Empirical Kinetic Modelling and Mechanisms of Quercetin Thermal Degradation in Aqueous Model Systems: Effect of Ph and Addition of Antioxidants

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Article Empirical Kinetic Modelling and Mechanisms of Quercetin Thermal Degradation in Aqueous Model Systems: Effect of pH and Addition of Antioxidants

Abdessamie Kellil 1, Spyros Grigorakis 1, Soﬁa Loupassaki 1 and Dimitris P. Makris 2,*

1 Food Quality & Chemistry of Natural Products, Mediterranean Agronomic Institute of Chania (M.A.I.Ch.), International Centre for Advanced Mediterranean Agronomic Studies (CIHEAM), P.O. Box 85, 73100 Chania, Greece; [email protected] (A.K.); [email protected] (S.G.); soﬁ[email protected] (S.L.) 2 Department of Food Science & Nutrition, School of Agricultural Sciences, University of Thessaly, N. Temponera Street, 43100 Karditsa, Greece * Correspondence: [email protected]; Tel.: +30-24410-64792

Abstract: Quercetin (Qt) is a natural flavonoid of high biological significance, and it occurs in a wide variety of plant foods. Although its oxidation by various means has been extensively studied, its behavior with regard to thermal treatments remains a challenge. The study described herein aimed at investigating Qt thermal decomposition, by proposing an empirical sigmoidal model for tracing degradation kinetics. This model was employed to examine the effect of addition of antioxidants on Qt thermal degradation, including ascorbic acid, L-cysteine, and sulfite. Furthermore, degradation pathways were proposed by performing liquid chromatography-tandem mass spectrometry analyses. Upon addition of any antioxidant used, the sigmoidal course of Qt thermal degradation was pronounced, evidencing the validity of the empirical model used in the study of similar cases. Citation: Kellil, A.; Grigorakis, S.; The antioxidants retarded Qt degradation in a manner that appeared to depend on Qt/antioxidant Loupassaki, S.; Makris, D.P. Empirical molar ratio. No major differentiation in the degradation mechanism was observed in response to the Kinetic Modelling and Mechanisms addition of various antioxidants, and in all cases protocatechuic acid and phloroglucinol carboxylic of Quercetin Thermal Degradation in acid were typical degradation products identified. Furthermore, in all cases tested the solutions Aqueous Model Systems: Effect of pH resulted after thermal treatment possessed inferior antioxidant properties compared to the initial and Addition of Antioxidants. Appl. Qt solutions, and this demonstrated the detrimental effects of heating on Qt. The empirical model Sci. 2021, 11, 2579. https://doi.org/ proposed could be of assistance in interpreting the degradation behavior of other polyphenols, but 10.3390/app11062579 its validity merits further investigation.

Academic Editor: Piotr Latocha Keywords: antioxidants; degradation kinetics; oxidation; quercetin

Received: 17 February 2021 Accepted: 11 March 2021 Published: 14 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral Polyphenols are ubiquitous plant secondary metabolites, and they are undisputedly a with regard to jurisdictional claims in highly significant field of interest in human nutrition. A growing body of research over the published maps and institutional affil- last two decades suggests that polyphenol intake through consumption of plant foods may iations. play a vital role in maintaining a good health status, by regulating metabolism, battling chronic degenerative diseases, and controlling cell proliferation. To date, accumulated epidemiological evidence indicates that various polyphenols may exhibit strong antioxidant and anti-inflammatory properties, which could be implicated in the prevention of Copyright: © 2021 by the authors. cardiovascular disease, neurodegenerative disorders, cancer, etc. [1]. Licensee MDPI, Basel, Switzerland. Amongst several polyphenol classes, flavonols are probably one of the most prominent This article is an open access article ones, because they are abundant at significant amounts in many commonly consumed fruit distributed under the terms and and vegetables, but also in foods of plant origin, such as fruit juices, tea and wine [2]. The conditions of the Creative Commons major flavonol representative is quercetin, which occurs in plant tissues mainly as glycoside, Attribution (CC BY) license (https:// although various types of processing may result in glycoside decomposition, releasing the creativecommons.org/licenses/by/ aglycone [3]. Quercetin has powerful antioxidant properties, and its biological significance 4.0/).

Appl. Sci. 2021, 11, 2579. https://doi.org/10.3390/app11062579 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 2579 2 of 14

has been appraised by numerous investigations [4,5]. On the other hand, quercetin is a molecule susceptible to chemical and enzymic attacks, which may result in decomposition, skeleton rearrangement, and dimerization [6]. Such modifications can inevitably lead to alteration of the biological properties of quercetin, but this does not necessarily entail loss of biological activity. Decomposition or oxidation may end up in obtaining products of inferior antioxidant potency [7], but quercetin and other structurally related flavonoids were also shown to retain antioxidant activity, despite chemical or enzymic oxidation [8,9]. Quercetin oxidation may also result in generating compounds with enhanced biological activity [10]. Therefore, the changes in quercetin structure brought about through oxidative modification should be a subject of thorough appraisal. The issue of food thermal processing on flavonols has been long before addressed, on the basis of findings which indicated that regular domestic practices (frying, boiling) could have an important impact on flavonol content and composition of certain plant tissues [11]. Latter studies confirmed that onion boiling may provoke notable decreases in flavonol glycosides and antioxidant activity, the extent of which was linked to glycoside structure [12]. More recent studies were in concurrence, highlighting the importance of domestic processing (frying, baking, steaming) on onion flavonol glycosides and its nutritional consequences [13]. Polyphenol thermal degradation has also been observed in black rice flour [14] and apple juice [15] thermal processing. However, other investigations suggested that the changes traced for flavonols in processed foods may depend on the mode of processing, yielding either increased or decreased flavonol content [16]. Indeed, it has been supported that heat treatments may contribute to increasing polyphenolic content in vegetables, as a result of thermal destruction of cell walls and sub cellular compartments during the cooking process, which would allow for polyphenol release [17]. Considering the above concepts, the current study aimed at assessing the effect of thermal treatment on quercetin degradation in model solutions. The phenomenon was approached by implementing a novel kinetic model, through which the effects of pH and addition of antioxidants were evaluated. The elucidation of quercetin degradation mechanism was carried out with liquid chromatography-tandem mass spectrometry (LC- MS/MS), and putative consequences on the antioxidant activity were discussed on the ground of the results from representative in vitro assays. To the best of the authors’ knowl- edge, such a kinetic approach is, for the first time, proposed for the study of polyphenol thermal degradation.

2. Materials and Methods 2.1. Chemicals Potassium phosphate dibasic trihydrate (≥99%), L-ascorbic acid, 2,2-diphenylpicrylhy drazyl (DPPH), sodium sulﬁte (>98%), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), quercetin di- hydrate (>95%), 3,4-dihydroxybenzoic acid (protocatechuic acid), 2,4,6-trihydroxybenzoic acid (phloroglucinol carboxylic acid), and citric acid were from Sigma-Aldrich (St. Louis, MO, USA). L-Cysteine was from BioChemica AppliChem (Darmstadt, Germany). Trolox™ was from Sigma-Aldrich (Steinheim, Germany). Iron(III) chloride hexahydrate was from Honeywell-Fluka (Harvey St, Muskegon, MI, USA). Quercetin stock solution with concentration of 20 mM was prepared in acetone and stored at −17 ◦C. Stock solution of L-ascorbic acid (5 mM), L-cysteine (5 mM), and sodium sulﬁte (5 mM mM) were prepared exactly before used in double-distilled water. Solvents used for liquid chromatography were of HPLC grade.

2.2. Model Solutions and Thermal Treatment A volume of 50 mL of citrate-phosphate dibasic buffer, adjusted at pH 6.5, 7.0, and 7.5, was placed in 100-mL dark glass vial and heated up at 90 ◦C, by means of an oil bath, placed on a hotplate (VELP Scientiﬁca, Bohemia, NY, USA). The solution was then set under stirring, at 400 rpm, and spiked with appropriate volume of quercetin stock solution, to give a ﬁnal concentration of 100 µmol L−1. To test the effect of L-ascorbic acid, Appl. Sci. 2021, 11, 2579 3 of 14

sodium sulﬁte, and L-cysteine, Qt solutions were also spiked with suitable volumes of stock solutions of these compounds, to provide the desired concentration.

2.3. Degradation Kinetics To trace kinetics, sampling was accomplished at predetermined intervals by withdraw- ing 0.5 mL of model solution. This aliquot was then mixed with 0.5 mL methanol containing 2% HCl and absorbance readings were obtained in a double-beam JASCO V-530 UV/Vis spectrophotometer (Tokyo, Japan). For monitoring quercetin degradation, absorbance was measured at wavelength corresponding to λmax of the Band I of the ﬂavonol skeleton [18]. The λmax was determined by obtaining Qt UV–VIS spectrum within the range 200–600 nm, in a 50% aqueous methanol containing 1% HCl. A calibration curve was constructed by −1 measuring the absorbance at λmax and plotting it against concentration (5–50 µmol L ).

2.4. Antioxidant Activity To assess the antioxidant activity of the model systems, two complementary tests were performed, the antiradical activity (AAR) and the ferric-reducing power (PR)[19]. Results for AAR and PR were expressed as mmol Trolox equivalents (TRE) and µM ascorbic acid equivalents (AAE), respectively.

2.5. Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS) The equipment used was a chromatograph TSQ Quantum Access LC/MS/MS, a surveyor pump (Thermo Scientiﬁc, Walltham, MA, USA), interfaced by XCalibur 2.1, TSQ 2.1 software. Chromatography was carried out on a Superspher RP-18 column, 125 mm × 2 mm, 4 µm, at 40 ◦C, with 10 µL injection volume and a ﬂow rate of 0.3 mL min−1. Eluent (A) was 1% aqueous acetic acid and eluent (B) methanol containing 1% acetic acid. The elution program was a linear gradient, as follows: 0 min, 5% B; 35 min, 100% B. Mass spectra were acquired in negative ionization mode, after performing a full scan within 100–700 amu (scan time = 1 s), using the following settings: auxiliary gas pressure, 15 mTorr; sheath gas pressure, 30 mTorr; collision pressure at 1.5 mTorr; capillary temperature, 300 ◦C.

2.6. Statistical Analysis Curve ﬁtting for kinetic models, and all linear and non-linear regressions were performed with SigmaPlot™ 12.5 (Systat Software Inc., San Jose, CA, USA). Distribution analysis was performed with JMP™ Pro 13 (SAS, Cary, NC, USA). Determinations were carried out in triplicate and the values reported are average (±standard deviation).

3. Results and Discussion 3.1. The Effect of pH The thermal degradation of Qt at 90 ◦C was ﬁrst assessed as a function of pH near neutrality, that is, within the range 6.5–7.5. The evolution of the decrease of Qt concentration, C (µmol L−1), was plotted against time, t (s), and at all three pH values tested (6.5, 7.0, and 7.5) it was observed that the curve obtained had a sigmoid trend (Figure1). Therefore, a sigmoidal model was implemented, as follows:

C0 Ct = t− t (1) −( 1/2 ) 1 + e b

where C0 and Ct are the initial molar Qt concentration and the concentration at any time, 1 t, respectively, t1/2 is the half-life of the degradation reaction (s), and − b the apparent ﬁrst-order degradation rate, k (s−1). The examination was also carried out using ﬁrst-order kinetics (Figure2), as follows: C ln t = −kt (2) C0 Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 14

where C0 and Ct are the initial molar Qt concentration and the concentration at any time, t, respectively, t1/2 is the half-life of the degradation reaction (s), and − the apparent first- order degradation rate, k (s−1). The examination was also carried out using first-order kinetics (Figure 2), as follows:

ln = −kt (2)

The decline in absorbance is virtually stabilized after some time, which actually signified the end of the reaction within the time limits initially set. These points do not con- Appl. Sci. 2021, 11, 2579 tribute in tracing kinetic curves, and they were, therefore, excluded from the4 linear of 14 representation in Figure 1.

120

100

80 ) -1 pH 6.5 60

mol L pH 7.0 μ

( pH 7.5 C 40

0 0 2000 4000 6000 8000 10000

t (s)

0.5

0.0 pH 6.5 pH 7.0 pH 7.5 -0.5 )

0 -1.0 C / t C (

ln -1.5

-2.0

-2.5

-3.0 0 1000 2000 3000 4000 5000 6000

t (s) Figure 1.1. Kinetics ofof QtQt thermalthermal degradation degradation (100 (100µmol μmol L− L1)-1 at) at pH pH 7.5 7.5 and and 90 90◦C( °Cupper (upper plot plot). ). Veri- Veriﬁcationfication of the of the first-order ﬁrst-order kineti kineticscs are shownshown by by a a linear linear plot plot (lower (lower plot plot). ).

TheAs can decline be inseen absorbance in Table is1, virtually the implemen stabilizedtation after of some the time,sigmoidal which model actually exhibited signified the end of the reaction within the time limits initially set. These points do not higher R2, which indicated a better fitting to the experimental data compared to first-order contribute in tracing kinetic curves, and they were, therefore, excluded from the linear representation in Figure1. As can be seen in Table1, the implementation of the sigmoidal model exhibited higher R2, which indicated a better fitting to the experimental data compared to first-order model. However, for both sigmoidal and first-order models, the statistical significance was higher than 95% (p < 0.05). Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 14

Appl. Sci. 2021, 11, 2579model. However, for both sigmoidal and first-order models, the statistical significance 5 of 14 was higher than 95% (p < 0.05).

120

100

80 ) -1 60 pH 6.5

mol L pH 7.0 μ

( 40 pH 7.5 C Fitted model

0 2000 4000 6000 8000 10000 t (s)

Figure 2. FigureAdjustment 2. Adjustment of the sigmoidal of the sigmoidal model on model the kinetic on the data kinetic obtained data forobtained Qt thermal for Qt degradation thermal degrada- at various pH. tion at various pH. Table 1. Kinetic data generated after implementing ﬁrst-order and sigmoidal kinetic models. Table 1. Kinetic data generated after implementing first-order and sigmoidal kinetic models. First-Order Model Sigmoidal Model pHFirst-Order Model−1 Sigmoidal Model−1 pH k (s ) 2 k (s ) 2 −1 −3 ½ −3 t21 (s) −1R −3 −½3 t 1 (s)2 R k (s ) (×10 ) (×t10 (s)) R 2 k (s ) (×10 ) (×10t (s)) 2 R 6.5 0.306.5 2310 0.30 0.94 2310 0.76 0.94 0.762189 21890.99 0.99 7.0 0.407.0 1733 0.40 0.97 1733 0.92 0.97 0.921470 14700.99 0.99 7.5 1.607.5 1.60 433 0.93 433 3.65 0.93 3.65 673 1.00 673 1.00

Determination of degradation rate constants, k, and degradation half-lives, t1/2, re- Determination of degradation rate constants, k, and degradation half-lives, t1/2, re- vealed importantvealed discrepancies important between discrepancies models between (Table models1). In particular, (Table1). k In determined particular, thatk determined that the sigmoidal themodel sigmoidal were 2.3 model to 2.5 were higher 2.3 to than 2.5 higher those than determined those determined with the with first-order the first-order model. model. However, for pH 7.5, t1/2 determined with the sigmoidal model was 1.55-times However, for pH 7.5, t1/2 determined with the sigmoidal model was 1.55-times higher than higher than thatthat determined determined with with the the first-order first-order model. model. This This finding finding pointed pointed to toan an under- underestimation of estimation of thethe degradation degradation rate rate constant, constant, when when the the first-order first-order model model was was used, used, but but also also to a different 1/2 to a different relationshiprelationship between between k and t1/2 inin the the sigmoidal sigmoidal model, model, as as implied implied by EquationEqua- (1). At this tion (1). At thispoint, point, the the issue issue that that arises arises is the is the reason reason for whichfor which the sigmoidal the sigmoidal model, model, which rather traces which rather tracesQt degradation Qt degradation more more accurately, accurately, delivers delivers higher higherk. The k. monitoringThe monitoring of Qt of degradation is Qt degradationcarried is carried out out by by recording recording the the decrease decrease in inλmax λmax(370 (370 nm). nm). Yet, Yet, decrease decreaseof of absorbanceab- at 370 sorbance at 370nm nm occurs occurs not not when when Qt Qt starts starts toto breakbreak downdown intointo simplersimpler phenolics, but but as as soon as Qt is soon as Qt is convertedconverted into into the the initial initial oxidatio oxidationn product, product, which which is its is quinone its quinone (Figure (Figure 3) [20].3)[ 20]. Should this be the case, then initially, decrease in λmax might be slower. This is because Qt quinone exhibits a λmax at around 425–430 nm [21] and, thus, it may possess notable absorbance at 370 nm, which would affect monitoring of Qt disappearance. First-order kinetics fails to incorporate this stage, and the points included in tracing Qt degradation are those corresponding to rapid Qt disappearance, which takes place during quinone decomposition. Cleavage of the Qt quinone skeleton would lead to decreases at 370 nm, yielding degradation products (simpler phenolics) that possess practically no absorbance at this region of the spectrum [22]. This is most probably the rationale behind the differences Appl. Sci. 2021, 11, 2579 6 of 14

Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 14 observed when kinetic parameters were determined with the sigmoidal model instead of the ﬁrst-order model.

(1) (3) (5)

(2) (6)

(4) (7)

Figure 3. Figure 3. StructuresStructures of ofQt Qt degradation/oxidation/reaction degradation/oxidation/reaction productsproducts tentatively tentatively identified identified in thein modelthe model solutions solutions tested. tested. m z m z m z Assignments:Assignments: (1) (Protocatechuic1) Protocatechuic acid acid ( (m/z == 151); 151); ((22)) phloroglucinolphloroglucinol carboxylic carboxylic acid acid ( / (m=/ 169);z = 169); (3) Qt (3 quinone) Qt quinone ( / = ( 299);m/z = 299); (4), 2,3,4,-chalcan-trione(4), 2,3,4,-chalcan-trione o-quinone;o-quinone; (5 ()5 )3,4-dione-2-oxo-phenylacetic 3,4-dione-2-oxo-phenylacetic acid acid (m (/mz/=z = 179); 179); (6 )(6 Qt) Qt 3-sulfate 3-sulfate (m/ (zm=/z 381); = 381); (7) Qt (7) Qt 3- sulfate3-sulfate quinone quinone (m/z (=m 379)./z = 379). The determination of k with either model demonstrated an approximate 5-fold in- creaseShould from this pH 6.5be tothe 7.5, case, illustrating then initially, the effect decrease of pH. Although in λmax might information be slower. on thermal This is be- causedegradation Qt quinone kinetics exhibits of quercetin a λmax at 95around◦C was 425–430 previously nm provided[21] and, tothus, some it extentmay possess [23], no- tableno dataabsorbance regarding at the370 effect nm, ofwhich pH at would similar affect temperatures monitoring have of been Qt disappearance. reported. Early First- orderexaminations kinetics fails demonstrated to incorporate the dependence this stage, of and quercetin the points oxidative included degradation in tracing on Qt pH degra- dationwithin are the those range corresponding 4–7, suggesting ato logarithmic rapid Qt disappearance, increase in first-order which kinetics takes constantplace during as a qui- ◦ nonefunction decomposition. of pH, at 50 CleavageC[24]. In theof the same Qt line, quinone more recentskeleton studies would showed lead anto increasedecreases in at 370 first-order decomposition of quercetin as pH increased from 6.0 to 7.5 [25]. It was proposed nm, yielding degradation products (simpler phenolics) that possess practically no absorb- that quercetin deprotonation at alkaline pH accelerates its oxidation, affecting the C-ring anceand at the thiso-diphenol region structureof the spectrum of the B-ring, [22]. and This leading is most to the probably formation the of arationale benzofuranone behind the differencesderivative observed [26,27]. when kinetic parameters were determined with the sigmoidal model insteadThe of LC-MS/MSthe first-order analysis model. of a Qt solution after treatment at 90 ◦C for 50 min (Figure4) enabledThe determination the tentative identification of k with ofeither typical model degradation demonstrated products, an including approximate protocate- 5-fold in- creasechuic from acid (PCA) pH 6.5 and to phloroglucinol7.5, illustrating carboxylic the effect acid of (PGCA) pH. Although (Table2; Figureinformation3). on thermal degradation kinetics of quercetin at 95 °C was previously provided to some extent [23], no data regarding the effect of pH at similar temperatures have been reported. Early examinations demonstrated the dependence of quercetin oxidative degradation on pH within the range 4–7, suggesting a logarithmic increase in first-order kinetics constant as a function of pH, at 50 °C [24]. In the same line, more recent studies showed an increase in first-order decomposition of quercetin as pH increased from 6.0 to 7.5 [25]. It was proposed that quercetin deprotonation at alkaline pH accelerates its oxidation, affecting the C-ring and the o-diphenol structure of the B-ring, and leading to the formation of a benzofuranone derivative [26,27]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 14

The LC-MS/MS analysis of a Qt solution after treatment at 90 °C for 50 min (Figure Appl. Sci. 2021, 11, 2579 7 of 14 4) enabled the tentative identification of typical degradation products, including protocatechuic acid (PCA) and phloroglucinol carboxylic acid (PGCA) (Table 2; Figure 3).

2 5.11 100 5.16 5.04 5.28 90

80 7 70 21.66

50 1 2.90 40 Relative Abundance 30

20 10.92 2.04 22.63 10 5.71 25.58 0.02 6.43 11.21 15.37 15.59 20.53 25.80 30.85 34.92 0 0 5 10 15 20 25 30 Time (min) Figure 4. TotalFigure ion 4. chromatogram Total ion chromatogram (TIC) of a Qt(TIC) solution of a Qt (100 solutionµmol (100 L−1), μ treatedmol L−1), at treated 90 ◦C, forat 90 50 °C, min, for at 50 pH 7.5. For peak assignment,min, see at Table pH2 7.5.. For peak assignment, see Table 2.

Table 2. Mass spectralIdentification data of the of majorboth QtPCA degradation and PGCA products, was based detected on mass in Qt solutionsspectral afterdata treatmentpreviously for 50 min, at 90 ◦C. reported [28]. Qt quinone was also detected, which confirmed its formation as an intermediate in Qt degradation. The typical oxidation product 2-(hydroxybenzoyl)-2-hy- Other Rt droxybenzofuran-3(2Molecular Ion H)-one (benzofuranone), which may arise as a result of Qt oxidationQt + Peak # Fragments Tentative Identity Formula Qt a Qt + AsA b Qt + Sulfite d (min) (m/z) CySH c by various means [28],(m/z) was not detected. This fact might be attributed to the relatively high temperature of the treatment,Phloroglucinol which carboxylic most probably triggered benzofuranone ther- acid 1 2.90 169 151 (-H O) C H O + + + + mal decomposition. On2 the other(2,4,6,-trihydroxybenzoic hand, alternative oxidation7 5 5 pathways might have taken place as previously proposed [18,29], resultingacid) in PCA and PGCA formation. It should be Protocatechuic acid 2 5.16 153 109 (-CO ) C H O + + + + noted that Qt oxidation 2 in the(2,4-dihydroxybenzoic absence of H2 acid)O2 did 7not5 4yield PCA and PGCA [30,31]. 3,4-dione-2-oxo-phenylacetic 3 12.10Therefore, 179 the degradation 119 pathway(s) involved wouldC H beO likely- to include + formation - of - acid 8 3 5 4 12.41H2O2 too, 381 as intermediate. - This could Quercetin arise 3-sulfate through reaction C15H13O7S of O -2 with 2H - atoms offered - + 5 14.22 379 - Quercetin 3-sulfate quinone C15H11O7S - - - + 6 20.27by Qt, hence 301 the formation - of Qt quinone. Quercetin Such a Creaction15H10O7 would- be facilitated + at +the + 7 21.66mildly alkaline 299 pH used - (7.5). Then Quercetin H2O2 quinonenucleophilic C attack15H8O7 at the+ C2 position + would lead + + a Treatmentto of 100crossµmol ring L− 1cyclicQt; b Treatment peroxylation, of 100 µresultingmol L−1 Qt an + 200intermediateµmol L−1 AsA; depside.c Treatment Decomposition of 100 µmol L− 1ofQt this + 200 µmol L−1 d −1 −1 CySH; Treatmentdepside of 100wouldµmol LeventuallyQt + 200 µ yieldmol L PCAsulfite. and PGCA [31]. Such a reaction would also be feasible through radical formation, as previously suggested [29]. Identification of both PCA and PGCA was based on mass spectral data previously Table 2. Mass spectral data of the major Qt reporteddegradation [28 ].products, Qt quinone detected was in also Qt detected,solutions after which treatment confirmed for 50 its min, formation at as an intermedi- 90 °C. ate in Qt degradation. The typical oxidation product 2-(hydroxybenzoyl)-2-hydroxybenzof uran-3(2H)-one (benzofuranone), which may arise as a result of Qt oxidation by vari- Molecular Ion Other Fragments Peak # Rt (min) Tentative Identity Formula Qt a Qt + AsA b Qt + CySH c Qt + Sulfite d (m/z) (m/z) ous means [28], was not detected. This fact might be attributed to the relatively high temperaturePhloroglucinol of the carboxylic treatment, acid which most probably triggered benzofuranone thermal de- 1 2.90 169 151 (-H2O) C7H5O5 + + + + composition.(2,4,6,-trihydroxybenzoic On the other hand,acid) alternative oxidation pathways might have taken place as previouslyProtocatechuic proposed [ 18acid,29 ], resulting in PCA and PGCA formation. It should be noted 2 5.16 153 109 (-CO2) C7H5O4 + + + + (2,4-dihydroxybenzoic acid) that Qt oxidation in the absence of H2O2 did not yield PCA and PGCA [30,31]. Therefore, 3 12.10 179 119 3,4-dione-2-oxo-phenylacetic acid C8H3O5 - + - - the degradation pathway(s) involved would be likely to include formation of H2O2 too, as 4 12.41 381 - Quercetin 3-sulfate C15H13O7S - - - + intermediate. This could arise through reaction of O2 with 2H atoms offered by Qt, hence 5 14.22 379 - Quercetin 3-sulfate quinone C15H11O7S - - - + 6 20.27 301 - the formation of Quercetin Qt quinone. Such C a15H reaction10O7 - would +be facilitated + at the mildly + alkaline pH 7 21.66 299 - used (7.5). ThenQuercetin H2 Oquinone2 nucleophilic C15 attackH8O7 at+ the C2 + position would + lead to + cross ring cyclic peroxylation, resulting an intermediate depside. Decomposition of this depside would eventually yield PCA and PGCA [31]. Such a reaction would also be feasible through radical formation, as previously suggested [29]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 14

Appl. Sci. 2021, 11, 2579 8 of 14 a Treatment of 100 μmol L-1 Qt; b Treatment of 100 μmol L-1 Qt + 200 μmol L-1 AsA; c Treatment of 100 μmol L-1 Qt + 200 μmol L-1 CySH; d Treatment of 100 μmol L-1 Qt + 200 μmol L-1 sulfite.

3.2.3.2. The Effect ofof AsAAsA AdditionAddition −1 ToTo testtest thethe effecteffect ofof AsA, AsA, increasing increasing AsA AsA amounts amounts varying varying from from 10 10 to to 200 200µ μmolmol L −L1 werewere addedadded into Qt solutions, solutions, to to provide provide a aQt/AsA Qt/AsA molar molar ratio ratio range range from from 10 to 10 0.5. to 0.5.In this In thiscase, case, the thesigmoidal sigmoidal course course of degradation of degradation was was more more evident evident as AsA as AsA concentration concentration in- increasedcreased (Figure (Figure 5).5). Although Although AsA AsA at levels ofof 1010 andand 2525 µμmolmol LL−−1 resultedresulted in lower kk,, anan increasingincreasing trendtrend observedobserved forfor kk,, fromfrom 1010 andand up up to to 100 100µ μmolmol L L−−1 AsA (Table 3 3).). However,However, thethe differencesdifferences inin kk comparedcompared toto controlcontrol treatmenttreatment (no(no AsAAsA addition)addition) werewere aboutabout 14%,14%, atat 1/2 most.most. OnOn thethe otherother hand,hand, tt1/2 displayeddisplayed a a consistent consistent increasing tendency withinwithin thethe wholewhole rangerange ofof AsAAsA concentrationsconcentrations tested.tested. This outcomeoutcome suggestedsuggested that the effecteffect ofof AsAAsA onon kk waswas dependentdependent onon Qt/AsAQt/AsA molar ratio, but aa riserise inin AsAAsA concentrationconcentration upup toto a a Qt/AsA Qt/AsA molarmolar rationration ofof 0.50.5 providedprovided aa proportionalproportional QtQt stability,stability, as as implied implied by by the the increased increasedt 1/2t1/2.

120 0 μmol L-1 AsA 10 100 25 50 100 80 200 ) -1 60 mol mol L μ

( 40 C

0 500 1000 1500 2000 2500 t (s) FigureFigure 5. ImplementationImplementation of of the the sigmoidal sigmoidal kinetic kinetic mode modell toto trace trace Qt Qt thermal thermal degradation degradation in the in the presencepresence ofof variousvarious concentrationsconcentrations ofof AsA.AsA.

TableSuch 3. The a effectphenomenon of antioxidants could on be Qt attributed degradation to a kinetics stabilizing using effect the sigmoidal on Qt quinone, model. or, if Qt radical formation has preceded, donation of a H atom to convert Qt radical back to Qt. −1 TheAntioxidant latter would Added prevent (µmol O2 L-mediated) oxidation of Qt, Kinetic which Constants would further * decompose −1 −3 2 into simpler phenolics [29]. The fact thatk (s Qt)( was×10 oxidized) firstt 1 (s)could be supportedR by its lower oxidation potential, compared to AsA [32,33]. 2 No Addition 3.65 673 1.00 Table 3. The effectAsA of antioxidants on Qt degradation kinetics using the sigmoidal model. 10 3.51 942 0.99 Antioxidant25 Added (μmol L−1) 3.58Kinetic 969 Constants * 0.99 50 4.09k (s−1) (×10−3 1042) t½ (s) 0.99 R2 No100 Addition 4.18 3.65 1151 673 0.99 1.00 200 3.86 1402 0.99 CySHAsA 1010 3.363.51 709942 1.000.99 2525 2.843.58 749969 0.990.99 5050 2.374.09 8801042 0.990.99 100 2.11 875 1.00 200100 2.104.18 8551151 0.990.99 Na2SO2003 3.86 1402 0.99 10CySH 3.09 901 0.99 2510 3.513.36 885709 0.991.00 50 3.42 865 0.99 10025 3.212.84 910749 0.990.99 200 2.78 980 0.99 * Signiﬁcance of model ﬁtting was at a level higher than 99.9% (p < 0.0001). Appl. Sci. 2021, 11, 2579 9 of 14

Such a phenomenon could be attributed to a stabilizing effect on Qt quinone, or, if Qt radical formation has preceded, donation of a H atom to convert Qt radical back to Qt. The latter would prevent O2-mediated oxidation of Qt, which would further decompose into simpler phenolics [29]. The fact that Qt was oxidized ﬁrst could be supported by its lower oxidation potential, compared to AsA [32,33]. Therefore, it would be likely that, in a mixture of AsA and Qt, the latter is preferably oxidized, giving rise to Qt radicals and/or Qt quinone. Then, AsA might scavenge O2 (and/or H2O2), delaying to some extent further Qt oxidation and decomposition. This is likely the reason for determining increasing t1/2 upon addition of increasing AsA concentration. Upon depletion of AsA, Qt would react with O2 (or H2O2) generating eventually PCA and PGCA. To verify whether the presence of AsA could alter the mechanism of Qt decomposition, the mixture of Qt treated in the presence of 200 µmol L−1 AsA for 50 min, at 90 ◦C, was analyzed by LC-MS/MS. Once again, as in the case of Qt alone, the major compounds detected were PCA and PGCA, but also another one with m/z = 179. The latter was tentatively assigned to 3,4-dione-2-oxo-phenylacetic acid (Figure3), and it could derive from decomposition of 2,3,4-chalcan-trione o-quinone, as previously proposed [31]. In such a case, formation of Qt quinone should precede and, therefore, it could be supported that, in the presence of AsA, Qt quinone formation and subsequent decomposition could be a plausible predominant pathway.

3.3. The Effect of CySH Addition As in the case of AsA addition, the sigmoidal course of Qt decomposition was also pronounced by the addition of increasing amounts of CySH (Figure6), compared to control, although less so compared to AsA. The addition of increasing amounts of CySH resulted in a consistently decreasing tendency in k, and when CySH was added at a level of 200 µmol L−1, Qt displayed the least k, which was by almost 43% lower compared to control −1 (no CySH addition). On the other hand, t1/2 increased up to 50 µmol L CySH, but then it exhibited a rather stabilizing trend (Table3). Based on the hypothesis set out for AsA, it would appear that CySH stabilized Qt quinone by preventing quinone reaction with oxygen, or by acting as radical scavenger, up to 50 µmol L−1; additional increase in CySH had no or little effect. Stabilizing effect of CySH against thermally-induced, copper- catalyzed oxidative Qt degradation has been previously reported to occur at 1:1 molar ratio [34]. The authors attributed this protective action to the ability of CySH to react with oxygen, giving stable products, such as L-cystine, L-cysteine sulphinic acid, and L-cysteine sulphonic acid. On the other hand, under different conditions, CySH might accelerate Qt degradation through the involvement of cysteinyl radicals [18]. However, given the stabilizing effect observed, the latter case would be rather unlikely. The chromatographic proﬁle of a Qt mixture with 200 µmol L−1 CySH, after 50 min of treatment at 90 ◦C, did not show any difference compared to that of Qt alone, and the major products detected were PCA, PGCA, and Qt quinone (Table2). Unlike previous examinations [18,34], the appearance of peaks that would manifest Qt reaction with CySH was not evidenced. Such a ﬁnding would strongly point to a non-radical degradation mechanism, as also supported for Qt/AsA mixtures.

3.4. The Effect of Sulfite Addition Similarly to what was observed by addition of either AsA or CySH, the sigmoidal course of Qt degradation was more evident by the addition of sodium sulfite (Figure7). At a concentration of 10 µmol L−1, sulfite provoked a decrease in k by almost 15% compared to control (no sulfite addition), but at 25 µmol L−1 an increase was recorded (Table3). Thereafter, k followed a constant decline, as in the case of CySH. Likewise, although t1/2 decreased up to 50 µmol L−1, from 100 to 200 µmol L−1 an increase was seen. This outcome indicated that sulfite showed a consistent stabilizing effect from 50 µmol L−1 onwards. Appl.Appl. Sci.Sci. 20212021,, 1111,, 2579x FOR PEER REVIEW 1010 of 1414

120 0 μmol L-1 CySH 10 100 25 50 100 80 200 ) -1 60 mol L μ ( 40 C

Appl. Sci. 2021, 11, x FOR PEER REVIEW 11 of 14

0 500 1000 1500 2000 2500 t (s) the reduction was still significant compared to non-treated Qt solution. Regarding PR, the Figure 6. ImplementationImplementation of of the the sigmoidal sigmoidal kinetic kinetic mode modell to totrace trace QtQt thermal thermal degradation degradation in the in the reduction was significant irrespective of the antioxidant added. presencepresence ofof variousvarious concentrationsconcentrations ofof CySH.CySH.

3.4. The120 Effect of Sulfite Addition μ -1 0 mol L Na2SO3 Similarly to what was observed by addition of either10 AsA or CySH, the sigmoidal course of Qt100 degradation was more evident by the addition of25 sodium sulfite (Figure 7). At a concentration of 10 μmol L−1, sulfite provoked a decrease in k50 by almost 15% compared to control (no 100 −1 sulfite80 addition), but at 25 μmol L an increase was recorded200 (Table 3). Thereafter, k followed a constant decline, as in the case of CySH. Likewise, although t1/2 decreased up to 50 μmol L−1, ) from-1 60100 to 200 μmol L−1 an increase was seen. This outcome indicated that sulfite showed a consistent stabilizing effect from 50 μmol L−1 onwards. mol mol L μ

( Sulfite40 may act as oxygen scavenger and reducing agent [35]. Therefore, the protec- tiveC action it could exert would be through preventing reaction of Qt (or Qt quinone) with oxygen,20 or reduction of the Qt quinone back to Qt. Sulfur dioxide (SO2) has been proven to prevent Qt electrochemical oxidation [36], and the authors claimed that the antioxidant

protection0 seen was a result of quinone reduction or semiquinone radical reduction. Judg- ing by the degradation products generated, it was also concluded that Qt oxidative degradation follows the same pathway, irrespective of the presence of SO2. To test this hypothesis,0 as well as 500 to shed more 1000 light onto 1500 the effect 2000 of sulfite on 2500 Qt oxidative degradation, a Qt solution treated for 50 min, at 90 °C, in the presence of 200 μmol L−1 sodium t (s) sulfite, was subjected to LC-MS/MS analysis. Apart from the typical degradation products Figure 7. Implementation of the sigmoidal kinetic model to trace Qt thermal degradation in the FigurePCA, PGCA7. Implementation and Qt quinone, of the sigmoidal two other kinetic peaks mode withl to m trace/z = 381 Qt thermaland 379 degradation were also indetected the presence of variousvarious concentrationsconcentrations ofof sulfite.sulfite. (Table 3). These compounds were encountered neither in Qt solutions, nor in Qt solutions treated with AsA and CySH. The peak with m/z = 381 might correspond to Qt 3-sulfate, as SulfiteQt is considered may act as a oxygen powerful scavenger antioxidant, and reducing due to unique agent [structural35]. Therefore, features, the protectivewhich are previously proposed [37,38], while the peak with m/z = 379 could be its quinone (Figure actionnot encountered it could exert in other would flavonoids, be through such preventing as the combination reaction ofof Qtthe(or o-diphenol Qt quinone) in B-ring, with 3). This finding raised evidence for plausible reaction(s) between Qt and sulfite. oxygen,the C2–C3 or double reduction bond, of thethe Qt4-oxo quinone group back and tothe Qt. 5-hydroxyl Sulfur dioxide group (SO[39].2) Once has been the skeleton proven tois cleaved, prevent these Qt electrochemical features are abrogated oxidation and [36 ],thus and it thewould authors be reasonable claimed thatto anticipate the antioxi- re- 3.5. Impact on Antioxidant Activity dantduced protection antioxidant seen activity. was a resultFurthermore, of quinone the reductiontwo major or degradation semiquinone products, radical reduction. PCA and JudgingPGCA,To havetest by thewhether been degradation proven Qt decomposition to be products of considerab generated, couldly impact lower it was itsefficiency alsoantioxidant concluded [22]. propertiesAsA, that CySH, Qt to oxidative anda signif- sul- −1 degradationfiteicant may extent, also a followsdegrade Qt solution theand/or same of oxidize100 pathway, μmol to someL irrespective was extent assayed and of apparently thebefore presence and couldafter ofSO treatmentnot2. compensate To test at this pH hypothesis,7.5 and 90 °C, as for well 50 as min. to shed As can more be lightseen ontoin Figure the effect 8, both of sulfiteAAR and on P QtR were oxidative significantly degra- for the antioxidant activity lost. ◦ − dation,reduced a after Qt solution treatment treated (p < for0.05). 50 This min, outcome at 90 C, clearly in the presence pointed ofto 200a detrimentalµmol L 1 effectsodium of sulfite,Qt thermal was break subjected down to LC-MS/MSon its antioxidant analysis. potency. Apart The from examination the typical degradation of solutions productswith 200 3.0 PCA,μmol PGCAL−1 of either and Qt AsA, quinone, CySH two and other sulfite peaks showed with thatm/ zA=AR 381 may and be 379lesswere affected, also although detected (Table3). These compoundsa were encountered neither in Qt solutions, nor in Qt solutions treated2.5 with AsA and CySH. The peakc with m/cz = 381 might correspond to Qt 3-sulfate, as

) c -1 2.0 b

1.5 (mmol TRE L TRE (mmol

AR 1.0 A

0.5

0.0 (i) f) A H te t t ( As yS lfi Q Q C su µM M M 00 0 µ 0 µ 2 20 20 + + + Appl. Sci. 2021, 11, x FOR PEER REVIEW 11 of 14

the reduction was still significant compared to non-treated Qt solution. Regarding PR, the reduction was significant irrespective of the antioxidant added.

120 μ -1 0 mol L Na2SO3 10 100 25 50 100 80 200 ) -1 60 mol mol L μ

( 40 C

0 Appl. Sci. 2021, 11, 2579 11 of 14

0 500 1000 1500 2000 2500 t (s) previously proposed [37,38], while the peak with m/z = 379 could be its quinone (Figure3). ThisFigure finding 7. Implementation raised evidence of the forsigmoidal plausible kinetic reaction(s) model to between trace Qt thermal Qt and degradation sulfite. in the presence of various concentrations of sulfite. 3.5. Impact on Antioxidant Activity Qt is considered a powerful antioxidant, due to unique structural features, which are To test whether Qt decomposition could impact its antioxidant properties to a signifi- not encountered in other flavonoids, such as the combination of the o-diphenol in B-ring, cant extent, a Qt solution of 100 µmol L−1 was assayed before and after treatment at pH the C2–C3 double bond, the 4-oxo group and the 5-hydroxyl group [39]. Once the skeleton 7.5 and 90 ◦C, for 50 min. As can be seen in Figure8, both A and P were significantly is cleaved, these features are abrogated and thus it would beAR reasonableR to anticipate re- reduced after treatment (p < 0.05). This outcome clearly pointed to a detrimental effect duced antioxidant activity. Furthermore, the two major degradation products, PCA and of Qt thermal break down on its antioxidant potency. The examination of solutions with PGCA, have− 1been proven to be of considerably lower efficiency [22]. AsA, CySH, and sul- 200 µmol L of either AsA, CySH and sulfite showed that AAR may be less affected, al- thoughfite may the also reduction degrade wasand/or still oxidize significant to some compared extent toand non-treated apparently Qt could solution. not compensate Regarding for the antioxidant activity lost. PR, the reduction was significant irrespective of the antioxidant added.

3.0 a 2.5 c c

) c -1 2.0 b

1.5 (mmol TRE L TRE (mmol

AR 1.0 A

0.5

0.0 Appl. Sci. 2021, 11, x FOR PEER REVIEW 12 of 14 (i) f) A H te t t ( As yS lfi Q Q C su µM M M 00 0 µ 0 µ 2 20 20 + + + 180

160 a

140 )

-1 120 b 100 b b 80 b mol mol AAE L μ ( R 60 P

0 (i) f) A H te t t ( s yS lfi Q Q A C su µM M 0 µ µM 20 00 00 + + 2 + 2

Figure 8. Antiradical activity (upper plot) and ferric-reducing power (lower plot) of a Qt solution (100Figureµmol 8. Antiradical L−1), at pH activity 7.5, prior (upper to thermal plot) and treatment ferric-reducing (Qt(i)), after power 50 min(lower at 90plot◦C) of (Qt(f)) a Qt andsolution after (100 μmol L-1), at pH 7.5, prior to thermal treatment (Qt(i)), after 50 min at 90 °C (Qt(f)) and after 50 min at 90 ◦C in the presence of 200 µmol L−1 AsA, CySH and sulﬁte. Bars designated with 50 min at 90 °C in the presence of 200 μmol L-1 AsA, CySH and sulfite. Bars designated with differ- different letters indicate statistically different value (p < 0.05). ent letters indicate statistically different value (p < 0.05).

4. Conclusions The thermal degradation of Qt in an aqueous model system, at pH 7.5, was shown to obey a sigmoidal curve, and on this ground a novel empirical kinetic model was proposed, to investigate the decomposition behavior of Qt. This sigmoidal trend was even more evident when Qt solutions were treated in the presence of compounds that express antioxidant effect, including AsA, CySH, and sulfite. In all cases it was found that addition of antioxidants may delay Qt thermal degradation, but this depended on the molar ratio Qt/antioxidant. Some reaction products between Qt and sulfite tentatively identified may also play a role in this regard. However, the typical degradation products detected were PCA and PGCA, irrespective of the antioxidant added. In any case, the solution resulted after thermal treatment possessed inferior antioxidant properties compared to the initial Qt solution, and this demonstrated the detrimental effects of heating on Qt. The kinetic model proposed should be considered strictly as empirical, and it may be of assistance in interpreting the degradation behavior of other polyphenols. Thus, testing of a wider range of structures could reveal its validity in assessing various factors (pH, antioxidants) in polyphenol thermal degradation, but also its limitations, which are yet to be defined.

Author Contributions: Conceptualization: D.P.M.; methodology: D.P.M., S.G.; validation: A.K., S.G.; formal analysis: A.K., S.G., S.L.; data curation: D.P.M., S.G., A.K.; writing—original draft preparation: D.P.M.; visualization: D.P.M., S.G., A.K.; supervision: D.P.M. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable Conflicts of Interest: The authors declare no conflict of interest

Appl. Sci. 2021, 11, 2579 12 of 14

Qt is considered a powerful antioxidant, due to unique structural features, which are not encountered in other flavonoids, such as the combination of the o-diphenol in B-ring, the C2–C3 double bond, the 4-oxo group and the 5-hydroxyl group [39]. Once the skeleton is cleaved, these features are abrogated and thus it would be reasonable to anticipate reduced antioxidant activity. Furthermore, the two major degradation products, PCA and PGCA, have been proven to be of considerably lower efficiency [22]. AsA, CySH, and sulfite may also degrade and/or oxidize to some extent and apparently could not compensate for the antioxidant activity lost.

4. Conclusions The thermal degradation of Qt in an aqueous model system, at pH 7.5, was shown to obey a sigmoidal curve, and on this ground a novel empirical kinetic model was proposed, to investigate the decomposition behavior of Qt. This sigmoidal trend was even more evident when Qt solutions were treated in the presence of compounds that express antioxidant effect, including AsA, CySH, and sulfite. In all cases it was found that addition of antioxidants may delay Qt thermal degradation, but this depended on the molar ratio Qt/antioxidant. Some reaction products between Qt and sulfite tentatively identified may also play a role in this regard. However, the typical degradation products detected were PCA and PGCA, irrespective of the antioxidant added. In any case, the solution resulted after thermal treatment possessed inferior antioxidant properties compared to the initial Qt solution, and this demonstrated the detrimental effects of heating on Qt. The kinetic model proposed should be considered strictly as empirical, and it may be of assistance in interpreting the degradation behavior of other polyphenols. Thus, testing of a wider range of structures could reveal its validity in assessing various factors (pH, antioxidants) in polyphenol thermal degradation, but also its limitations, which are yet to be defined.

Author Contributions: Conceptualization: D.P.M.; methodology: D.P.M., S.G.; validation: A.K., S.G.; formal analysis: A.K., S.G., S.L.; data curation: D.P.M., S.G., A.K.; writing—original draft preparation: D.P.M.; visualization: D.P.M., S.G., A.K.; supervision: D.P.M. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

−1 AAR Antiradical activity (mmol TRE L ) −1 C0 Initial concentration of quercetin (µmol L ) −1 Ct Concentration of quercetin at time t (µmol L ) k Quercetin ﬁrst-order degradation constant (s−1) −1 PR Ferric-reducing power (µmol AAE L ) T Temperature (◦C) t Time (s)

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