Part2: Interactions Of Ascorbic Acid, 5-Caffeoylquinic Acid, And Quercetin-3-Rutinoside in The Presence And Absence Of Iron During Thermal Processing And The Influence On Antioxidant Activity

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3. Discussion

3.1.Structure-Activity Relationship of Ascorbic Acid,5-Caffeoylquinic Acid, and Quercetin-3-Rutinoside

A first indicator of the AOA, measured for different substances depending on the structure-activity relationship, resulted from the number of functional groups. Comparing all three analyzed substances, ascorbic acid had the lowest AOA, followed by 5-caffeoylquinic acid, while quercetin-3-rutinoside reached the highest AOA. Csepregi et al. [14] found the same order when comparing the AOA of these three compounds. This ranking may be explained by the total amount of hydroxy groups: quercetin-3-rutinoside has ten, 5-caffeoylquinic acid has five, and ascorbic acid has four. For flavonoids, the total amount of hydroxy groups and their impact on mechanisms of AOA was previously shown by Burda and Oleszek[15]. Hydroxy groups are particularly valuable in enediol structures, as they can easily oxidize to diketones [8]. In the analyzed substances the endiol structure is also important for the ability to form complexes with metal ions [16-18]. The endiol structure occurs in quercetin-3-rutinoside and 5-caffeoylquinic acid molecules in the phenyl ring, which may also influence the stronger AOA of these compounds. Further studies found that the AOA may also be influenced by other molecule structures. Phenolic acids are possibly affected by the carboxylic acid groups, e.g., hydroxyphenyl acetic acid (R-CH=CH-COOH) is a weaker electron-withdrawing group, compared to hydroxycinnamic acid (R-CH,-COOH) such as caffeic acid in 5-caffeoylquinic acid [19]. In flavonoids, aglycones had higher AOA than the corresponding glycosides [20,21], in the case of quercetin-3-rutinoside, a glycoside, the AOA of the aglycone quercetin is consequently higher [14]. There are many different functional groups that can influence the AOA and, consequently, it is important to use different test assays with different mechanisms, such as single-electron transfer (SET), hydrogen atom transfer(HAT), and sequential proton-loss electron transfer(SPLET) for detection. Each mechanism and even the used assay reagent can detect different structures of bioactive compounds [22].

3.2.Influence of Thermal Processing and Interaction of Structurally Different Antioxidants on the Antioxidant Activity in the Absence of the Mineral Iron

Stable AOA over an extended time of 40 min of thermal processing indicates that thermal degradation is of less importance than initially hypothesized. Furthermore, HPLC data showed that pure 5-caffeoylquinic acid and quercetin-3-rutinoside were stable, with a maximum degradation of 20%, during thermal processing. Previous studies proved the stability of5-caffeoylquinic acid up to the normal boiling point of water [23], while Dawidowicz and Typek[24] found nine derivative compounds, after heating 5-caffeoylquinic acid for 5 h under reflux. For ascorbic acid concentration, a degradation after 40 min of cooking was found. Influencing factors, resulting in ascorbic acid degradation without iron in an aqueous solution, could be pH values, light exposure, oxidation, temperature, and different concentrations [25,26]. In this study, the factors temperature and concentration most likely played the main role, as oxidative processes were reduced to a minimum due to minimal gas space in the microtubes. It is possible that the remaining gas phase is still enough for degradation under aerobic conditions. The different conditions result in different products [27,28], so in this study, after 40 min of cooking, products of both conditions could be found. Yuan and Chen [28] reported that furfural, 2-furoic acid, 3-hydroxy-2-pyrrone, and an unknown substance are major degradation products of ascorbic acid in an aqueous solution depending on pH value. Shinoda et al. [29,30] found in orange juice the degradation products furfural, 2-furoic acid, 5-hydroxymaltol, 3-hydroxy-2-pyrrone, and 5-(hydroxymethyl)furfural. Hsu et al. [31] analyzed ascorbic acid in ethanolic solutions and detected 2-furoic acid and 3-hydroxy-2-pyrrone. Depending on aerobic or anaerobic conditions, the detected ascorbic acid derivatives (peaks 3 and 4) might be furfural, 2-furoic acid, or 3-hydroxy-2-pyrrone or intermediates. The ascorbic acid decreased in a dose-response relation, and higher concentrations of ascorbic acid showed lower degradation ratios during cooking. Ascorbic acid seems to stabilize itself in higher concentrations, presumably due to hydrogen bonds and van de Waals energy [32].

In binary and ternary mixtures, mainly additional effects on AOA were found. In binary mixtures, if the substance with higher AOA increased in its concentration, the AOA of their combination also increased. Other studies also found additive effects between (+)-catechin (200 um) with ascorbic acid (50-200 mg/L)[33], and between binary mixtures of different monoterpenes[34]. Only in the DPPH assay, antagonistic effects were found in 5-caffeoylquinic acid combined with quercetin-3-rutinoside and in ternary mixtures with doubled quercetin-3-rutinoside concentrations. This could be caused by the orientation of the molecules in space, especially quercetin-3-rutinoside, as a quite large molecule, and the steric accessibility of the DPPH radical molecule [35]. In the TPC assay, the combination of ascorbic acid and 5-caffeoylquinic acid resulted in strong antagonistic effects in a 1:2 ratio, while reversed mixtures with a 2:1 ratio resulted in strong synergistic effects, exceeding the sum of the respective AOAs. Double concentration ascorbic acid may give, under these conditions, an additional boost to AOA, due to self-stabilization followed by the stabilization of other molecules, demonstrated by 5-caffeoylquinic acid in this experiment. In pomegranate-nectarine juice, between the natural phenols and ascorbic acid, the same interaction was found, while in grape juice increasing antagonistic effects by increasing ascorbic acid concentration were observed [36]. These results suggest that mixtures of ascorbic acid, 5-caffeoylquinic, and quercetin-3-rutinoside achieve the highest AOA potential when the most potent antioxidants are abundantly available.

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3.3. Influence of the Mineral Iron

The addition of iron to the samples had the most influence on changes in their AOA. Contrary to the hypothesis, based on the pro-oxidative activity of iron, the AOA increased, compared to the same substance or substance mixture without iron. Iron may act like a catalyst itself or forms metal chelates, which are effective catalysts. The changed stoichiometry of the chelates can form additional radical-scavenging metal centers [18], which explains the increased AOA. Further tests showed that reduced ferrous iron (50-100%,v/o)itself interacts with the TEAC(0.266-0.538 mol TE/mol iron), DPPH(0.210-0.495 mol TE/mol iron), and TPC(16.65-31.82g GAE/mol iron) assay reagents, while oxidized ferric iron does not (data not shown). In the presence of iron, 5-caffeoylquinic acid had the highest AOA, followed by quercetin-3-rutinoside and ascorbic acid in TEAC and DPPH assays. This may be due to the changed stoichiometry by metal chelation. The AOA ranking detected by the TPCassay stayed the same, as in the absence of iron: quercetin-3-rutinoside >5-caffeoylquinic acid>ascorbic acid. However, the addition of iron to the mixtures had an influence on TEAC and DPPH assay results, while TPC assays were widely unaffected. For this reason, it is important to use different test assays with different working mechanisms when working with iron-rich samples.

Only in the DPPH assay the AOA of pure quercetin-3-rutinoside, and in combination with ascorbic acid, equimolar or non-equimolar with doubled quercetin-3-rutinoside concentrations, did have lower values in the presence of iron. Boligon et al. [351 explained that the DPPH assay detects smaller antioxidants better, due to the steric accessibility of these radicals. Presumably, quercetin-3-rutinoside with the two binding sides can build quite large complexes with iron, and thus be inaccessible for the assay. As mentioned by Kejic et al. [8], this might be explained by the formation of supramolecular complexes via the coordination of metal ions. In combination with ascorbic acid, which is able to build mixed-valence complexes in a 1:2 ratio [12], presumably, these larger mixed complexes cannot interact with the DPPH radical. Contrary to the results in this study, other studies [18,37,38] found, by using the DPPH assay, that quercetin-3-rutinoside complexes are more effective antioxidants than pure quercetin-3-rutinoside. It is known that chelate complexes of flavonoids and metal ions can negate the radical activity of complexed metal ions [39]. The only difference was that in this study ammonium iron (II) sulfate was used, instead of iron (ⅡI) chloride or sulfate, used in the work of Symonowics and Kolandek [39].

Synergistic effects between ascorbic acid and 5-caffeoylquinic acid in the presence of iron in the 2:1 ratio, and antagonistic effects in the 1:2 ratio, were found. The combination of 5-caffeoylquinic acid with iron was not recommended in vivo [6]. However, this study showed that, in vitro, the doubled amount of ascorbic acid in comparison to 5-caffeoylquinic acid can even have synergistic effects on AOA. Further investigations on ratios could show whether positive effects can also be achieved in vivo.

According to the HPLC data, adding iron to the sample will have a minimal effect on its catalytic activity, as only the ascorbic acid concentration in 0 min cooked samples decreased. In the cooked ascorbic acid samples, contrary to samples without iron, higher ascorbic acid concentrations led to a higher degradation ratio.5-Caffeovlquinic acid and quercetin-3-rutinoside need the additional factors temperature and time, as well as interactions in mixtures, for the catalytical activity of iron to work. Quercetin-3-rutinoside, in the presence of iron, only decreased in binary mixtures with ascorbic acid after thermal processing. This could be due to the fact that the flavonoid acts as a primary antioxidant and then the resulting compound radical reacts with ascorbic acid, regenerating the original compound [18]. 5-Caffeoylquinic acid seems to be special, because it protects quercetin-3-rutinoside, while ascorbic acid is not able to protect quercetin-3-rutinoside. Hence, 5-caffeoylquinic acid may be the key molecule for stabilizing the system in combination with iron and thermal processing. This hypothesis of 5-caffeoylquinic acid as a stabilizing molecule was further confirmed in the ternary mixtures. Here, quercetin-3-rutinoside was always stabilized by 5-caffeoylquinic acid, so that even in the presence of ascorbic acid no reduction in quercetin-3-rutinoside concentration was observed. In another study, 5-caffeoylquinic acid demonstrated protective properties against the degradation of anthocyanins through a co-pigmentation mechanism [40]. New degradation products, with possibly higher AOA, appeared from all three substances in the presence of iron. Caffeic acid (peak 6) was identified as a 5-caffeoylquinic acid breakdown product (data not shown). This led to the assumption that the other substance may be quinic acid or one of the nine possible derivatives described by Dawidowicz and Typek [24]: quinic acid;(1S,3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-2-hydroxypropanoyl]-1,4,5-trihydroxy-cyclohexane carboxylic acid;(1S3R,4R,5R)-5-[3-(3,4-dihydroxyphenyl)-3-hydroxypropanoyl]-1,4,5 trihydroxycyclohex-anecarboxylic acid; trans 3-O-caffeoylquinic acid; trans 5-O-caffeoylquinic acid; trans 4-O-caffeoylquinic acid; caffeic acid; cis-5-O-caffeoylquinic acid;4,5-dicaffeoylquinic acid. For quercetin-3-rutinoside, a degradation product appears in the chromatogram, which could not be identified.

3.4. Ability to Form Chelates with Ferric (Fe3+)and Ferrous Iron(Fe2+)

In all samples containing ascorbic acid, ferric iron was reduced to ferrous iron. In this process, ascorbic acid takes an electron from the ferric iron and reduces it to ferrous iron, and becomes a radical itself. The unstable radical converts rapidly to dehydroascorbic acid and further degradation products [12]. Due to missing hydroxy peroxide, a reaction back to ferric iron via the Fenton cycle is not possible. In 0 min cooked binary mixtures, more than 20 % of iron was bound in the presence of ascorbic acid. It is known that ascorbic acid forms complexes with iron species and other metal ions by chelation via the 3-O and 2-O nuclei following hydrogen displacement from the 3-OH and 2-OH groups [16,17,41. It can also form mixed-valence iron-ascorbate complexes [42]. However, ascorbic acid is a weak chelating agent and, after cooking, only traces of bound iron were detected in pure and mixed samples when ascorbic acid was present. Furthermore, due to the cooking process, ascorbic acid breaks down to degradation products, which seem unable to chelate with iron.

5-Caffeoylquinic acid is a relatively poor reductant. Ferric iron was reduced by 5-caffeoylquinic acid, and with prolonged cooking, reduction increased.5-Caffeoylquinic acid chelates with ferric iron in a ligand to metal charge transfer [17].5-Caffeoylquinic acid carries one possible binding side at the3,4 endiol structure of the caffeic acid. This could be an indicator as to why caffeic acid is the bioactive part of 5-caffeoylquinic acid, while quinic acid has almost no AOA [43]. Endiols inhibited OH formation due to the formation of an iron complex[41]in a one-to-one ratio [44,45]. Lamy et al.[46]conclude that 5-caffeoylquinic acid forms monomeric complexes, whereas Kiss et al. [47] found oligomeric species. Contrary to previous studies [17,48], only traces of bound iron were found in the presence of 5-caffeoylquinic acid pre-and post-thermal processing. This can be explained by the neutral pH conditions in this study, while other studies worked in an acidic medium, based on a pH value from 1-2.5in the human stomach [48]. A black precipitate was found in stored samples after several hours, which is an indicator of 5-caffeoylicquinic acid-ferric iron complexes. Recently mixed samples were used for the analysis, so the formation of these complexes at a neutral pH requires a longer period of time. Iron complexes with caffeic acid showed little scavenging activity [49]. Furthermore, there is no spectrophotometric evidence for a reaction between quinic acid and ferric iron [48]. Contrary to 5-caffeoylquinic acid, in the quercetin-3-rutinoside sample, bound iron was found, even after thermal processing.

Quercetin-3-rutinoside reduced ferric iron to ferrous iron with a minimum increase by a prolonged cooking time. This moderate reducing activity of quercetin-3-rutinoside was previously described by Mira et al. [50]. The ferric reducing activity of quercetin-3-rutinoside was detected in 3-rutinoside, 5,7,3',4'-OH [50]. The moderate interaction with ferric iron can be explained by a lower number of -OH groups, which resulted in lower negative charge density at the chelation side [50]. Flavonoids can chelate with metal ions at three potential coordination sides:(i)between 5-hydroxy and 4-carbonyl groups, (ii) between 3-hydroxy and 4-carbonyl groups, and (ii) between3', A'-hydroxy groups in Bring [39]. Quercetin-3-rutinoside uses the binding sides (i) and (i)[9], and at the 3-hydroxy group, the rutinoside is attached. Spectral data even showed that metal ions are only bound to the 3', A'-hydroxy group [18]. Chelates are more effective with iron in its bivalent form [50]. In binary mixtures, quercetin-3-rutinoside is not able to form complexes when ascorbic acid is present, contrary in ternary mixtures. If 5-caffeoylquinic acid is present, small amounts of bound iron are found. This indicates that 5-caffeoylquinic protects quercetin-3-rutinoside molecules in the presence of ascorbic acid, so quercetin-3-rutinoside can form complexes with iron.

4. Materials and Methods 

4.1.Chemicals

ABTS(2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt)(≥98%)was obtained from Sigma-Aldrich (Steinheim, Germany), DPPH*(2,2-diphenyl-1-picrylhydrazyl) radical (95%) and Trolox(97%)were obtained from Thermo Fisher (Kandel, Germany). Folin-Ciocalteu phenol reagent was purchased from Merck(Darmstadt, Germany). HPLC grade methanol, acetonitrile(HPLC grade), glacial acetic acid (100%, p.a.), sodium acetate trihydrate (≥99.5% p.a.), potassium thiocyanate (≥98.5%, p.a., ACS),2,2'-dipyridyl (≥95%), hydrochloric acid (≥25%, p.a., ISO), gallic acid monohydrate (≥99%), potassium persulfate (≥99%), rutin trihydrate (working standard), chlorogenic acid (working standard), L-(+)-ascorbic acid (working standard), sodium carbonate(>99%), ferric ammonium sulfate dodecahydrate, and ferrous ammonium sulfate hexahydrate were purchased from Carl Roth (Karlsruhe, Germany).

4.2.Samples

Aqueous solutions and mixtures of authentic standards of ascorbic acid, 5-caffeoylquinic acid, and quercetin-3-rutinoside were prepared as pure solutions and mixtures in different ratios. All possible binary mixtures were made in equimolar ratios, and non-equimolar ratios with one compound doubled. Ternary mixtures were also prepared in equimolar ratios, as well as in non-equimolar ratios with one or two compounds doubled, respectively. The antioxidants' final concentrations were 0.3 mM for each test solution. Additionally, all experiments were repeated after the addition of 0.3 mM iron(0.15 mM ferrous iron and 0.15 mM ferric iron) in total per mixture. The chosen amounts of antioxidants and iron are not based on physiological or food levels, they are based on molar masses to study the effect of molecular interaction. Consequently, an equimolar ratio between total antioxidants and iron was established. All mixtures were cooked for 0, 10, 20, and 40 min in boiling water, and afterward cooled on ice to stop the heating process. This was done for three independent replicates.

4.3.Photometric Measurements

Antioxidants (pure or mixed) in the absence and presence of iron were measured for their total reducing activity and antioxidant activity in three independent technical replicates using a high-throughput method in 96-well plates (SynergyTM HTX Multi-Mode Microplate Reader, BioTek Instruments, Winooski, VT, USA). Different test assays were used, because of the different reaction mechanisms: single-electron transfer(SET) and hydrogen atom transfer(HAT). While the TEAC and TPC assay is based on SET [17,18], for the DPPH test assay, the literature is not quite clear if it is based on SET, HAT, or even a combination of these two mechanisms [21-23]. A recent study by Foti[51] discovered that phenols can react with DPPH via sequential proton-loss electron transfer (SPLET), a combination of the two mechanisms. Factors, such as medium polarity and ionization potential, influence the predominant mechanism.

4.3.1. Total Phenolic Content (TPC)

Total phenol content(TPC) was determined using the Folin-Ciocalteu method in a 96-well plate, being previously described by Bobo-Garcia et al.[52] with some modifications.

Briefly， 10 μL Folin-Ciocalteu reagents were mixed with a 50 μL sample, and afterward 100 μL Na， CO3 was added. The 96-well plate was incubated at 37 °C（±0.2°C） and with constant orbital shaking at a moderate speed (237 CPM, 4 mm) for 14 min. After a 1 min resting period, the absorbance was measured at 736 nm. Results were expressed as gallic acid equivalents (mg GAE/ mol Antioxidant), using a standard curve ranging from 5.97 to 59.7 ug gallic acid/mL(R~>0.99). The common name of this test is misleading because the Folin-Ciocalteu reagent also reacts on non-phenolics, such as vitamins and minerals [22]. It describes better the"total reducing activity" of bioactive compounds. 

4.3.2. Trolox Equivalent Antioxidant Capacity (TEAC)

The antioxidant activity was determined using the TEAC assay in a 96-well plate with some modifications. A stock solution with 9.6 mg ABTS and 1.66 mg potassium persulfate filled up with H2O to 25 mL was prepared and incubated in the dark at room temperature for 12-16 h. From this stock solution, a TEAC working solution, containing a 5mL stock solution filled up to 25 mL with 100% MeOH, was prepared.

Briefly， 10 μL of the sample was mixed with 150 μL of the TEAC working solution. After a 5 min incubation, the plate was shaken orbitally at a moderate speed for 1 min, followed by a 1 min resting period. The absorbance was measured at a wavelength of 734 nm. The TEAC was expressed as Trolox equivalents (mol TE/mol Antioxidant), using a standard curve ranging from 0.025-0.8 mM Trolox(R->0.98).

4.3.3. DPPH*Radical Scavenging

The antioxidant activity was determined using the modified DPPHmethod for 96-well plates. A DPPHworking solution with 7.88 mg DPPH filled up to 100 mL was prepared. Briefly， 20 μL of the sample was mixed with 180 ul of the DPPH working solution and incubated in the dark for 28 min at room temperature. After 1 min orbital shaking at a moderate speed and a 1 min resting time, the absorbance was measured at a wavelength of 515 nm. Results were expressed as Trolox equivalents (mol TE/mol Antioxidant), using a standard curve ranging from 0.025-0.8 mM Trolox(R2>0.98).

4.4. Synergism and Antagonism

For analysis of synergistic and antagonistic effects of the antioxidant activity, a comparison of the results obtained experimentally with the theoretical values calculated by the sum of the effects of individual components at the corresponding concentration was made [53]. Synergism describes an interaction of two or more substances so that the combined action is greater than the sum of each acting separately. Contrary to this, antagonism is a phenomenon where the interaction of two or more substances in combination has an overall effect that is less than the sum of their individual effects.

4.5.Determination of Ionic Iron

The colorimetric determination of ferrous and ferric iron was modified according to Niedzielski et al. 【54】 for 96-well plates. Briefly， for ferrous iron detection， 20 μL acetate buffer(90 g sodium acetate trihydrate and 48 g acetic acid glacial filled up to 200 mL)and 20 μL 2，2'dipyridyl （0.5%， m/m） and， for ferric iron detection， 20 μL hydrochloric acid （2 M） and 20 μL potassium thiocyanate （5%， m/m）were pipetted into a 200 μL sample in the 96-well plate, incubated there for 10 min at room temperature, and the absorbance was measured for ferrous iron at520nm and for ferric iron at 470nm. Results were expressed in mM ionic iron/mM total iron, using a standard curve ranging for ferrous iron from 0.024-0.214mM(R'>0.99) and for ferric iron from0.005-0.178 mM(R->0.99). The difference between total iron and ionic iron, the sum of ferrous and ferric iron, is the bound iron.

4.6.HPLC-DAD

To quantify the antioxidants ascorbic acid, 5-caffeoylquinic acid, and quercetin-3-rutinoside and degradation products thereof in the same extracts used for the photometric

measurements, a Shimadzu Prominence 20 high-performance liquid chromatography (HPLC) system equipped with a refrigerated SIL-20AC HT autosampler, CTO-10AS VP column oven, DGU-20A5 degasser, LC-20 AT liquid chromatograph quaternary pump, and an SPD-M20A diode array detector (DAD) was used. As a column for separation, a Supelco Ascentis⑧Express an F5 column (150× 3.0 mm, 5 um)equipped with a Supelco Guard column （5×3.0 mm， 5 μm） and a 0.2 micron SST Frit for UltraLite was used. The column temperature was set to 30°C. UV detection was at 245 nm for ascorbic acid, 320 nm for 5-caffeoylquinic, and 360 nm for quercetin-3-rutinoside. The mobile phase consisted of Eluent A (1% acetic acid (v/o), pH 2.5) and Eluent B(100% ACN). The separation was achieved using the following gradient program: 0-2.5 min.5% B:2.5-15 min.5-20%B:15-20 min,20%B;20-22.5 min, 20-5%B;22.5-30 min,5%B.The flow rate was 0.3 mL/min, and the sample injection volume was 30 μL. Standard calibration curves for the three substances 5-caffeoylquinic （0.5-0.15 μM; R2>0.99）， ascorbic acid （0.35-0.025 uM; R2>0.99）， and quercetin-3-rutinoside（0.35-0.025 μM; R2>0.99） were prepared. Derived compounds were tentatively identified by analyzing the pure standards in the presence and absence of iron pre- and post-thermal processing. Therefore, the new peak must derive from the insert standard. Furthermore, selected mixtures were measured via HPLC-MS to verify the proposed structure.

4.7. Statistical Analysis

Microsoft Excel 2016(Microsoft, Redmond, USA) and R Statistics (version 3.6.3, Hold-ing the Windsock, 2020) were used for biostatistics tests and presenting and drawing data results. Inferential statistics for assessing and linking treatments were carried out by using a three-way analysis of variance(ANOVA), a post hoc Tukey's HSD test, and Pearson correlation. The R packages used were ggplot2 [55], means [56], and multcomp [57].

5. Conclusions

Based on the above-described findings, the AOA of ascorbic acid, 5-caffeoylquinic acid, and quercetin-3-rutinoside was influenced by their molecule structure, concentration, ratio, and interactions with other antioxidants and iron. Interaction especially seems to play a role in AOA when combining ascorbic acid and 5-caffeoylquinic acid. Here, synergistic and antagonistic effects were detected. The temperature had a minimal influence on AOA, while at the same time temperature influenced the stability of all antioxidants in certain mixtures, especially in the presence of iron. Only the ascorbic acid concentration decreased in the absence of iron with prolonged cooking time and 5-caffeoylquinic acid concentration decreased only in the presence of iron, while quercetin-3-rutinoside concentration decreased only in combination with ascorbic acid in the presence of iron. In combination with iron, 5-caffeoylquinic acid was able to protect other molecules from being reduced in their concentration by thermic processing.

In plants, combinations of ascorbic acid, 5-caffeoylquinic acid, and quercetin-3-rutinoside are not only possible but common. Hence, these results give basic knowledge on the processes that occur during the cooking of vegetables. Food matrices are more complex and contain countless bioactive compounds, including enzymes, other minerals, or acids, that change reaction conditions or are reactants themselves. Those complex interactions are far beyond the scope of this study and beneficial concentrations and interactions of antioxidants in cooked vegetables have to be addressed in the future.

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