesquiterpene Lactones from Chicory Roots. J. Agric. Food Chem. 1990, 38, 1035–1038. [CrossRef] 15. Malarz, J.; Stojakowska, A.; Kisiel, W. Sesquiterpene lactones in a hairy root culture of Cichorium intybus. Z. Für Nat. C 2014, 57, 11–12. [CrossRef] 16. Cavin, C.; Delannoy, M.; Malnoe, A.; Debefve, E.; Touché, A.; Courtois, D.; Schilter, B. Inhibition of the expression and activity of cyclooxygenase-2 by chicory extract. Biochem. Biophys. Res. Commun. 2005, 327, 742–749. [CrossRef][PubMed] 17. Ferioli, F.; D’Antuono, L.F. An update procedure for an effective and simultaneous extraction of sesquiterpene lactones and phenolics from chicory. Food Chem. 2012, 135, 243–250. [CrossRef] 18. Foster, J.G.; Clapham, W.M.; Belesky, D.P.; Labreveux, M.; Hall, M.H.; Sanderson, M.A. Influence of cultivation site on sesquiterpene lactone composition of forage chicory (Cichorium intybus L.). J. Agric. Food Chem. 2006, 54, 1772–1778. [CrossRef][PubMed] 19. Michalska, K.; Stojakowska, A.; Malarz, J.; Dolezalova, I.; Ales Lebeda, W.K. Systematic implications of sesquiterpene lactones in Lactuca species. Biochem. Syst. Ecol. 2009, 37, 174–179. [CrossRef] 20. Delgado, G.; Garcia, P.E.; Roldan, R.I.; Bye, R.; Linares, E. New Eremophilane Sesquiterpene Lactones from the Roots of the Medicinal Plant Roldana sessilifolia (Asteracea). Nat. Prod. Lett. 2006, 37–41. [CrossRef] 21. David, J.P.; Santos, A.J.D.O.; Guedes, M.L.S.; David, J.M.; Chai, H.; Pezzuto, J.M.; Angerhofer, C.K.; Cordell, G.A. Sesquiterpene Lactones from ambrosia artemisiaefolia (Asteraceae). Pharm. Biol. 1999, 37, 165–168. [CrossRef] 22. Sakamoto, H.T.; Laudares, E.P.; Crotti, A.E.; Lopes, N.P.; Vichnewski, W.; Lopes, J.L.; Heleno, V.C. Sesquiterpenes lactones and flavonoids from Eremanthus argenteus (Asteraceae). Nat. Prod. Commun. 2010, 5, 681–684. [CrossRef][PubMed] 23. Pourmortazavi, S.M.; Hajimirsadeghi, S.S. Supercritical fluid extraction in plant essential and volatile oil analysis. J. Chromatogr. A. 2007, 1163, 2–24. [CrossRef][PubMed] 24. Lang, Q.; Wai, C.M. Supercritical fluid extraction in herbal and natural product studies—A practical review. Talanta 2001, 53, 771–782. [CrossRef] 25. Khaw, K.Y.; Parat, M.O.; Shaw, P.N.; Falconer, J.R. Solvent Supercritical Fluid Technologies to Extract Bioactive Compounds from Natural Sources: A Review. Molecules 2017, 22, 1186. [CrossRef] 26. Reverchon, E.; De Marco, I. Supercritical fluid extraction and fractionation of natural matter. J. Supercrit. Fluids 2006, 38, 146–166. [CrossRef] 27. da Silva, R.P.F.F.; Rocha-Santos, T.A.P.; Duarte, A.C. Supercritical fluid extraction of bioactive compounds. Trac-Trends Anal. Chem. 2016, 76, 40–51. [CrossRef] 28. Fornari, T.; Vicente, G.; Vázquez, E.; García-risco, M.R.; Reglero, G. Isolation of essential oil from different plants and herbs by supercritical fluid extraction. J. Chromatogr. A 2012, 1250, 34–48. [CrossRef] 29. Pereira, C.G.; Meireles, M.A.A. Supercritical Fluid Extraction of Bioactive Compounds: Fundamentals, Applications and Economic Perspectives. Food Bioprocess. Tech. 2010, 340–372. [CrossRef] 30. Kisiel, W.; Zielin, K. Guaianolides from Cichorium intybus and structure revision of Cichorium sesquiterpene lactones. Phytochemistry 2001, 57, 523–527. [CrossRef] Molecules 2021, 26, 2583 17 of 17

31. Hailes, H.C. Reaction Solvent Selection: The Potential of Water as a Solvent for Organic Transformations Abstract. Org. Process. Res. Dev. 2007, 11, 521–527. [CrossRef] 32. Arbera, Ä.S.; Erreres, F.E.F. Lettuce and Chicory Byproducts as a Source of Antioxidant Phenolic Extracts. J. Agric. Food. Chem. 2004, 5109–5116. [CrossRef] 33. Graziani, G.; Ferracane, R.; Sambo, P.; Santagata, S.; Nicoletto, C.; Fogliano, V. Profiling chicory sesquiterpene lactones by high resolution mass spectrometry. Food Res. Int. 2015, 67, 193–198. [CrossRef] 34. Matos, M.S.; Anastácio, J.D.; Allwood, J.W.; Carregosa, D.; Marques, D.; Sungurtas, J.; McDougall, G.J.; Menezes, R.; Matias, A.A.; Stewart, D.; et al. Assessing the intestinal permeability and anti-inflammatory potential of sesquiterpene lactones from chicory. Nutrients 2020, 12, 3547. [CrossRef] 35. Saadane, A.; Masters, S.; DiDonato, J.; Li, J.; Berger, M. Parthenolide inhibits IkappaB kinase, NF-kappaB activation, and inflammatory response in cystic fibrosis cells and mice. Am. J. Respir Cell Mol. Biol 2007, 36, 728–736. [CrossRef][PubMed] 36. Kang, S.; Li, H.; Rao, A.; Hogan, P.G. Inhibition of the calcineurin-NFAT interaction by small organic molecules reflects binding at an allosteric site. J. Biol. Chem. 2005, 280, 37698–37706. [CrossRef] 37. King, M.M. Modification of the calmodulin-stimulated phosphatase, calcineurin, by sulfhydryl reagents. J. Biol. Chem. 1986, 261, 4081–4084. [CrossRef] 38. Thormann, U.; Hänggi, R.; Kreuter, M.; Imanidis, G. Membrane transport of nobilin conjugation products and use of the extract of Chamomillae romanae flos influence absorption of nobilin in the Caco-2 model. Eur. J. Pharm. Sci. 2015, 70, 92–106. [CrossRef] 39. Araki, Y.; Wu, H.; Kitagaki, H.; Akao, T.; Takagi, H.; Shimoi, H. Ethanol stress stimulates the Ca2+-mediated calcineurin/Crz1 pathway in Saccharomyces cerevisiae. J. Biosci. Bioeng. 2009, 107, 1–6. [CrossRef] 40. Rodrigues, L.; Silva, I.; Poejo, J.; Serra, A.T.; Matias, A.A.; Simplício, A.L.; Bronze, M.R.; Duarte, C.M.M. Recovery of antioxidant and antiproliferative compounds from watercress using pressurized fluid extraction. RSC Adv. 2016, 6, 30905–30918. [CrossRef] 41. Khuri, I.; Mukhopadhyay, S. Response surface methodology. Wires 2010, 2, 128–149. [CrossRef] 42. Ba, D.; Boyaci, I.H. Modeling and optimization i: Usability of response surface methodology. J. Food Eng. 2007, 78, 836–845. [CrossRef] 43. Almeida, M.; Erthal, R.; Padua, E.; Silveira, L.; Am, L. Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 2008, 76, 965–977. [CrossRef] 44. Miller, J. Experiments in molecular genetics. Astrophys. Space Sci. 1972. 45. Menezes, R.; Foito, A.; Jardim, C.; Costa, I.; Garcia, G.; Rosado-Ramos, R.; Freitag, S.; Alexander, C.J.; Outeiro, T.F.; Stewart, D.; et al. Bioprospection of Natural Sources of Polyphenols with Therapeutic Potential for Redox-Related Diseases. Antioxid 2020, 9, 789. [CrossRef]