(Poly)phenol characterisation in white and red cardoon stalks: could the sous-vide technique improve their bioaccessibility?

Abstract This study aims to evaluate whether sous-vide cooking better preserves the (poly)phenol content and profile of red and white cardoon stalks versus traditional boiling, both before and after simulated oral-gastro-intestinal digestion. Thirty one (poly)phenols were quantified in red and white cardoon by HPLC-MS/MS, phenolic acids being >95%, and 5–caffeoylquinic and 1,5–dicaffeoylquinic acids the major ones. Although both varieties showed a similar profile, raw red cardoon had 1.7-fold higher (poly)phenol content than raw white cardoon. Culinary treatments decreased (poly)phenol content, but sous-vide cooked cardoon had a greater content than the boiled one, suggesting a protective effect. After gastrointestinal digestion, (poly)phenol bioaccessibility of boiled and sous-vide cooked cardoon (52.6–90.5%) was higher than that of raw samples (0.2–0.7%), although sous-vide system no longer played a protective effect compared to boiling. In summary, red cardoon was a richer source of bioaccessible (poly)phenols than white cardoon, even sous-vide cooked or boiled.


Introduction
(Poly)phenols are secondary metabolites of plants and are subject of increasing scientific interest for their great abundance in diet, antioxidant capacity, and possible role in the prevention of various chronic human diseases, such as some types of cancer (Scalbert et al. 2005;Galanakis 2018). The potential health benefits of (poly)phenols depend on both their respective intakes and their bioavailability, which in turn are influenced by several factors such as the food matrix, vegetable heat treatments and industrial processing, and their bioaccessibility in the gastrointestinal tract (Manach et al. 2004;Galanakis 2018).
Heat treatments have been shown to directly influence the content of (poly)phenols and antioxidant activity of vegetables (Murador et al. 2018). Boiling is the most common cooking method used as hydrothermal treatment for vegetables (Shahidi and Yeo 2016). However, along with chemical and structural matrix changes, boiling generally results in losses of (poly)phenol content and antioxidant activity in vegetables, mainly due to their leaching into the cooking water (Murador et al. 2018). With the aim of avoiding these negative effects, sous-vide cooking technique was developed to preserve food compounds, properties, and flavours. It consists of introducing the food in a vacuum bag and later applying the heat treatment in a humid or liquid medium with controlled temperature, such as a water bath. Thus, the isolation of vegetables from oxygen and the cooking media (water) during the heat treatment may prevent oxidation and leaching of their bioactive compounds. Previous studies on artichokes, green beans, potato, broccoli and carrots show positive results with sous-vide cooking, since higher levels of ascorbic acid and (poly)phenols, and higher antioxidant capacity, were observed in sousvide cooked vegetables than in boiled ones (Iborra-Bernad et al. 2015;Guill en et al. 2017).
Bioaccessibility is defined as the amount of a food constituent that is released from its matrix in the gastrointestinal tract and becomes potentially available for absorption. Among other factors, the amount of bioaccesible (poly)phenols depends on the action of digestive enzymes (upper gastrointestinal tract), bacterial microflora (lower gastrointestinal tract), pH conditions, and the food matrix (Thakur et al. 2020;Wojtunik-Kulesza et al. 2020). Furthermore, evidence is emerging that in vitro bioaccessibility of (poly)phenols is enhanced when some vegetables are previously subjected to a heat treatment (Ju aniz et al. , 2017De Santiago et al. 2018). Up to our best knowledge, the impact of sous-vide cooking on the bioaccessibility of vegetable (poly)phenols has not been deeply investigated.
Cultivated cardoon [Cynara cardunculus L. var. altilis (DC)] remains of regional importance in Northern Spain, Italy and Southern France, where is consumed in traditional dishes and plays an important role in their agricultural economy (Pinelli et al. 2007;Gatto et al. 2013). Although the white or green variety of cardoon is popularly better known in the Mediterranean area, the red variety is also highly regarded in some regions of Northern Spain. The stalks are the edible part of white and red cardoon, which are generally eaten fresh in salads or boiled in typical recipes (Iborra-Bernad et al. 2015). In the current literature, there are few studies reporting the (poly)phenolic profile of white cardoon stalks (Ramos et al. 2014;Ju aniz et al. , 2017Petropoulos et al. 2018), but there is none on red cardoon stalks. Moreover, there is only one study that investigates the influence of heat treatment (frying and griddling) on the bioaccessibility of white cardoon (poly)phenols by high-performance liquid chromatography (HPLC) (Ju aniz et al. 2017). Therefore, this study aimed to evaluate for the first time whether the impact of an innovative cooking technique (i.e. sous-vide cooking) better preserves the (poly)phenol content and profile of red and white cardoon stalks in comparison to traditional boiling, both before and after a simulated oral-gastro-intestinal digestion (bioaccessibility).

Chemicals and reagents
For the in vitro digestion, human a-amylase, pepsin and pancreatin were purchased from Sigma-Aldrich (Darmstadt, Germany). CaCl 2 was obtained from Merck (Darmstadt, Germany), and HCl and NaOH were purchased from Panreac Qu ımica SLU (Barcelona, Spain). For the extraction of (poly)phenols and HPLC with tandem mass spectrometry (MS/MS) analysis, methanol of liquid chromatography-mass spectrometry (LC-MS) grade was adquired from Panreac AppliChem (Darmstadt, Germany).

White and red cardoon samples preparation
White and red cardoon stalks [Cynara cardunculus L. var. altilis (DC)] were obtained from Spanish retail stores during winter season. First, cardoon stalks from the same variety (red or white) were washed and the spiny skin was removed manually and cut into rectangular homogeneous pieces. Then, fresh cut stalks were divided in three different samples of 400 g each one: raw cardoon, boiled cardoon, and sous-vide cooked cardoon. Raw cardoon samples were immediately stored in the freezer and the others were cooked as given bellow: Boiling: 200 g of cardoon stalks were added to 100 mL of boiling water (98 C) in a stainless-steel pan and maintained at 80-90 C for 60 min, until they were completely cooked. This procedure was carried out twice obtaining a total of 400 g of boiled cardoon.
Sous-vide cooking: 200 g of cardoon stalks were packaged in a vacuum bag with a thin layer of water (45 mL) and then, vacuum sealed (vacuum sealer VP-3710.10, AK-Ramon, Vilassar de Dalt, Barcelona, Spain). Two vacuum bags were added to boiling water (98 C) and maintained at 80-90 C during 45-50 min, until they were completely cooked. This procedure was carried out twice obtaining a total of 400 g of sous-vide cooked cardoon.
Finally, all samples were lyophilised in a freeze dryer Cryodos-80 (Telstar, Terrassa, Spain) and stored at À 18 C until further analysis.

Simulated oral-gastro-intestinal digestion in vitro
A three step in vitro digestion model was carried out in a bioreactor, according to Minekus et al. (2014) adapted to our laboratory. So, 2 g of each lyophilised sample were added to a 100 mL three-neck round bottom vessel. Three sequential steps were carried out in the absence of light. Firstly, the oral digestion phase was performed by adding 250 lL of a human a-amylase solution (75 U/mL in final oral-phase volume), 0.10 mL of 0.3 M CaCl 2, and distilled water up to 20 mL. The sample was then magnetically shaken for 30 min at 37 C. Then, the gastric digestion step was carried out by adding 1 mL of pepsin solution (200 U/mL in final gastric-phase volume), 0.01 mL of 0.3 M CaCl 2, and distilled water up to 20 mL. Then, the sample was magnetically shaken for 2 h at 37 C maintaining pH 3 by adding 1 M HCl. Then, the intestinal digestion step was carried out by adding 10 mL of pancreatin solution (100 U/mL in final intestinal-phase volume), 0.08 mL of 0.3 M CaCl 2, and distilled water up to 40 mL. The samples were magnetically shaken for 2 h at 37 C at pH 7 by the addition of 1 M CaCl 2 . Finally, an enzymatic inhibition was carried out by thermal shock, which consisted on a brief heat treatment (30 s, 100 C) and subsequent cooling in ice. Then, cooled samples were immediately frozen, lyophilised in a freeze dryer Terrasa,Spain), and stored at À 18 C until further analysis. Two digestions per cardoon sample were performed to digest 4 g in total.
Both lyophilised digested samples were mixed and homogenised.

Extraction of (poly)phenols
Extraction of (poly)phenols from raw and cooked cardoon, both non-digested and digested, was performed according to S anchez- Salcedo et al. (2015) method, with some modifications. Briefly, 25 mg of each lyophilised sample were mixed with 0.5 mL of methanol/acidified water (0.1% formic acid) (80:20 v/v) and vortexed for 1 min. Then, the samples were sonicated for 90 min and centrifuged for 10 min at 14,000 rpm (Mikro 200, Hettich, Tuttlingen, Germany). The supernatant was collected and the residue was reextracted by adding 0.25 mL methanol/acidified water (80:20 v/v), followed by 1 min vortexing, 25 min sonication and 10 min centrifugation at 14,000 rpm. The resulting supernatant was collected and combined with the first one. Three (poly)phenolic extracts were prepared per sample and stored at À 18 C.
The analysis of potential (poly)phenols was carried out first in full scan, scanning m/z from 100 to 1000, followed by a consecutively selective product ion mode (MS2) analysis with specific m/z. Finally, for the identification and quantification of the (poly)phenols, the ion multiple reaction monitoring mode was used. The MS functioned in negative ionisation mode, with the turbo heater maintained at 700 C and Ion Spray voltage set at À 3500 V. Nitrogen was used as nebulising, turbo heater and curtain gas, and it was set at the pressure of À 65, À 60 and À 35 psi, respectively. The declustering potential and entrance potential were set at À 20 and À 11 V, respectively. The collision energy was optimised for each compound using the same standards as for (poly)phenols identification. (Poly)phenols were identified by comparing the MS/ MS fragmentation pattern and retention time with pure reference standards, when available. In the absence of available standards, (poly)phenols were tentatively identified by comparing the MS/MS fragmentation pattern with the literature fragmentation pathway. One, two or three transitions were studied for each phenolic compound. The most abundant transition was used for quantification and the second and third ones for confirmation. Mass spectrometric characteristics of (poly)phenols identified as well as the pure reference standards used are reported in Table S1 (Appendix 1).
For (poly)phenols quantification, calibration curves of 5-CQA, 1,5-diCQA and rutin reference standards were prepared, and a linear relationship was obtained in each of them (r > 0.992). Results for repeatability showed a good precision of the method with coefficient of variation values below 7%, and a good accuracy with relative error values below 14%. The limit of quantification (LOQ) was 0.25 mg/mL for 5-CQA and 1,5-diCQA, and 0.025 mg/mL for rutin. Phenolic acids were quantified using 5-CQA linear equation; with the exception of diCQAs and derivatives that were quantified using 1,5-diCQA linear equation. Luteolin derivates, apigenin and pinoresinol derivates were quantified using rutin linear equation.
Chromatograms and spectral data were acquired using Analyst software 1.6.3 (AB SCIEX). Three (poly)phenolic extracts were analysed per sample and results are expressed as the mean of micrograms of each (poly)phenolic compound per gram of dry matter (mg/g dm) ± standard deviation (SD). Total (poly)phenol content of each sample was obtained by adding the amount of each individual (poly)phenol quantified, and was expressed as the mean of mg of total (poly)phenolic compounds/g dm ± SD.

Determination of (poly)phenol bioaccessibility
The bioaccessibility of (poly)phenols after simulated gastrointestinal digestion in vitro of each sample was calculated as percentage as follows:

Statistical analysis
One-way analysis of variance (ANOVA) in combination with post-hoc Tukey test, ajusted by multiplecomparison test, was applied to study differences in the individual and total content of (poly)phenols among raw and cooked samples, before and/or after gastrointestinal digestion. A significance level of p < 0.05 was used for all statistical analyses. Principal Component Analysis (PCA), based on Pearson's correlation matrix, was applied in order to study the differences among all digested and non-digested samples. Statistical analyses were performed using STATA v.12.0 software package.

Results and discussion
Red vs white cardoon (poly)phenol profile A total of 31 (poly)phenols were identified and quantified in white and red cardoon stalks under raw, boiled and sous-vide cooked conditions (Table 1). Phenolic acids, and specifically hydroxycinnamic acids, were the most abundant family of (poly)phenols in both white and red cardoon stalks, which accounted for more than 95% of the total content of (poly)phenols in all samples. Flavonoids and pinoresinol derivatives represented only few quantities of the total (poly)phenolic amount in cardoon samples.
In agreement with Ju aniz et al. (2017), two succinyl diCQAs and caffeic acid were found in cardoon stalks. Surprisingly, an isomer of caffeic acid and two diCQA glucosides were also identified, being reported neither by Petropoulos et al. (2018)  On the other hand, 5 flavonoids (apigenin and four luteolin derivatives) were quantified in both white and red cardoon samples (Table 1). Ramos et al. (2014) also detected luteolin and apigenin derivatives in white cardoon stalks; whereas Ju aniz et al. (2017) only reported luteolin derivatives. Furthermore, 2 pinoresinol derivatives (pinoresinol glucoside and pinoresinol acetylhexoside) were also found, which are present in C. cardunculus species according to Abu-Reidah et al. (2013) and Petropoulos et al. (2018).
White and red cardoon showed a similar (poly)phenolic profile, but clear differences regarding the content of total and individual (poly)phenols. Raw red cardoon had 1.7-fold higher total content than raw white cardoon, mainly due to significantly (p < 0.05) greater content of total diCQAs and their derivatives (10.409 ± 0.763 and 3.080 ± 0.054 mg/g dm respectively) with a very marked difference in 1,5-diCQA (2.9-fold) and 3,5-diCQA (5.9-fold) contents (Table  1). In addition, most flavonoids were present in significantly (p < 0.05) higher amounts in raw red cardoon than in white one (i.e. apigenin, luteolin, luteolin acetylglucoside and luteolin 7-O-glucoside) becoming total flavonoids in raw red cardoon significantly (p < 0.05) higher (0.328 ± 0.022 and 0.086 ± 0.007 mg/g dm respectively). On the contrary, the total content of monoCQAs and derivatives and, mainly 5-CQA, was similar in both varieties of raw cardoon stalks, even some monoCQAs (e.g. 3-CQA Results are expressed in mean mg of (poly)phenolic compound per g of cardoon dry matter ± standard deviation (n ¼ 3). Different letters indicate significant differences (p < 0.05) in individual or total (poly)phenolic compounds among samples. and CQA derivatives I-IV) were found in significantly (p < 0.05) greater amounts in the red variety. To our best knowledge, this is the first study that characterises individual (poly)phenols in two different cardoon varieties. In line with our results, Lahoz et al. (2011) reported greater (poly)phenol content (measured by the Folin assay) in the Spanish red cardoon variety "Rojo Agreda" than in other Spanish white varieties.

Impact of heat treatment on white and red cardoon (poly)phenols
Within each cardoon variety, the boiled samples had significantly (p < 0.05) the lowest total content of (poly)phenols in comparison to raw and sous-vide cooked ones (Table 1). Raw red cardoon showed the highest total content of (poly)phenols (17.272 ± 1.040 mg/g dm), followed by sous-vide cooked red cardoon (15.151 ± 0.222 mg/g dm), and then boiled red cardoon (13.480 ± 0.552 mg/g dm). However, the total content of the white cardoon variety varied between 7.375 ± 0.050 and 10.537 ± 0.166 mg/g dm, with similar contents (p > 0.05) in raw and sous-vide cooked white cardoon. Concerning individual compounds, both boiling and sous-vide cooking induced significantly (p < 0.05) lower content of the most abundant mono and diCQAs (5-CQA, 1,5-diCQA and 3,5-diCQA), as well as other chlorogenic and hydroxycinnamic acids, in both varieties of cardoon stalks, whereas other minor (poly)phenols (3-CQA, 4-CQA, 1,3-diCQA and 3,4-diCQA) significantly (p < 0.05) increased. In the case of sous-vide cooking, contents of some chlorogenic acids also significantly increased (p < 0.05) as compared to raw (i.e. 1,4-CQA and 4,5-CQA). It has been reported that CQAs may be degraded or isomerised by temperature, pH and light (Xue et al. 2016). Li et al. (2015) showed that 5-CQA in a boiling water bath, heated up to 2 h, was transformed into 4-CQA. In other study, 3,5-diCQA gave rise to 3,4-diCQA, 4,5-diCQA and 5-CQA after 30 min of boiling (Gouveia and Castilho 2012). Thus, mono and diCQAs undergo transformations such as isomerisation, while diCQAs could also be degraded to monoCQAs (Li et al. 2015). Therefore, events of isomerisation and hydrolysis during the boiling and sous-vide cooking of cardoon stalks may explain the differences and redistributions of mono and di-CQAs observed between raw and cooked samples, as well as the increase in caffeic acid. In addition, some diCQAs, caffeic acid, and luteolin glucoside have been previously reported bound to the food matrix of white cardoon stalks, where are released by the thermal destruction of cell walls and sub-cellular compartments during the cooking processes, like frying and griddling (Ju aniz et al. 2017).
Comparing both cooking treatments, sous-vide cooked cardoon showed significantly (p < 0.05) greater amounts of (poly)phenols than boiled one in both cardoon varieties, and even significantly (p < 0.05) higher   Results are expressed as mean mg of (poly)phenolic compounds per g of cardoon dry matter ± standard deviation, and percentage of bioaccessibility after in vitro gastrointestinal digestion (n ¼ 3). Different letters indicate significant differences (p < 0.05) in individual or total (poly)phenols among digested samples.
than raw white cardoon. Traditional boiling implies that the food matrix is totally submerged in the cooking water, and requires a longer duration (60 min) than sous-vide cooking (45-50 min) for the cardoon stalks to be fully cooked. According to our results, the sous-vide technique seems to prevent the loss of (poly)phenols by leaching into the cooking water, as well as by thermal degradation due to its shorter duration.

Impact of heat treatments on (poly)phenol bioaccessibility
Comprehensive studies on the effects of food processing on (poly)phenol content are essential to define the nutritional quality of food, but those focussed on bioaccessibility are required to assess the health impact of food (Ribas-Agust ı et al. 2018). Figure 1 and Table 2 show the impact of a simulated gastrointestinal digestion in vitro on the initial content of (poly)phenols in raw and cooked (boiled and sous-vide cooked) white and red cardoon stalks. The bioaccessibility percentage of each compound is also reported ( Table 2).
The total amount of (poly)phenols significantly (p < 0.05) decreased after gastrointestinal digestion in all cardoon samples, except for the boiled ones ( Figure 1). Indeed, the total amount of (poly)phenols and their bioaccessibility was highly influenced by the previous aplication or not of a heat treatment. Total (poly)phenols of raw white and red cardoon stalks decreased drastically (p < 0.05) after simulated gastrointestinal digestion (Figure 1), which resulted in <1% of (poly)phenols bioaccessibility (0.2-0.7%) ( Table 2). In raw cardoon stalks of the white variety, only luteolin acetylglucoside and pinoresinol derivatives remained quantificable after gastrointestinal digestion, while in the red cardoon variety, some monoCQAs (1-CQA, 3-CQA, 5-CQA), as well as flavonoid aglycones (apigenin and luteolin) were also bioaccessible in low amounts, probably due to the higher initial content of (poly)phenols in the red variety than in the white one. On the contrary, the (poly)phenols bioaccessibility of neither boiled nor sous-vide cooked cardoon showed the drastic reduction observed in the raw samples. Digested boiled cardoon samples had a total (poly)phenol content of 5.940 ± 0.403 and 12.197 ± 0.536 mg/g dm, in the white and red variety, respectively (Table 2), which was not significantly different (p > 0.05) to the content of their respective undigested boiled samples (Figure 1). Regarding the sous-vide cooked samples, the total amount of (poly)phenols after gastrointestinal digestion (5.542 ± 1.018 and 13.104 ± 0.432 mg/g dm in digested sous-vide cooked white and red cardoon, respectively) significantly (p < 0.05) decreased as compared with the undigested sous-vide cooked samples (Figure 1), but it was similar (p > 0.05) to the digested boiled samples (Figure 1, Table 2). However, because the total amount of (poly)phenols was initially higher (p < 0.05) in undigested sous-vide cooked cardoon (white and red) than in the boiled one (Table 1), the bioaccessibility percentages were lower in sous-vide cooked samples (52.6% and 86.5%) than in digested boiled ones (80.5 and 90.5%) ( Table 2). In a similar way to our findings, Ju aniz et al. (2017) found a significant loss of total (poly)phenols in raw and cooked (fried and griddled) white cardoon stalks after simulated gastrointestinal digestion, and the bioaccessibility of total (poly)phenols was much higher in cooked samples (60-67%) than in raw ones (2%).
The increase or decrease in bioaccessibility in a heat-treated food, compared to its raw material, could be explained by the balance between the degradation of (poly)phenols during both heat treatment and digestion, and the release of compounds that were bound to the food matrix (Ribas-Agust ı et al. 2018). Moreover, it is known that heat treatment can induce the formation of polymeric structures to which (poly)phenols are linked (Ju aniz et al. 2017;Ribas-Agust ı et al. 2018). Taking this into account, higher amounts of (poly)phenols in digested boiled and sous-vide cooked samples than in digested raw samples could be due to the following: (1) a limited thermal degradation during cooking treatments, (2) the rupture during digestion of molecular interactions that enhance the release of bound compounds, and (3)  The application of a simulated gastrointestinal digestion on white and red cardoon (raw and cooked), not only induced changes in the total content of (poly)phenols, but also on the (poly)phenolic profile of the vegetables. Most of the individual (poly)phenols of cooked cardoon decreased after gastrointestinal digestion remaining in significant (p < 0.05) higher amounts than in raw samples (Tables 1 and 2). However, some (poly)phenols increased their contents (>100% bioaccessibility) in cooked samples. Thus, a significant (p < 0.05) decrease of 5-CQA, 1,5-diCQA and 3,5-diCQA, and the simultaneous significant (p < 0.05) increase of 3-CQA, 4-CQA, cis 5-CQA, 1,3-diCQA and 3,4-diCQA, in the digested cooked samples from one or both cardoon varieties suggest isomerisation reactions and hydrolysis of di-to mono-CQAs during gastrointestinal digestion. A previous study reported that neutral or mild alkaline pH during the intestinal step of the digestion process causes partial degradation of native CQAs and partial formation of caffeoylquinic isomers (D'Antuono et al. 2015). More in detail, in vitro gastrointestinal digestion on 5-CQA led to 4-CQA; 1,5-diCQA digestion led to 1,3-and 1,4-diCQAs; and 3,5-diCQA digestion led to 4,5-and 3,4-diCQAs (D'Antuono et al. 2015). Furthermore, the release of some (poly)phenols from cardoon matrix during digestion might also explain the increase of other compounds such as luteolin and luteolin acetylglucoside in digested cooked samples.
Additionally, the release of (poly)phenols is reported to depend on the nature of the food matrix (Papillo et al. 2014). Both cardoon varieties (red and white) showed a similar pattern of changes in the (poly)phenolic profile in response to the analysed heat treatments, supporting the hypothesis that heat treatment might have a greater influence on bioaccessibility than the cardoon variety. However, dissimilarities in (poly)phenolic content between varieties were still present after gastrointestinal digestion of the vegetable, mainly due to different initial amount of (poly)phenols in each variety. Thus, 5-CQA remained as the most abundant compound in cooked white cardoon (around 28% of total content), followed by 3-CQA (around 25% of total content), whereas cooked red cardoon was richer in 1,3-diCQA (around 23%) than in 5-CQA (16%) and 3-CQA (14.5%) after digestion.
To sum up, (poly)phenol bioaccessibility may depend little on the cardoon variety, but more strongly on the presence or the absence of heat treatment. Cooking improved the bioaccessibility of cardoon (poly)phenols after gastrointestinal digestion, although sous-vide system seems to no longer play a protector effect versus boiling treatment.

Correlation between cardoon variety, culinary technique and (poly)phenol bioaccessibility
Principal Component Analysis (PCA) was applied to evaluate the influence of cardoon variety, culinary technique applied and individual (poly)phenolic compounds bioaccessibility at a glance. Figure 2 shows the bidimentional representation of the (poly)phenols detected (Figure 2(a)) and the distribution of the digested and non-digested white and red cardoon samples under the studied culinary techniques (raw, boiled and sous-vide cooked) (Figure 2(b)) based on their correlation matrix.
The resulting PCA explained 60.98% of the total variance by two components. Principal component (PC) 1, accounting for 31.12% of the total variance, discerned between white (left) and red (right) cardoon, except for digested raw red cardoon. PC1 is characterised on the right part of the graphic by most of the (poly)phenols identified and quantified in cardoon samples (monoCQAs, diCQAs, other hydroxycinnamic acids and some flavonoids) (Figure 2(a)). This graphically shows how red cardoon samples (except digested raw one) had much higher content of these compounds as compare to white cardoon samples that are in the left side of the graph. Furthermore, from right to left, it can be observed that non-digested raw red cardoon (RR) is located more to the right side of the graph than the other red cardoon samples (Figure 2(b)) as it had the highest content of (poly)phenols among all the studied samples (Tables 1 and 2). Additionally, among cooked samples of cardoon (both white and red variety as well as non-digested and digested), those having sousvide cooking treatment are further to the right than those having a traditional boiling. This fact is in concordance with results reported in Tables 1 and 2, being total (poly)phenol amount the highest in both varieties of raw cardoon, followed by sous-vide cardoon and then by boiled cardoon. Finally, the total content of (poly)phenols of both varieties of cardoon decreased after the in vitro gastrointestinal digestion, being more pronounced in raw red cardoon (Table 2). This is in line with what can be observed in Figure  2(b), where digested samples are located in the left side of their corresponding non-digested samples and the distance between non-digested and digested raw red cardoon samples is the biggest compared with white cardoon samples.
On the other hand, PC2, accounting for 29.86% of the total variance, clearly distinguished between raw (on the top) and cooked samples. Both raw white and red cardoon samples are mainly characterised by the highest content of 5-CQA and other compounds, while sous vide cooked and more intensively boiled samples (on the bottom) increased the presence of 3-CQA and 4-CQA isomers (negative side PC2, Figure  2(a)) at expenses of 5-CQA. Figure 2(b) clearly shows how sous vide technique was less detrimental in terms of (poly)phenols than boiling. Similarly, both digested white and red cardoon raw samples are clearly shown above digested cooked ones.

Conclusion
In summary, red cardoon is shown to be a richer source of (poly)phenols than the white one, having both varieties a similar profile of (poly)phenols. On the other hand, despite the fact that sous-vide cooking prevented the loss of (poly)phenols to a greater extent than traditional boiling, it seems to no longer play a protector effect after gastrointestinal digestion. Nevertheless, cooked cardoon, and especifically the red variety, provides a higher content of bioaccessible (poly)phenols than raw one. Future studies including also the action of gut microbiota are needed to obtain a more reliable picture of the bioaccessible compounds of cardoon in the entire gastrointestinal tract. Furthermore, research on the bioavailability of raw and cooked cardoon (poly)phenols and their subsequent bioactivity through in vitro and in vivo studies would be of a great interest to establish the relationship between this vegetable and human health.