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Article Stability of Anthocyanins and Their Degradation Products from Cabernet Sauvignon Red Wine under Gastrointestinal pH and Temperature Conditions

Ping Yang 1,†, Chunlong Yuan 1,2,3,†, Hua Wang 1,2,3,*, Fuliang Han 1,2,3,*, Yangjie Liu 1, Lin Wang 1 and Yang Liu 1

1 College of Enology, Northwest A&F University, Yangling 712100, China; [email protected] (P.Y.); [email protected] (C.Y.); [email protected] (Y.L.); [email protected] (L.W.); [email protected] (Y.L.) 2 Shaanxi Engineering Research Center for Viti-Viniculture, Northwest A&F University, Yangling 712100, China 3 Heyang Viticulture Experimental Station, Northwest A&F University, Heyang 715300, China * Correspondence: [email protected] (H.W.); hanfl@nwsuaf.edu.cn (F.H.); Tel.: +86-29-8709-2107 (H.W. & F.H.) † These authors contributed equally to this work.

Received: 14 January 2018; Accepted: 5 February 2018; Published: 7 February 2018

Abstract: This study investigated the stability of wine anthocyanins under simulated gastrointestinal pH and temperature conditions, and further studied the evolution of anthocyanin degradation products through simulated digestive conditions. The aim of this study was to investigate the relation between anthocyanins’ structure and their digestive stability. Results showed that a total of 22 anthocyanins were identified in wine and most of these anthocyanins remained stable under simulated gastric digestion process. However, a dramatic concentration decrease happened to these anthocyanins during simulated intestinal digestion. The stability of anthocyanins in digestive process appeared to be related to their structure. The methoxy group in the B-ring enhanced the stability of anthocyanins, whereas hydroxyl group resulted in a reduction of their stability. Acylation decreased the stability of malvidin 3-O-glucoside. Pyruvic acid conjugation enhanced the structural stability of pyranoanthocyanins, whereas acetaldehyde attachment weakened their stability. A commercial malvidin 3-O-glucoside standard was used to investigate anthocyanin degradation products under simulated digestion process, and syringic acid, protocatechuic acid and vanillic acid were confirmed to be the degradation products via anthocyanin chalcone conversion path. Gallic acid, protocatechuic acid, vanillic acid, syringic acid, and p-coumaric acid in wine experienced a significant concentration decrease during digestion process. However, wine model solution revealed that phenolic acids remained stable under gastrointestinal conditions, except gallic acid.

Keywords: Cabernet Sauvignon wine; anthocyanins; degradation; stability; gastrointestinal pH and temperature

1. Introduction Anthocyanins are the major colorants in grapes and wine, and they have been confirmed to play important roles in affecting the appearance and sensory attributes of grapes and wine [1–5]. More importantly, anthocyanins can serve as one of the most important antioxidants in grapes and wine to provide multiple health promoting properties [6–8]. For example, it has been reported that anthocyanins possess the anti-cancer, anti-inflammatory, antimicrobial features, and the consumption of these antioxidants can lower the incidence of cardiovascular, diabetic, and obesity diseases [9–15].

Molecules 2018, 23, 354; doi:10.3390/molecules23020354 www.mdpi.com/journal/molecules Molecules 2018, 23, x FOR PEER REVIEW 2 of 19 Molecules 2018, 23, 354 2 of 20 anthocyanins possess the anti-cancer, anti-inflammatory, antimicrobial features, and the consumption of these antioxidants can lower the incidence of cardiovascular, diabetic, and obesity diseases [9–15]. Anthocyanins can can be be synthesized as as secondary metabolites in in grapes during the grape development period period [16,17]. [16,17]. These These colorants colorants can can be be extracted extracted into into red red wine wine during during the wine maceration process and they can further be metabolized during the wine fermentation and aging period [1,18,19]. [1,18,19]. Therefore, Therefore, the composition and co concentrationncentration of anthocyanins mainly determine the appearance of of red red wine. wine. It Ithas has been been reported reported that that the thelevel level of anthocyanins of anthocyanins in red in wine red wineranges ranges from 10from to 102000 to mg/L 2000 [19–21], mg/L [ 19and–21 their], and level their in levelwine inis winemainly is determined mainly determined by grape by variety, grape climate variety, conditions,climate conditions, management management system, and system, fermentation and fermentation technique technique [3,19,22–26]. [3,19 Regarding,22–26]. Regarding their chemical their nature,chemical anthocyanins nature, anthocyanins can be divided can beinto divided monomeri intoc anthocyanins monomeric anthocyanins (Figure 1), acylated (Figure anthocyanins,1), acylated pyranoanthocyanins,anthocyanins, pyranoanthocyanins, and polymeric anthocyanins and polymeric [27–31]. anthocyanins In Vitis vinifera [27–31 grapes]. In Vitisand wine, vinifera thegrapes major monomericand wine, theanthocyanins major monomeric include pelargonidin anthocyanins 3- includeO-glucoside, pelargonidin delphinidin 3-O 3--glucoside,O-glucoside, delphinidin cyanidin 3-O-glucoside, cyanidinpetunidin 3-O -glucoside,3-O-glucoside, petunidin peonidin 3-O-glucoside, 3-O-glucoside, peonidin and 3- Omalvidin-glucoside, 3- andO-glucoside malvidin (Figure3-O-glucoside 1) [2,26,31]. (Figure The1)[ monomeric2,26,31]. The anthocyanins monomeric anthocyaninscan be further can converted be further into converted the acetylated, into the coumaroylated,acetylated, coumaroylated, and caffeoylated and caffeoylatedanthocyanins anthocyanins [2,30,31]. Pyranoanthocyani [2,30,31]. Pyranoanthocyaninsns can be synthesized can be throughsynthesized conjugating through conjugatingmonomeric monomericor acylated oranthoc acylatedyanins anthocyanins with low molecu with lowlar molecular compounds compounds in wine, whereasin wine, whereasthe interaction the interaction among among anthocyanins anthocyanins or between or between anthocyanins anthocyanins and and other other phenolic compounds couldcould resultresult in in the the formation formation of of polymeric polymeric anthocyanins anthocyanins [30 –[30–333]. Pyranoanthocyanins3]. Pyranoanthocyanins and andpolymeric polymeric anthocyanins anthocyanins normally normally are formed are formed during du winering wine aging aging process, process, which which could could shift wineshift wine color colorfrom brightfrom bright red color red tocolor brick to andbrick dark and reddark color red [color34,35 [34,35].].

Anthocyanins R1 R2 DelphinidinAnthocyanins 3-O-glucoside R OH1 R OH2 DelphinidinCyanidin 3- 3-OO-glucoside-glucoside OHOH OHH CyanidinPetunidin 3-3-OO-glucoside-glucoside OHOCH3 HOH PetunidinPeonidin 3-O-glucoside OCHOCH3 3 OHH PeonidinMalvidin 3-3-OO-glucoside-glucoside OCHOCH3 3 OCHH 3 Malvidin 3-O-glucoside OCH3 OCH3 Figure 1. Five monomeric anthocyanins in red wine of Vitis Vinifera L. Figure 1. Five monomeric anthocyanins in red wine of Vitis Vinifera L. It has been reported that anthocyanins, like other phenolic compounds, can be absorbed through the humanIt has beengastrointestinal reported that tract. anthocyanins, The absorption like other rate of phenolic anthocyanins compounds, is repo canrted be to absorbed be around through 0.1% tothe 2%, human and gastrointestinal they could be tract.readily The distributed absorption rateto different of anthocyanins organs, isincluding reported toliver, be aroundlung, kidney, 0.1% to prostate,2%, and theyheart, could and brain be readily [6,36–38]. distributed The major to different digestive organs, segments including in the human liver, lung, gastrointestinal kidney, prostate, tract includeheart, and stomach brain [and6,36 small–38]. Theintestine. major Nutrient digestive digestion segments mainly in the happens human gastrointestinal in the stomach, tractwhereas include the smallstomach intestine and small is the intestine. major place Nutrient where digestion nutrients mainlyare absorbed happens [39,40]. in the It stomach,has been whereasreported thethat small 10% tointestine 25% of isanthocyanins the major place were where degraded nutrients in stomach, are absorbed whereas [39 the,40 small]. It has intestin beene reported was responsible that 10% for to 30% 25% toof anthocyanins50% of the anthocyanins’ were degraded degradation in stomach, [41–45]. whereas Anthocyanin the small intestine degradation was responsible can happen for 30%at their to 50% A- and/orof the anthocyanins’B-ring. The degradationA-ring degradation [41–45]. Anthocyaninhas been reported degradation to canresult happen in the at their formation A- and/or of formylphloroglucinalB-ring. The A-ring degradation or phlorogluc hasinol been carboxylic reported acid, to result whereas in the syri formationngic, vanillic of formylphloroglucinal and protocatechuic acidsor phloroglucinol were the major carboxylic products acid, from whereas the anthocya syringic,nins vanillicdegradation and protocatechuic via B-ring cleavage acids [46–48]. were the major productsIt has from been the anthocyaninsaccepted that degradation the percentage via B-ring of cleavageanthocyanins’ [46–48 degradation]. in the human gastrointestinalIt has been tract accepted is closely that thecorrelated percentage to the of anthocyanins’anthocyanins’ degradationstructure. However, in the human such investigationsgastrointestinal have tract isnot closely been correlatedwell documented to the anthocyanins’ to our beststructure. knowledge, However, especially such regarding investigations the acylatedhave not anthocyanins, been well documented pyranoanthocyanins, to our best knowledge, and polymeric especially anthocyanins regarding found the acylated in wine. anthocyanins, Therefore,

Molecules 2018, 23, 354 3 of 20 pyranoanthocyanins, and polymeric anthocyanins found in wine. Therefore, the present study selected Molecules 2018, 23, x FOR PEER REVIEW 3 of 19 anthocyanins in Cabernet Sauvignon wine, and further investigated their stability under simulated humanthe gastrointestinal present study selected pH and anthocyanins temperature in conditions.Cabernet Sauvignon Furthermore, wine, and anthocyanin further investigated standards their and their potentialstability degradation under simulated products human were alsogastrointestinal investigated pH under and temperature the simulated conditions. gastrointestinal Furthermore, conditions to elucidateanthocyanin the degradationstandards and pathway their potential of anthocyanins. degradation products The findings were fromalso investigated this study couldunder providethe usefulsimulated information gastrointestinal on the elucidation conditions ofto theelucidate anthocyanins’ the degradation stability pathway and metabolism of anthocyanins. in the The human gastrointestinalfindings from tract. this study could provide useful information on the elucidation of the anthocyanins’ stability and metabolism in the human gastrointestinal tract. 2. Results and Discussion 2. Results and Discussion 2.1. Anthocyanins and Phenolic Acids in Red Wine 2.1. Anthocyanins and Phenolic Acids in Red Wine A total of 22 anthocyanins were identified in the wine samples, including five monomeric A total of 22 anthocyanins were identified in the wine samples, including five monomeric anthocyanins, six acylated anthocyanins, eight pyranoanthocyanins, and three polymeric anthocyanins anthocyanins, six acylated anthocyanins, eight pyranoanthocyanins, and three polymeric (Figureanthocyanins2a and Table (Figure1). 2a and Table 1).

mAU 5 mAU 10 175 10.0 13 150 7.5 9 125 5.0 11 12

2.5 100 14 30.0 32.5 min

75

50 20 6 25 8 3 7 10 1 4 13 15 17 2 9 11 12 16 18 19 0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 min (a)

mAU

125

100

1 75

4 50

25 5 2 3

0 0 10 20 30 40 50 min (b)

FigureFigure 2. HPLC 2. HPLC chromatography chromatography of of ( a()a) identified identified anthocyanins and and (b) ( bphenolic) phenolic acids acids in Cabernet in Cabernet Sauvignon red wine. Sauvignon red wine.

Molecules 2018, 23, 354 4 of 20

Table 1. Anthocyanins found in Cabernet Sauvignon red wine.

Peak No. Compounds Rt (min) λmax Precursor Ion Product Ion Concentration (mg/L) 1 Delphinidin 3-O-glucoside 7.978 524 465 303 2.801 ± 0.011 2 Cyanidin 3-O-glucoside 11.297 524 449 287 0.814 ± 0.010 3 Petunidin 3-O-glucoside 13.226 524 479 317 5.047 ± 0.014 4 Peonidin 3-O-glucoside 17.137 523 463 301 2.295 ± 0.001 5 Malvidin 3-O-glucoside 18.921 276, 525 493 331 137.815 ± 0.396 6 Vitisin A 22.183 510 561 399 6.743 ± 0.015 7 Vitisin B 24.669 - 517 355 4.106 ± 0.032 8 Malvidin 3-O-(6-O-acetyl)-glucoside-pyruvic acid 26.168 514 603 399 5.916 ± 0.026 9 Malvidin 3-O-glucoside-ethyl-catechin (1) 29.29 525 809 357 1.999 ± 0.357 10 Malvidin 3-O-(6-O-acetyl)-glucoside-acetaldehyde 29.926 495 559 355 4.012 ± 0.032 11 Malvidin 3-O-glucoside-ethyl-catechin (2) 30.847 532 809 357 0.337 ± 0.008 12 Malvidin 3-O-glucoside-ethyl-catechin (3) 32.762 532 809 357 0.404 ± 0.008 13 Peonidin 3-O-(6-O-acetyl)-glucoside 34.396 523 505 301 3.530 ± 0.464 14 Malvidin 3-O-(6-O-acetyl)-glucoside 35.799 528 535 331 80.068 ± 0.419 15 Malvidin 3-O-(6-O-p-coumaryl)-glucoside-pyruvic acid 36.989 517 707 399 1.382 ± 0.018 16 Malvidin 3-O-(6-O-caffeoyl)-glucoside 40.239 531 655 331 1.510 ± 0.382 17 Malvidin 3-O-(6-O-p-coumaryl)-glucoside-acetaldehyde 42.046 - 663 355 1.624 ± 0.208 18 Malvidin 3-O-(6-O-cis-p-coumaryl)-glucoside 42.615 532 639 331 1.354 ± 0.034 19 Peonidin 3-O-(6-O-trans-p-coumaryl)-glucoside 45.814 524 609 301 1.175 ± 0.433 20 Malvidin 3-O-(6-O-trans-p-coumaryl)-glucoside 46.45 531 639 331 16.081 ± 0.570 21 Malvidin 3-O-glucoside-4-vinylphenol adduct - - 609 447 - 22 Malvidin 3-O-(6-O-acetyl)-glucoside-4-vinylphenol adduct - - 651 447 - Total anthocyanins - - - - 299.752 ± 2.352 Note: “-”Not detected. Molecules 2018, 23, 354 5 of 20 Molecules 2018, 23, x FOR PEER REVIEW 5 of 19

ItIt shouldshould bebe notednoted thatthat malvidinmalvidin 3-O-glucoside-4-vinylphenol adduct adduct and and malvidin malvidin 3- 3-OO-(6--(6-OO-- acetyl)-glucoside-4-vinylphenol adduct were not quan quantifiedtified due to their limited concentration concentration in in the the wine sample. TheThe totaltotal anthocyaninsanthocyanins concentration concentration in in the the wine wine sample sample was was about about 299.75 299.75 mg/L mg/L (Table (Table1). 1).Malvidin Malvidin 3-O 3--glucosideO-glucoside appeared appeared to be to the be highest the highest individual individual anthocyanin anthocyanin in the in wine the sample wine sample and its andconcentration its concentration represented represented more than more 49% than of the 49% total of anthocyaninthe total anthocyanin concentration. concentration. Besides, malvidinBesides, malvidin3-O-(6-O-acetyl)-glucoside 3-O-(6-O-acetyl)-glucoside and malvidin and malvidin 3-O-(6-O -3-transO-(6--p-coumaryl)-glucosideO-trans-p-coumaryl)-glucoside were also were found also to foundbe the to predominant be the predominant anthocyanins anthocyanins in the wine in sample,the wine and sample, their and concentration their concentration accounted accounted for about for29% about and 6%29% of and the 6% total of anthocyaninsthe total anthocyanins content, conten respectively.t, respectively. Gallic acid, Gallic protocatechuic acid, protocatechuic acid, vanillic acid, vanillicacid, syringic acid, syringic acid, and acid,p-coumaric and p-coumaric acid were acid all were detected all detected in the wine in the sample wine (Figuresample2 (Figureb and Table 2b and2). TableIt should 2). It be should noted be that noted syringic that acidsyringic exhibited acid ex thehibited highest the concentration highest concentr in theation wine in the sample, wine whereassample, whereasp-coumaric p-coumaric acid existed acid in existed the wine in the with wine the lowestwith the concentration. lowest concentration.

Table 2. Phenolic acids found in Cabernet Sauvignon red wine.

Peak No.Peak No. Compounds Compounds Rt Rt (min) (min) λmaxλmax Concentration Concentration (mg/L) (mg/L) 1 Gallic acid 4.243 271 11.676 ± 0.321 1 Gallic acid 4.243 271 11.676 ± 0.321 2 2 Protocatechuic Protocatechuic acid acid 7.319 7.319 259, 259, 293 293 4.771 ± 4.771 0.208± 0.208 33 Vanillic Vanillic acid acid 15.363 15.363 291 291 8.156 ± 8.156 0.853± 0.853 44 Syringic Syringic acid acid 16.671 16.671 274 274 21.350 21.350± 0.412± 0.412 5 5 p-Coumaricp-Coumaric acid acid 26.818 26.818 309 309 2.846 ± 2.846 0.162± 0.162

2.2. Evolution of Anthoc Anthocyaninsyanins and Phenolic Acids under Simulated Gastrointestinal pH and Temperature ConditionsConditions

2.2.1. Total Anthocyanins Figure3 3 shows shows thethe evolutionevolution ofof thethe totaltotal winewine anthocyaninsanthocyanins inin thethe simulatedsimulated gastrointestinalgastrointestinal pHpH and temperature conditions. It was observed that incubating the wine sample in the simulated gastric condition significantly significantly resulted in an apparent 8.22%8.22% increase of the total anthocyanin concentration after 6 hh ((pp << 0.05).0.05). However,However, its its concentration concentration decreased decreased significantly significantly by by about about 49% 49% after after digesting digesting the winethe wine in the in simulatedthe simulated intestinal intestinal pH conditionpH condition (after (after 12 h) 12 (p h)< ( 0.05).p < 0.05). Our Our result result was was consistent consistent with with the previousthe previous report report [44]. [44].

400

360 + 8.22% 320 b b b 280 a

240

200 c - 48.75% 160 d e 120 Total anthocyanins (mg/L)

80

40

0 W 0h S 2h S 4h S 6h I 2h I 4h I 6h Time

Figure 3. ContentContent alteration alteration of of total total anthocyanins anthocyanins in in red red wine wine during during stimulated simulated digestion process (wine(Wine sample:sample: WW 00 h;h; SS 22 h,h, SS 44 h,h, SS 66 h:h: samples underunder the gastric condition;condition; II 22 h,h, II 4 h, I 6 h: samples under the intestinal condition; different lowercase letterletter meansmeans significantsignificant differencedifference atat 0.050.05 level).level).

Molecules 2018, 23, 354 6 of 20

It has been known that anthocyanin molecules possess an equilibrium under different pH conditions, and lower pH environment could result most anthocyanins in the flavylium cation conformation [49]. Such a conformation shift led to an apparent increase on the total anthocyanins concentration under the simulated gastric condition. In the simulated intestinal tract, the decrease of the total anthocyanins concentration mainly resulted from the degradation of anthocyanins via cleaving their aromatic ring as suggested from previously studies [47,50]. Additionally, basic condition in the simulated intestinal tract was also reported to trigger the interactions between anthocyanin molecules and other compounds to yield polymeric compounds [43,51]. Such interactions could also result in the decrease of the total anthocyanins concentration.

2.2.2. Monomeric Anthocyanins Five monomeric anthocyanins (Figure1), including malvidin 3- O-glucoside, petunidin 3-O-glucoside, peonidin 3-O-glucoside, delphinidin 3-O-glucoside, and cyanidin 3-O-glucoside, were detected in the wine sample. Malvidin 3-O-glucoside, petunidin 3-O-glucoside, peonidin 3-O-glucoside, and delphinidin 3-O-glucoside exhibited the similar evolution pattern under the simulated gastrointestinal conditions (Figure4). These four monomeric anthocyanins increased their apparent concentration in the simulated gastric condition, and then significantly decreased their level under the simulated intestinal tract (p < 0.05). Our results were in accordance with the previous reports [44,45,52]. Flavylium cation conformation has been confirmed to be the dominant structure for monomeric anthocyanins under the acidic condition, and this conformation can benefit the stability of anthocyanins [49]. This could explain the apparent increase on these monomeric anthocyanins during the incubation at the simulated gastric tract. The basic condition in the simulated intestinal condition could facilitate the cleavage of the C-ring in anthocyanins structure, which led to a concentration decrease of these monomeric anthocyanins [42]. It should be worth noting that cyanidin 3-O-glucoside exhibited an opposite evolution during the incubation at the simulated gastrointestinal condition compared to the other monomeric anthocyanins. A significant concentration decrease of cyanidin 3-O-glucoside was found under the simulated gastric conditions, whereas incubating the wine under the simulated intestinal condition resulted in a concentration increase and then a decrease of this anthocyanin (p < 0.05) (Figure4). Acidic conditions might cause the release of the sugar moiety from its aglycone due to hydrolysis, whereas flavylium cation conformation could stabilize anthocyanins under acidic conditions [40]. Therefore, we speculated that hydrolysis and/or polymeric reaction might take place more rapidly on cyanidin 3-O-glucoside than the formation of its flavylium cation structure, which caused its concentration decrease under the simulated gastric tract [43,51]. During the incubation at the simulated intestinal condition, cyanidin 3-O-glucoside exhibited a concentration increase initially, followed by a decrease. Its concentration increase might result from the degradation of polymeric anthocyanins or bound anthocyanins or other unknown reason [51,53]. However, the basic condition eventually caused cyanidin 3-O-glucoside to be degraded, which lowered its concentration in the simulated intestinal condition. It should be noted that after the digestion process the concentrations of delphinidin 3-O-glucoside, petunidin 3-O-glucoside, malvidin 3-O-glucoside, peonidin 3-O-glucoside, and cyanidin 3-O-glucoside decreased significantly by 92.9%, 84.3%, 51.1%, 49.7% and 46.7%, respectively (p < 0.05) (Figure4). Their stability in the digestive system appeared to be related to their molecular structure. The increase on the substituent groups in the B-ring of anthocyanins resulted in a significant decrease on the stability of the monomeric anthocyanins in the simulated gastrointestinal tract, whereas the methoxy group presence on the B-ring could stabilize the anthocyanins structure in the digestive system [54,55]. It should be aware that more hydroxyl groups in the anthocyanins structure accelerated the degradation of anthocyanins in the digestive system [54,55]. Molecules 2018, 23, 354 7 of 20 Molecules 2018, 23, x FOR PEER REVIEW 7 of 19

180 6.0 + 12.39% Malvidin 3-O-glucoside Cyanidin 3-O-glucoside b Molecules 2018, 23, x FOR PEER162 REVIEW b b Petunidin 3-O-glucoside 5.4 7 of 19 Peonidin 3-O-glucoside c a bc Delphinidin 3-O-glucoside 180144 b + 13.70% 6.04.8 + 12.39% Malvidin 3-O-glucoside a Cyanidin 3-O-glucoside 126 b 4.2 162 b b Petunidin 3-O-glucoside 5.4 Peonidin 3-O-glucoside c 108 a bc Delphinidin 3-O-glucoside 3.6 144 b ++ 13.70%12.21% 4.8 d a b b 12690 b 4.23.0 a b c b + 25.95% e - 51.13% 10872 b 3.62.4 + 12.21% a da f

Anthocyanins (mg/L) Anthocyanins 54 1.8 (mg/L) Anthocyanins 90 b b b - 46.69% 3.0 a b d 36 b c e - 51.13%c 1.2 72 a b + 25.95% a c 2.4 a bc b - 84.31% f e 18 bc c a 0.6 Anthocyanins (mg/L) Anthocyanins 54 c 1.8 (mg/L) Anthocyanins - 49.71%- 46.69%c - 48.04% d d - 92.90% 0 c 0.0 36 a 1.2 Wa 0h S 2h S 4h S 6h I 2h I 4hc I 6h e bc bc b - 84.31% 18 Timec c 0.6 - 49.71% c - 48.04% d - 92.90% d Figure 4. Content alteration0 of monomeric anthocyanins in red wine during 0.0simulated digestion Figure 4. Content alterationW 0h of monomeric S 2h S 4h anthocyanins S 6h I 2h in red I 4h wine I 6h during simulated digestion process (Left axis: malvidin 3-O-glucoside; right axis: other anthocyanins; Wine sample: W 0 h; S 2 h, process (Left axis: malvidin 3-O-glucoside; rightTime axis: other anthocyanins; wine sample: W 0 h; S 2 h, S 4 h, S 6 h: samples under the gastric condition; I 2 h, I 4 h, I 6 h: samples under the intestinal condition; S 4 h, S 6 h: samples under the gastric condition; I 2 h, I 4 h, I 6 h: samples under the intestinal condition; Figuredifferent 4. lowercase Content alteration letter means of signmonomericificant difference anthocyani at ns0.05 in level). red wine during simulated digestion differentprocess lowercase (Left axis: letter malvidin means 3-O significant-glucoside;difference right axis: other at 0.05 anthocya level).nins; Wine sample: W 0 h; S 2 h, 2.2.3.S Acylated 4 h, S 6 h: samplesAnthocyanins under the gastric condition; I 2 h, I 4 h, I 6 h: samples under the intestinal condition; 2.2.3. Acylateddifferent Anthocyanins lowercase letter means significant difference at 0.05 level). Peonidin 3-O-(6-O-trans-p-coumaryl)-glucoside exhibited an apparent concentration increase Peonidin2.2.3.(about Acylated 7.62%) 3-O under-(6-AnthocyaninsO- trans-pthe simulated-coumaryl)-glucoside gastric conditions, exhibited whereas the an simulated apparent intestinal concentration conditions increase (aboutreduced 7.62%) its under concentration the simulated by 26.27% gastric (p < 0.05) conditions, (Figure 5a). whereas Its evolution the simulatedpattern was intestinal similar to that conditions of peonidinPeonidin 3-O -glucoside3-O-(6-O-trans-p under-coumaryl)-glucoside the digestive conditions. exhibited However, an apparent a continuous concentration concentration increase reduced(about its 7.62%) concentration under the bysimulated 26.27% gastric (p < 0.05)conditio (Figurens, whereas5a). Its the evolution simulated patternintestinal was conditions similar to that ofdecrease peonidin happened 3-O-glucoside to peonidin under 3-O-(6- theO digestive-acetyl)-glucoside conditions. during However, the whole a continuousdigestion process concentration (p < reduced0.05). For its instance, concentration about bya 7.3% 26.27% concentration (p < 0.05) (Figure decrease 5a). wasIts evolution observed pattern under thewas simulated similar to gastricthat of decrease happened to peonidin 3-O-(6-O-acetyl)-glucoside during the whole digestion process (p < 0.05). peonidinconditions, 3- whereasO-glucoside its concentration under the digestivedecreased conditions. by 68.58% afterHowever, the incubati a continuouson under concentrationthe simulated For instance,decreaseintestinal about happenedconditions. a 7.3% to Thesepeonidin concentration indicated 3-O-(6-decrease thatO-acetyl)-glucoside the waspresence observed of during an underacetyl the thewholegroup simulated digestionin the peonidin gastric process conditions, 3-(pO <- whereas0.05).glucoside its For concentration structureinstance, mightabout decreased lowera 7.3% the concentration structural by 68.58% stability decrease after theunder was incubation acidic observed and underbasicunder conditions. the simulated simulated In addition, gastric intestinal conditions.conditions,peonidin These 3- whereasO-(6- indicatedO-acetyl)-glucoside its concentration that the or presencedecreased its chalcone ofby 68.58% an stru acetylcture after groupmight the incubati react in the onwith peonidin under other the compounds 3-simulatedO-glucoside structureintestinal(such might as acetaldehyde, conditions. lower the These structuralpyruvic indicated acid, stability etc.) that to undertheform presence the acidic new andofunknown an basic acetyl compounds conditions. group in the[43,51]. In addition,peonidin Compared peonidin3-O- glucoside structure might lower the structural stability under acidic and basic conditions. In addition, 3-O-(6-to Othe-acetyl)-glucoside acetyl group, the coumaryl or its chalcone group addition structure stabilized might the react structure with of other peonidin compounds 3-O-glucoside, (such as peonidin 3-O-(6-O-acetyl)-glucoside or its chalcone structure might react with other compounds acetaldehyde,leading to a pyruvic lesser degradation acid, etc.) rate to formunder the the newsimulated unknown intestinal compounds conditions. [ 43,51]. Compared to the (such as acetaldehyde, pyruvic acid, etc.) to form the new unknown compounds [43,51]. Compared acetyl group, the coumaryl4.0 group addition stabilized the structure of peonidin 3-O-glucoside, leading to the acetyl group, the coumaryl group addition stabilized Peonidin 3-O the-(6-O-acetyl)-glucoside structure of peonidin 3-O-glucoside, to a lesser degradation rate under the simulated intestinal Peonidin 3- conditions.O-(6-O-trans-p-coumaryl)-glucoside leading to a lesser degradation3.6 rate under the simulated Peonidin intestinal 3-O-glucoside conditions. a 4.03.2 a - 7.30% a Peonidin 3-O-(6-O-acetyl)-glucoside b b a Peonidin 3-O-(6-O-trans-p-coumaryl)-glucoside 3.62.8 b + 25.95% Peonidin 3-O-glucoside a a - 7.30% 3.22.4 a a a b b a 2.82.0 b + 25.95% b a 2.41.6 a c + 7.62% c - 46.69% Anthocyanins (mg/L) Anthocyanins 2.01.2 c b b b d ab b - 68.58% ac 1.60.8 c cd d - 26.27% + 7.62% c - 46.69% Anthocyanins (mg/L) Anthocyanins 1.20.4 c b b d ab b - 68.58% ac 0.80.0 cd d - 26.27% W 0h S 2h S 4h S 6h I 2h I 4h I 6h 0.4 Time 0.0 (a) W 0h S 2h S 4h S 6h I 2h I 4h I 6h Time (a)

Figure 5. Cont.

Molecules 2018, 23, 354 8 of 20 Molecules 2018, 23, x FOR PEER REVIEW 8 of 19

200 Malvidin 3-O-(6-O-cis-p-coumaryl)-glucoside 4.0 Malvidin 3-O-(6-O-caffeoyl)-glucoside 180 Malvidin 3-O-(6-O-acetyl)-glucoside 3.6 Malvidin 3-O-(6-O-trans-p-coumaryl)-glucoside Malvidin 3-O-glucoside 160 3.2

bc c 140 b + 13.70% 2.8 a 120 c 2.4 b + 30.29%b

100 d 2.0 bb a + 4.83% b 80 1.6 a + 13.94% b e - 51.13% b b

Anthocyanins (mg/L) Anthocyanins 60 a d f 1.2 (mg/L) Anthocyanins

c e 40 - 60.07% 0.8 d f + 9.15% c e a ab bc c - 65.13% 20 d e 0.4 d e - 72.54% f 0 - 58.38% 0.0 W 0h S 2h S 4h S 6h I 2h I 4h I 6h Time (b)

Figure 5. Content alteration of acylated anthocyanins in red wine during simulated digestion process Figure 5. Content alteration of acylated anthocyanins in red wine during simulated digestion (Left axis: malvidin 3-O-(6-O-acetyl)-glucoside, malvidin 3-O-(6-O-trans-p-coumaryl)-glucoside, process (Left axis: malvidin 3-O-(6-O-acetyl)-glucoside, malvidin 3-O-(6-O-trans-p-coumaryl)-glucoside, malvidin 3-O-glucoside; Right axis: malvidin 3-O-(6-O-cis-p-coumaryl)-glucoside, malvidin 3-O-(6-O- malvidin 3-O-glucoside; Right axis: malvidin 3-O-(6-O-cis-p-coumaryl)-glucoside, malvidin caffeoyl)-glucoside; Wine sample: W 0 h; S 2 h, S 4 h, S 6 h: samples under the gastric condition; I 2 h, 3-O-(6-I 4O h,-caffeoyl)-glucoside; I 6 h: samples under wine the intestinal sample: condit W 0 h;ion; S different 2 h, S 4 lowercase h, S 6 h: samplesletter means under significant the gastric condition;difference I 2 h,at I0.05 4 h, level). I 6 h: samples under the intestinal condition; different lowercase letter means significant difference at 0.05 level). Similarly, the concentrations of malvidin 3-O-(6-O-acetyl)-glucoside, malvidin 3-O-(6-O-trans-p- Similarly,coumaryl)-glucoside, the concentrations malvidin 3-O-(6- ofO malvidin-cis-p-coumaryl)-glucoside, 3-O-(6-O-acetyl)-glucoside, and malvidin 3-O malvidin-(6-O-caffeoyl)- 3-O-(6-O- glucoside apparently increased by 4.83%, 9.15%, 13.94%, and 30.29% after 6 h incubation under the trans-p-coumaryl)-glucoside, malvidin 3-O-(6-O-cis-p-coumaryl)-glucoside, and malvidin 3-O-(6-O- simulated gastric conditions, respectively (p < 0.05) (Figure 5b). However, their concentration caffeoyl)-glucosidedramatically decreased apparently by 65.13%, increased 58.38%, by 4.83%, 72.45% 9.15%, and 60.07% 13.94%, under and 30.29%the simulated after 6 intestinal h incubation underconditions, the simulated respectively gastric ( conditions,p < 0.05). These respectively results indicated (p < 0.05) that (Figure different5b). substituen However,t groups their concentration affected dramaticallythe stability decreased of anthocyanins by 65.13%, in the 58.38%,simulated 72.45%digestive and system, 60.07% and underthe trans-p the-coumaryl simulated addition intestinal conditions,exhibited respectively the best enhancement (p < 0.05). on These the stability results indicatedof anthocyanin that differentstructure, substituentfollowed by groupsthe caffeoyl affected the stabilitygroup, acetyl of anthocyanins group, and the in least, the simulatedthe cis-p-coumaryl digestive group. system, and the trans-p-coumaryl addition exhibited the best enhancement on the stability of anthocyanin structure, followed by the caffeoyl 2.2.4. Pyranoanthocyanins group, acetyl group, and the least, the cis-p-coumaryl group. The pyranoanthocyanins formed in the wine sample of the present study mainly resulted from 2.2.4.the Pyranoanthocyanins interactions through malvidin 3-O-glucoside or its acylated anthocyanins with pyruvic acid (Figure 6a). For example, vitisin A is synthesized by conjugating malvidin 3-O-glucoside with The pyranoanthocyanins formed in the wine sample of the present study mainly resulted from the pyruvic acid. This pyranoanthocyanin significantly increased its apparent concentration in the O interactionssimulated through gastric malvidin condition 3-(p <-glucoside 0.05). Acetylvitisin or its acylated A and coumarylvitisin anthocyanins with A after pyruvic the incubation acid (Figure in 6a). For example,the simulated vitisin gastric Ais condition synthesized remained by conjugating at similar concentrations, malvidin 3- Ohowever,-glucoside their with concentration pyruvic acid. This pyranoanthocyanindecreased by 17.73%, significantly19.77% and increased27.15% after its the apparent incubation concentration under the insimulated the simulated intestinal gastric conditionconditions, (p < 0.05).respectively Acetylvitisin (p < 0.05). ACompared and coumarylvitisin to the evolution A of after malvidin the incubation3-O-glucoside in during the simulated the gastricsimulated condition digestion remained process, at similarthe presence concentrations, of pyruvic acid however, in the malvidin their concentration 3-O-glucoside decreasedstructure by 17.73%,enhanced 19.77% its and stability. 27.15% However, after the this incubation structural under addition the reduced simulated the intestinalstability of conditions, malvidin 3-O respectively-(6-O- (p < 0.05).acetyl)-glucoside Compared toand the malvidin evolution 3- ofO malvidin-(6-O-p-coumaryl)-glucoside 3-O-glucoside during under the the simulated simulated digestion digestive process, the presenceconditions. of pyruvicAdditionally, acid invitisin the malvidin B and malvidin 3-O-glucoside 3-O-(6- structureO-p-coumaryl)-glucoside-acetaldehyde enhanced its stability. However, exhibited an apparent concentration increase under the simulated gastric conditions, whereas a this structural addition reduced the stability of malvidin 3-O-(6-O-acetyl)-glucoside and malvidin concentration decrease was observed in malvidin 3-O-(6-O-acetyl)-glucoside-acetaldehyde (p < 0.05) 3-O-(6-(FigureO-p-coumaryl)-glucoside 6b). These pyranoanthocyanins under the simulatedexperienced digestive a dramatic conditions. concentration Additionally, decrease vitisin(61.30%, B and malvidin79.08%, 3-O and-(6- 75.31%,O-p-coumaryl)-glucoside-acetaldehyde respectively) under the simulated intestinal exhibited conditions an apparent (p < 0.05). concentration These indicated increase underthat the acetaldehyde simulated gastricconjugation conditions, weakened whereas the stability a concentration of vitisin B and decrease it derivatives. was observedIt should be in noted malvidin 3-O-(6-thatO-acetyl)-glucoside-acetaldehyde vitisin A and its derivatives were (p

9.0 Vitisin A 220 Malvidin 3-O-(6-O-acetyl)-glucoside-pyruvic acid 8.1 Malvidin 3-O-(6-O-p-coumaryl)-glucoside-pyruvic acid 198 Malvidin 3-O-glucoside b 7.2 a ab + 4.31% 176 a c b bc + 13.70% 6.3 c - 17.73% 154 - 1.48% cd a d a a 5.4 a a 132

b - 27.15% 4.5 110 d c d 3.6 e 88 - 51.13% f

Anthocyanins (mg/L) Anthocyanins 2.7 66 (mg/L) Anthocyanins

- 0.62% 1.8 aaba a - 19.77% 44 c bc c 0.9 22

0.0 0 W 0h S 2h S 4h S 6h I 2h I 4h I 6h Time (a) 6.0 Vitisin B 200 Malvidin 3-O-(6-O-acetyl)-glucoside--acetaldehyde 5.4 Malvidin 3-O-(6-O-p-coumaryl)-glucoside-acetaldehyde 180 Malvidin 3-O-glucoside c 4.8 b bc + 13.70% 160 a 4.2 + 7.62% c 140 a ab bc a 3.6 b 120 c c d - 9.74% 3.0 100

d e 2.4 - 51.13% 80 f

+ 9.91% Anthocyanins (mg/L)

Anthocyanins (mg/L) 1.8 e 60 - 61.30% f a d 1.2 a aa 40 e - 79.08% 0.6 f 20 b b -75.31% b 0.0 0 W 0h S 2h S 4h S 6h I 2h I 4h I 6h Time (b)

Figure 6. Content alteration of pyranoanthocyanins in red wine during simulated digestion process (wine(Wine sample:sample: WW 00 h;h; SS 22 h,h, SS 44 h,h, SS 66 h:h: samples underunder thethe gastricgastric condition;condition; II 22 h,h, II 44 h,h, II 66 h:h: samples under the intestinal condition; different lowercaselowercase letterletter meansmeans significantsignificant differencedifference atat 0.050.05 level).level).

2.2.5. Polymeric Anthocyanins 2.2.5. Polymeric Anthocyanins Only three polymeric anthocyanins, including malvidin 3-O-glucoside-ethyl-catechin (1), malvidin Only three polymeric anthocyanins, including malvidin 3-O-glucoside-ethyl-catechin (1), 3-O-glucoside-ethyl-catechin (2), and malvidin 3-O-glucoside-ethyl-catechin (3), were tentatively malvidin 3-O-glucoside-ethyl-catechin (2), and malvidin 3-O-glucoside-ethyl-catechin (3), were identified in the wine sample. These polymeric anthocyanins exhibited the different evolution tentatively identified in the wine sample. These polymeric anthocyanins exhibited the different patterns in the simulated digestive condition (Figure 7). Malvidin 3-O-glucoside-ethyl-catechin (1) evolution patterns in the simulated digestive condition (Figure7). Malvidin 3- O-glucoside- showed a decreasing trend on its concentration, whereas an increase happened to malvidin 3-O- ethyl-catechin (1) showed a decreasing trend on its concentration, whereas an increase happened to glucoside-ethyl-catechin (3) (p < 0.05). Malvidin 3-O-glucoside-ethyl-catechin (2) showed a concentration malvidin 3-O-glucoside-ethyl-catechin (3)(p < 0.05). Malvidin 3-O-glucoside-ethyl-catechin (2) showed increase under the simulated gastric condition but a concentration decrease in the simulated intestinal a concentration increase under the simulated gastric condition but a concentration decrease in the environment (p < 0.05). Under acidic conditions, the concentration increase might result from the simulated intestinal environment (p < 0.05). Under acidic conditions, the concentration increase might reaction between malvidin 3-O-glucoside and catechin, whereas the concentration decrease might result from the reaction between malvidin 3-O-glucoside and catechin, whereas the concentration result from the further reaction between malvidin 3-O-glucoside-ethyl-catechin and other decrease might result from the further reaction between malvidin 3-O-glucoside-ethyl-catechin and compounds in red wine [56,57]. Under alkaline conditions, the concentration variation might be due other compounds in red wine [56,57]. Under alkaline conditions, the concentration variation might be to their degradations or the degradations of other bigger molecular polymeric anthocyanins [56,57]. due to their degradations or the degradations of other bigger molecular polymeric anthocyanins [56,57]. The mechanism behind these polymeric anthocyanins evolution needs to be further investigated. The mechanism behind these polymeric anthocyanins evolution needs to be further investigated.

Molecules 2018,, 23,, 354x FOR PEER REVIEW 10 of 2019 Molecules 2018, 23, x FOR PEER REVIEW 10 of 19 3.0 Malvidin 3-O-glucoside-ethyl-catechin(1) 3.0 Malvidin 3-O-glucoside-ethyl-catechin(2) 2.7 Malvidin 3-O-glucoside-ethyl-catechin(1) Malvidin 3-O-glucoside-ethyl-catechin(3) Malvidin 3-O-glucoside-ethyl-catechin(2) 2.7 Malvidin 3-O-glucoside-ethyl-catechin(3) 2.4 2.4 2.1 a a - 11.28% a a 2.1 a a 1.8 a - 11.28% a 1.8 1.5 1.5 1.2 + 53.71% 1.2 c c c Anthocyanins (mg/L) Anthocyanins 0.9 + 53.71% + 39.67% c c c Anthocyanins (mg/L) Anthocyanins 0.9 d b 0.6 cd + 39.67% a a b b + 7.13% 0.6 cd d a ab b bcd + 7.13%ad 0.3 a acb b a ab + 15.34% ab b bcd - 84.5%ad 0.3 ac b a ab + 15.34% 0.0 - 84.5% 0.0 W 0h S 2h S 4h S 6h I 2h I 4h I 6h W 0h S 2h S 4hTime S 6h I 2h I 4h I 6h Time Figure 7. Content alteration of polymeric anthocyanins in red wine during simulated digestion Figureprocess 7.7.(WineContent Content sample: alteration alteration W 0 h; of S polymericof 2 polymeh, S 4 h, anthocyaninsric S 6anthocyanins h: samples in under red in wine red the duringwinegastric during simulatedcondition; simulated digestionI 2 h, I 4digestion h, process I 6 h: (winesamplesprocess sample: (Wineunder Wsample:the 0 intestinal h; S W 2 h,0 condition; Sh; 4S h,2 Sh, 6 S h: different4 samplesh, S 6 h:lowercase undersamples the letter under gastric means the condition; gastric significant condition; I 2 difference h, I 4 I h, 2 Ih, 6at h:I 0.05 4 samplesh, level). I 6 h: undersamples the under intestinal the intestinal condition; condition; different different lowercase lowercase letter meansletter means significant significant difference difference at 0.05 at 0.05 level). level). 2.2.6. Phenolic Acids 2.2.6. Phenolic Acids It has been reported that anthocyanins can be degraded into phenolic acids during the digestion process,ItIt has and been gallic reported acid, thatprotocatechuicthat anthocyaninsanthocyanins acid, cancan vanillic bebe degradeddegraded acid, syringic into phenolic acid and acids p -coumaric during the acid digestion have process,beenprocess, reported andand gallic gallic to acid, beacid, the protocatechuic protocatechuic major anthocyanins acid, acid, vanillic vanillic de acid,gradation acid, syringic syringic products acid acid and and[46,47,58].p-coumaric p-coumaric Therefore, acid haveacid have been we reportedinvestigatedbeen reported to be the the to evolution major be the anthocyanins ofmajor these anthocyanins phenolic degradation ac idsde products gradationfrom the [46 ,wine47products,58 ].under Therefore, [46,47,58]. the simulated we investigatedTherefore, digestion thewe evolutionprocessinvestigated (Figure of thesethe 8). evolution phenolic acidsof these from phenolic the wine ac underids from the simulatedthe wine digestionunder the process simulated (Figure digestion8). process (Figure 8). 30 6.0 Gallic acid 30 Vanillic acid 6.0 27 Gallic acid 5.4 a Syringic acid ab Vanillic acid 27 abc Protocatechuic acid 5.4 a Syringic acid 24 ab p-Coumaric acid 4.8 abc Protocatechuic acid 24 a p-Coumaric acid 4.8 21 4.2 a - 22.90% a ab 21 abcd 4.2 a - 22.90% 18 ab 3.6 abcd 18 a 3.6 15 a b bd 3.0 cd - 50.28% a - 33.35% 15 a ac b bd 3.0 cd - 50.28% d 12 - -33.35% 27.80% 2.4 ac a a bc d 12 a - 27.80% c - 34.56% c 2.4

Phenolic acids (mg/L)acidsPhenolic ab (mg/L)acidsPhenolic 9 a a bc bc 1.8 a - 28.81% b c - 34.56% c

Phenolic acids (mg/L)acidsPhenolic ab (mg/L)acidsPhenolic 9 bc c 1.8 b b c - 72.55% 6 - 28.81% b c 1.2 b b - 72.55%bdc 6 bc c c - 55.75% 1.2 - 54.69% cd cd 3 bd 0.6 bc - 55.75% - 54.69% cd cd - 91.48% 3 c c c 0.6 0 - 91.48% 0.0 c c c 0 W 0h S 2h S 4h S 6h I 2h I 4h I 6h 0.0 W 0h S 2h S 4hTime S 6h I 2h I 4h I 6h Time Figure 8. Content alteration of phenolic acids in red wine during simulated digestion process (Wine (wine sample:Figure 8. W Content 0 h; S 2 alteration h, S 4 h, S of 6 h: phenolic samples acids under in the red gastric wine during condition; simulated I 2 h, I 4digestion h, I 6 h: samples process under(Wine thesample: intestinal W 0 h; condition;condition; S 2 h, S 4 differentdifferenth, S 6 h: samples lowercaselowercase under letterletter the meansmeans gastric significantsignificant condition; differencedifference I 2 h, I 4 ath,at 0.05I0.05 6 h: level). level).samples under the intestinal condition; different lowercase letter means significant difference at 0.05 level). As the simulated digestion proceeded, an increasing trend on these phenolic acids was expected As the simulated digestion proceeded, an increasing trend on these phenolic acids was expected since Asmost the simulatedanthocyanins digestion exhibited proceeded, a dramatic an increasi concentrationng trend on decrease. these phenolic However, acids awas continuous expected since most anthocyanins exhibited a dramatic concentration decrease. However, a continuous concentrationsince most anthocyanins decrease took exhibited place for a all dramatic the phenolic concentration acids during decrease. the digestion However, process a continuous (p < 0.05). concentration decrease took place for all the phenolic acids during the digestion process (p < 0.05). Forconcentration example, these decrease phenolic took acidsplace decreasedfor all the theirphenolic concentration acids during by 28.81%,the digestion 22.92%, process 54.69%, (p 33.35%,< 0.05). For example, these phenolic acids decreased their concentration by 28.81%, 22.92%, 54.69%, 33.35%, andFor example,27.80% after these the phenolic simulated acids gastric decreased digestion their process,concentration respectively by 28.81%, (p < 22.92%,0.05). Furthermore, 54.69%, 33.35%, the and 27.80% after the simulated gastric digestion process, respectively (p < 0.05). Furthermore, incubationand 27.80% under after thethe simulatedsimulated gastricintestinal digestion condition process, continued respectively to decrease (p < 0.05).their Furthermore,concentration theby the incubation under the simulated intestinal condition continued to decrease their concentration by 91.4%,incubation 50.28%, under 55.75%, the simulated 72.55%, and intestinal 34.56% (conditionp < 0.05). Thesecontinued indicated to decrease that these their phenolic concentration acids were by 91.4%, 50.28%, 55.75%, 72.55%, and 34.56% (p < 0.05). These indicated that these phenolic acids were

Molecules 2018, 23, 354 11 of 20

91.4%, 50.28%, 55.75%, 72.55%, and 34.56% (p < 0.05). These indicated that these phenolic acids were more stable under acidic conditions than the basic conditions. More importantly, these indicated that the degradation/transformation rate of these phenolic acids was faster than their rate of formation from the anthocyanins’ degradation under the simulated digestion process.

2.3. Evolution of Anthocyanin and Phenolic Acid Standards in Simulated Gastrointestinal pH and Temperature Conditions Wine is a complex system that consists of multiple nutrients and components. To elucidate the relations and degradation mechanism between anthocyanins and phenolic acids in wine, we further conducted the same digestion study using anthocyanin and phenolic acid standards (Table3).

Table 3. Degradation products from malvidin 3-O-glucoside standard under the simulated digestion process.

Compound λmax Precursor Ion Product Ion Syringic acid 274, 279 197 153 Peonidin 3-O-glucoside * - 463 301 Malvidin 3-O-glucoside * 525 493 331 Malvidin 3-O-glucoside chalcone * 343 511 349, 223 Malvidin 3-O-glucoside chalcone 343 509 347, 221 Note: * Positive electrospray ionization mode.

To ensure that phenolic acids are the products of the anthocyanins degradation during the digestion process, a commercial standard of malvidin 3-O-glucoside was incubated under the same digestive conditions, and its degradation products were identified using HPLC and UPLC-MS (Table3 and AppendixA, Figure A1a,b). A tiny amount of peonidin 3- O-glucoside was present in the commercial malvidin 3-O-glucoside standard ethanol solution. After incubating the standard solution under the simulated digestion conditions, a compound with a precursor ion of m/z 197 and a product ion of m/z 153 was detected at extraction ion mode (AppendixA Figure A2a,b). This degradation product was identified as syringic acid. Besides, malvidin 3-O-glucoside chalcone was also found due to the presence of its precursor ion of m/z 509 and product ion of m/z 347 in negative electrospray ionization mode, and the presence of its precursor ion of m/z 511 and product ion of m/z 349 in positive electrospray ionization mode [59–61] (AppendixA Figure A2c and Table3). Similarly, vanillic acid and protocatechuic acid were also identified as the malvidin 3-O-glucoside degradation products after the simulated digestion due to their retention time and maximal wavelength feature. Along with the digestion process, the commercial standards showed the similar concentration during the simulated gastric digestion process. Syringic acid and protocatechuic acid were found with a low level in the solution during the simulated gastric digestion. The simulated intestinal digestion significantly decreased the concentration of the anthocyanins, which resulted in a dramatic concentration increase of syringic acid, protocatechuic acid, and vanillic acid (Figure9a). Malvidin 3-O-glucoside chalcone exhibited a concentration decrease during the simulated gastric digestion process. During the simulated intestinal digestion, its concentration increased and then eventually decreased (Figure9b). This indicated that chalcone conformation was an intermediate stage during the anthocyanins’ degradation under the digestion process conditions [59]. It should be noted that the anthocyanin standard degradation percentage was not recovered by the formation of these phenolic acids, which was consistent with a previous report [51]. This indicated that further reactions might take place after the formation of phenolic acids. A further study needs to be conducted. Based on the structure of these three phenolics and the five basic anthocyanins (Figure1), it can be deduced that protocatechuic acid, vanillic acid and syringic acid result from the B-ring of cyanidin 3-O-glucoside, peonidin 3-O-glucoside and malvidin 3-O-glucoside, respectively [46,47,58]. In this study, the commercial standards didn’t contain cyanidin 3-O-glucoside. Thus, protocatechuic acid, Molecules 2018, 23, 354 12 of 20 the degradation product identified tentatively, might result from the other degradation pathway. However, this still needs to be further confirmed. Additionally, we purified four anthocyanin standards from Yan 73 grape skin, including delphinidin 3-O-glucoside, petunidin 3-O-glucoside, malvidin 3-O-glucoside and peonidin 3-O-glucoside. Their purity was 94.7%, 98.1%, 94.3%, and 99.0%, respectively [62]. These anthocyanin standards in the wine model solution exhibited a concentration decrease after the incubation under the simulated gastric conditions (13.10%, 20.81%, 35.10% and 6.25%, respectively). The simulated intestinal conditions significantly decreased their concentration by 100%, 94.45%, 86.56%, and 75.53%, respectively (Figure9c). These results indicated that peonidin 3- O-glucoside was the most stable monomeric anthocyanin, followed by malvidin 3-O-glucoside, petunidin 3-O-glucoside, and then delphinidin 3-O-glucoside. The evolution patterns of these standards were consistent with the evolution of these monomeric anthocyanins from the wine during the digestion process (Figure4). It should be worth noting that a much higher degradation rate was observed in these monomeric anthocyaninMolecules standards2018, 23, x FOR compared PEER REVIEW to those in the wine, indicating that the wine matrix and/or13 of 20 other anthocyaninother compounds fractions in (such the as wine anthocyanin, might help quinone, protect chalcone, monomeric or alcohols), anthocyanins resulting from in their degradation concentration under the digestiondecrease process [43,51]. conditions.

4 150 Malvidin 3-O-glucoside 5.0 8x10 a Peonidin 3-O-glucoside Syringic acid 135 4.5 4 Vanillic acid 7x10 Protocatechuic acid 120 + 3.03% 4.0 6x104 a 105 a a a 3.5 c 5x104 90 b 3.0 b cd d b 75 2.5 4x104

60 - 50.58% 2.0 c 3x104 b bc e chalcone-glucoside (peak area)

45 d 1.5 O + 3.82% c 4 -glucoside and its degradation products (mg/L) -glucoside and its degradation products (mg/L) 2x10 O 30 aa b 1.0 O a a b b aaa a 1x104 b b b 15 b 0.5 3- Malvidin a ab b b c c

Malvidin 3- Malvidin - 51.47% 3- Malvidin a aaa 0 0.0 0 0h S 2h S 4h S 6h I 2h I 4h I 6h 0h S 2h S 4h S 6h I 2h I 4h I 6h Time Time (a) (b)

35 Petunidin 3-O-glucoside 400 a - 0.94% 180 Delphinidin 3-O-glucoside a a Syringic acid Peonidin 3-O-glucoside a Gallic acid. a Protocatechuic acid 30 Malvidin 3-O-glucoside a a a a a a 360 a 160 a + 2.47% + 2.31% 25 ab

20 - 35.10% 320 a 140 abc abc - 4.72% b b b a c - 10.36% a - 6.25% cd d 15 a a 280 b 120 bcd bc

Anthocyanins (mg/L)Anthocyanins 10 cd c Phenolic acidstandards (mg/L) a - 42.15% Phenolic acid standards (mg/L) b 240 100 b - 20.80% b b 5 b a b - 75.53% - 13.10% bc d b ac c d - 86.56% d - 94.45% 200 80 0 0h S 2h S 4h S 6h I 2h I 4h I 6h 0h S 2h S 4h S 6h I 2h I 4h I 6h Time Time (c) (d)

Figure 9. Content alteration of anthocyanin standard (a,c), its degradation products (a,b) and phenolic Figure 9. Content alteration of malvidin anthocyanin standard (a,c), its degradation products (a,b) and acid standards (d) during simulated digestion process ((a): malvidin 3‐O‐glucoside located at left axis, phenolic acid standards (d) during simulated digestion process ((a): malvidin 3-O-glucoside located at other compounds located at right axis; (b): the content of chalcone malvidin 3‐O‐glucoside was left axis, other compounds located at right axis; (b): the content of chalcone malvidin 3-O-glucoside expressed as the peak area; (d): syringic acid located at left axis, protocatechuic acid and gallic acid was expressedlocated at as right the axis; peak 0 h: area; solution (d): syringicsample; S acid 2 h, S located 4 h, S 6 ath: leftsamples axis, under protocatechuic the gastric condition; acid and I gallic 2 acid locatedh, I 4 h, at I right 6 h: samples axis; wine under sample: the intestinal W 0 h; S condition; 2 h, S 4 h, different S 6 h: samples lowercase under letter the means gastric significant condition; I 2 h, Idifference 4 h, I 6 h: at samples 0.05 level). under the intestinal condition; different lowercase letter means significant difference at 0.05 level). 3. Materials and Methods

3.1. Evolution of Anthocyanin and Phenolic Acid Standards in Simulated Gastrointestinal pH Conditions Four anthocyanin standards used for the gastrointestinal condition stability study, including delphinidin 3‐O‐glucoside, petunidin 3‐O‐glucoside, malvidin 3‐O‐glucoside, and peonidin 3‐O‐ glucoside, were purified following a published method with some modifications [62]. Briefly, Yan 73 grape skin (5 g) was mixed with 1% HCl v/v methanol (20 mL). The mixture was sonicated for 30 min at 40 °C, and then centrifuged at 8000 rpm/min for 5 min to collect the supernatant. The residue was then extracted using the same method two more times, and the supernatants were pooled and then filtered through a 0.45 μm membrane filter. The resultant sample was purified using XAD‐7HP resin (Amberlite, Sigma‐Aldrich, Shanghai, China) and then each anthocyanin fraction was collected using a Shimadzu HPLC system (Shimadzu, Suzhou, China; pump: LC‐6AD; diode array detector: SPD‐M10‐ AVP; system controller: SCL‐10AVP) through a fraction collector (FRC‐10A). These four anthocyanins had a purity above 94%, and were stored at −20 °C before further study. A commercially available malvidin 3‐O‐glucoside standard (purity: 99.03% with peonidin‐3‐O‐glucoside as impurity) was used

Molecules 2018, 23, 354 13 of 20

Syringic acid, protocatechuic acid, or gallic acid standard in the solution was incubated under the same simulated digestive condition to investigate their stability (Figure9d). Syringic acid appeared to be stable to the digestive conditions. Similarly, the digestion process did not affect the stability of protocatechuic acid and its concentration only decreased by about 10% (p < 0.05). Gallic acid remained stable under the simulated gastric conditions. However, a dramatic concentration decrease (42.15%) happened to gallic acid in the simulated intestinal conditions. This result was identical with the previous report [63]. Under basic conditions, gallic acid might be changed into unstable quinone intermediates and/or other resonance forms which may ultimately oxidize in the presence of air to diketo derivatives or other degradation products [63]. However, the degradation mechanism is unclear at present. It should be noted that these phenolic acids experienced the significant concentration decrease in the wine during the same simulated digestion process (Figure8). This indicated that these phenolics (syringic acid, protocatechuic acid, or gallic acid) in the wine might further interact with other compounds (such as anthocyanin, quinone, chalcone, or alcohols), resulting in their concentration decrease [43,51].

3. Materials and Methods

3.1. Evolution of Anthocyanin and Phenolic Acid Standards in Simulated Gastrointestinal pH Conditions Four anthocyanin standards used for the gastrointestinal condition stability study, including delphinidin 3-O-glucoside, petunidin 3-O-glucoside, malvidin 3-O-glucoside, and peonidin 3-O-glucoside, were purified following a published method with some modifications [62]. Briefly, Yan 73 grape skin (5 g) was mixed with 1% HCl v/v methanol (20 mL). The mixture was sonicated for 30 min at 40 ◦C, and then centrifuged at 8000 rpm/min for 5 min to collect the supernatant. The residue was then extracted using the same method two more times, and the supernatants were pooled and then filtered through a 0.45 µm membrane filter. The resultant sample was purified using XAD-7HP resin (Amberlite, Sigma-Aldrich, Shanghai, China) and then each anthocyanin fraction was collected using a Shimadzu HPLC system (Shimadzu, Suzhou, China; pump: LC-6AD; diode array detector: SPD-M10-AVP; system controller: SCL-10AVP) through a fraction collector (FRC-10A). These four anthocyanins had a purity above 94%, and were stored at −20 ◦C before further study. A commercially available malvidin 3-O-glucoside standard (purity: 99.03% with peonidin-3-O-glucoside as impurity) was used to investigate the degradation products in the simulated gastrointestinal condition. Phenolic acid standards used for the digestion study included gallic acid, protocatechuic acid, vanillic acid, syringic acid, and p-coumaric acid, and these standards were purchased from Sigma-Aldrich.

3.2. Chemicals Formic acid (HPLC grade) was purchased from Kemiou Chemical Reagent Co. Ltd. (Tianjin, China), whereas hydrochloric acid (analytical grade) was purchased from Xilong Chemical Industry Co. Ltd. (Chengdu, China). Ethanol, acetic acid, and sodium hydroxide were obtained from Kelong Chemical Reagents (Chengdu, China). Acetonitrile and methyl alcohol were of HPLC grade and purchased from Tedia Company Inc. (Shanghai, China).

3.3. Grape Harvest and Wine-Making Ripen Cabernet Sauvignon (Vitis Vinifera L.) (total soluble solids: 21.5◦Brix; titrable acid: 7.6 g/L) grapes were harvested from the Caoxinzhuang vineyard (Yangling, China) in 2015 vintage. The harvested grapes were carefully handpicked and immediately transported back to our laboratory. The grape berries were manually de-stemmed and crushed into juice, and then thoroughly mixed with 50 mg/L SO2 in a 10 L glass bottle. After 2 h, 20 mg/L commercial pectinase (Ex-color, Lallemand Inc., Aurillac, France) was added for 24 h. Afterwards, 20 mg/L reactivated yeasts (RV002, Angel, Yichang, China) was inoculated into the juice to initiate wine alcoholic fermentation. During the fermentation Molecules 2018, 23, 354 14 of 20 process, temperature and must density were monitored three times daily. The fermentation was carried out on the skins at 25 to 28 ◦C for 7 days. After the pomace was separated, the fermentation continued 2 days. After the alcoholic fermentation, 50 mg/L SO2 was further mixed with the wine. The wine had a 12.0% alcohol level, 3.50 g/L reducing sugar content, 6.20 g/L acidity, and 0.48 g/L volatile acidity.

3.4. Digestion of Wine Sample, Anthocyanin Standards, and Phenolic Acid Standards in Simulated Gastrointestinal pH and Temperature Contidions Digestive enzymes were not included in the present simulated gastrointestinal condition since these enzymes have not been reported to digest phenolic compounds [64]. The wine sample was covered with tin foil to ensure dark conditions and was adjusted to pH 1.5 using 0.1% HCl solution. Then, the wine sample was incubated in a 37 ◦C incubator for up to 6 h. Afterwards, the resultant wine sample was immediately adjusted to pH 7.5 using 1 M NaOH solution and incubated at 37 ◦C for another 6 h. At each time interval (2 h) during the period of 12 h under the simulated gastrointestinal conditions, each series of wine samples were prepared in duplicate for the analysis of anthocyanins and phenolic acids, and their corresponding marks were labelled W0 as origin wine sample, S 2 h, S 4 h, S 6 h as treatments after 2, 4 and 6 h in simulated stomach conditions, I 2 h, I 4 h and I 6 h as treatments after 2, 4, 6 h in simulated intestinal conditions. Regarding anthocyanins or phenolic acid standards, each standard was initially dissolved in 12% ethanol solution at pH 3.5. The concentration of malvidin 3-O-glucoside standard was 111.48 mg/L with a 1.09 mg/L peonidin-3-O-glucoside concentration (impurity). The concentration of syringic acid, protocatechuic acid, and gallic acid were 354.78 mg/L, 136.16 mg/L, and 175.97 mg/L, respectively. The digestion of the standard solution followed the same procedure as the wine sample. Each series of these samples were also prepared in duplicate.

3.5. Anthocyanins and Phenolic Acids HPLC Analyses Anthocyanins and their degradation products were directly analyzed using a Shimadzu LC-20AT HPLC instrument coupled with a photodiode array detector (Shimadzu, Suzhou, China). A Synergi Hydro-RP C18 column (250 × 4.6 mm, 4 µm, Phenomenex, Torrance, CA, USA) was used to separate the anthocyanins in the samples under a flow rate of 1 mL/min. The mobile phase consisted of (A) 2.5% formic acid (v/v) in water: acetonitrile (8:1, v/v) and (B) 2.5% formic acid (v/v) in water: acetonitrile (4:5, v/v). The gradient was programed as follows: 0 to 45 min, 0% to 35% B; 45 to 46 min, 35% to 100% B; 46 to 50 min, 100% B isocratic; 50 to 51 min, 100% to 0% B; and 51 to 55 min, 0% B isocratic. The wavelength on the photodiode array detector was set at 520 nm. Malvidin 3-O-glucoside was used as the external anthocyanin standard for the quantitation of all the anthocyanins in the wine. Phenolic acids, including gallic acid, protocatechuic acid, vanillic acid, syringic acid, and p-coumaric acid were extracted according to a published method [65]. In brief, sample (1 mL) was mixed with ethyl acetate (1 mL) and acetonitrile (0.5 mL) in a 4-mL centrifuge tube. The mixture was vortexed for 10 s and then centrifuged at 5400× g for 5 min to collect the organic phase. The sample was extracted two more times with the same extraction procedure. Afterwards, the organic phase extracts were combined and then evaporated under nitrogen stream at room temperature. The dryness was finally dissolved into 0.5 mL methanol for HPLC analysis. A Shimadzu HPLC, equipped with a Synergi Hydro-RP C18 column (250 × 4.6 mm, 4 µm, Phenomenex), was used to analyze the phenolic acids under a flow rate of 1 mL/min. The mobile phase was comprised of (A) 0.1% acetic acid (v/v) in water: acetonitrile (8:1, v/v) and (B) 0.1% acetic acid (v/v) in water: acetonitrile (4:5, v/v). The gradient was as follows: 0 to 45 min, 0% to 35% B; 45 to 50 min, 35% to 100% B; 50 to 55 min, 100% B isocratic; 55 to 56 min, 100% to 0% B; and 56 to 62 min, 0% B isocratic. Gallic acid, protocatechuic acid, vanillic acid, and syringic acid were detected at 280 nm on the photodiode array detector, whereas p-coumaric acid was measured at 320 nm. These phenolic acids were quantified using their corresponding reference standard. Molecules 2018, 23, 354 15 of 20

Molecules 2018, 23, x FOR PEER REVIEW 15 of 19

3.6. UPLC-ESI-MS/MSwhereas p-coumaric acid was measured at 320 nm. These phenolic acids were quantified using their Anthocyaninscorresponding reference and their standard. degradation products after digestion in the simulated gastrointestinal tract were identified using a Waters Acquity UPLC system coupled with a BEH C18 column (100 × 2.1 mm, 3.6. UPLC-ESI-MS/MS 1.7 µm, Waters Corporation, Milford, MA, USA), a Waters 2489 UV-Visible detector, and a Synapt Q-TOF massAnthocyanins spectrometer and (Waterstheir degradatio Corporation).n products The after column digestion was maintainedin the simulated at 30 gastrointestinal◦C and the mobile tract were identified using a Waters Acquity UPLC system coupled with a BEH C18 column (100 × phase consisted of (A) 5% formic acid (v/v) in water and (B) 5% formic acid (v/v) in methanol under 2.1 mm, 1.7 µm, Waters Corporation, Milford, MA, USA), a Waters 2489 UV-Visible detector, and a a 0.3 mL/min flow rate. The elution gradient was programed as follows: 0 to 30 min, 50% B; 30 to Synapt Q-TOF mass spectrometer (Waters Corporation). The column was maintained at 30 °C and 35 min,the 100%mobile B; phase 35 to 37consisted min, 0% of B.(A) A 5% positive formicelectrospray acid (v/v) in ionwater mode and was(B) 5% used formic for theacid anthocyanins (v/v) in analysis,methanol whereas under phenolic a 0.3 mL/min acids flow was rate. ionized The elution under gradient a negative was mode programed with aas 3.5 follows: kV capillary 0 to 30 min, voltage, ◦ a 20 V50% cone B; voltage,30 to 35 min, and 100% 100 CB; dry35 to temperature. 37 min, 0% B. AA fullpositive scan electrospray mode from ionm/ modez 50 to was 1500 used was for recorded. the Masslynxanthocyanins 4.1 software analysis, (Waters whereas Corporation) phenolic acidswas was ionized used for under data a collectionnegative mode and with analyses. a 3.5 kV capillary voltage, a 20 V cone voltage, and 100 °C dry temperature. A full scan mode from m/z 50 to 1500 was 3.7. Statisticalrecorded. Masslynx Analysis 4.1 software (Waters Corporation) was used for data collection and analyses. ± Data3.7. Statistical were expressed Analysis as the mean standard deviation of duplicate tests. The analysis of variance (ANOVA) was carried out using SPSS22.0 software (SPSS Inc., Chicago, IL, USA) under Tukey’s honest significantData difference were expressed at a significant as the mean level ± standard of 0.05 (deviationp < 0.05). of duplicate tests. The analysis of variance (ANOVA) was carried out using SPSS22.0 software (SPSS Inc., Chicago, IL, USA) under Tukey’s 4. Conclusionshonest significant difference at a significant level of 0.05 (p < 0.05). In4. Conclusions conclusion, most of anthocyanins in wine remained stable under simulated gastric conditions. However,In they conclusion, experienced most of aanthocyanins dramatic concentration in wine remained decrease stable under in a simulated simulated gastric intestinal conditions. digestion process.However, Their structurethey experienced and matrix a dramatic played concentratio an importantn decrease role in theirin a simulated stability underintestinal digestive digestion system conditions.process. Syringic Their structure acid, protocatechuic and matrix played acid, an and import vanillicant role acid in weretheir stability found to under result digestive from anthocyanins’ system degradationconditions. and Syringic the anthocyanin acid, protocatechuic chalcone acid, was suggestedand vanillic to acid serve were as anfound important to result degradation from intermediateanthocyanins’ to form degradation degradation and the products. anthocyanin Phenolic chalcone acid was standards suggested solution to serve remained as an important stable under simulateddegradation digestion intermediate process conditions. to form degradation However, prod a dramaticucts. Phenolic decrease acid instandards their concentration solution remained happened to winestable during under the simulated simulated digestion digestion process process. conditions. However, a dramatic decrease in their concentration happened to wine during the simulated digestion process. Acknowledgments: This study was financially supported by the National Nature Science Foundation of China (GrantAcknowledgments: No. 31401479), National This study Key was R&D financially Program supported of China by the (Grant National No. Nature 2016YFD0400500), Science Foundation Key Researchof China and Development(Grant No. Plan 31401479), of Shaanxi National Province Key R&D (Grant Program Nos.2017NY-184, of China (Grant 2017NY-144), No. 2016YFD0400500), and Scientific KeyResearch Research Projectand of NorthwestDevelopment A&F University Plan of Shaanxi (Grant Provin No. Z109021703).ce (Grant Nos. 2017NY-184, 2017NY-144), and Scientific Research Project of Northwest A&F University (Grant No. Z109021703). Author Contributions: Hua Wang and Fuliang Han conceived and designed the experiments; Ping Yang and ChunlongAuthor Yuan Contributions: performed Hua the experiments;Wang and Fuliang Yangjie Han Liu, conceived Lin Wang and and desi Yanggned the Liu experiments; analyzed the Ping data; Yang Fuliang and Han wroteChunlong the manuscript. Yuan performed the experiments; Yangjie Liu, Lin Wang and Yang Liu analyzed the data; Fuliang ConflictsHan ofwrote Interest: the manuscript.The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appendix A

22.95 520 22.87 -3 -3 Range: 5.894×10 4.0×10 13.34 22.76 13.28 22.68 -3 13.22 3.0×10 22.98 23.11 2.0×10 -3 23.39 AU -3 23.46 1.0×10 23.74

0.0

-1.0×10 -3 Time -0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 (a)

Figure A1. Cont.

Molecules 2018, 23, x FOR PEER REVIEW 6 of 5

Article Stability of Anthocyanins and Their Degradation Molecules 2018, 23, 354 16 of 20 ProductsMolecules 2018, 23, x FORfrom PEER REVIEW Cabernet Sauvignon Red Wine under16 of 19

0.83 280 Gastrointestinal pH and Temperature ConditionsRange: 1.362×10 -2

-2 1.0×10 21.78 1,† 1,2,3,† 1,2,3, 1,2,3, 1 1 Ping8.0×10 Yang-3 , Chunlong Yuan , Hua Wang *, Fuliang Han *, Yangjie Liu , Lin Wang

-3 1 AU 6.0×10 8.53 and Yang Liu 22.75 23.23 13.34 4.0×10 -3 0.94 1 College-3 of Enology, Northwest A&F University, Yangling 712100, China; 2.0×10 1.30 15.26 1.932.45 3.05 6.43 18.36 21.38 [email protected] 4.99 (P.Y.); [email protected] (C.Y.);16.00 [email protected] 19.26 20.22 (Y.L.); 0.0 Time [email protected] 2.00 4.00 6.00 (L.W.); 8.00 [email protected] 10.00 12.00 (Y.L.) 14.00 16.00 18.00 20.00 22.00 24.00 2 Shaanxi Engineering Research Center for Viti‐Viniculture,(b) Northwest A&F University, Yangling 712100, China Figure A1. HPLC chromatography of (a) malvidin 3-O-glucoside standard at 520 nm and (b) its Figure3 Heyang A1. ViticultureHPLC chromatography Ex of (a) malvidin 3-O-glucoside standard at 520 nm and (b) its degradation products at 280 nm during the simulated digestion process. degradation products at 280 nm during the simulated digestion process.

22.91 197 23.22 78 22.79 0.86 23.25 22.91 197 8.60 22.58 23.43 0.92 23.22 78 22.4622.79 23.60 0.86 23.25 8.63 1.06 8.60 22.3922.58 23.43 0.92 20.51 23.69

% 22.2922.46 1.11 20.40 23.60 0.77 8.67 24.12 19.28 20.35 1.06 8.63 18.22 22.39 24.53 1.20 20.51 23.69 2.13 2.51 5.68 6.13 7.79 24.93

% 0.39 22.29 1.11 3.78 7.61 20.40 0.77 8.178.67 24.12 19.28 20.35 1.20 18.22 24.53 2.13 2.51 5.68 6.13 7.79 24.93 0.39 3.78 7.61 3 8.17 Time -0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

3 (a) Time -0.000.82 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 153 (a) 875 0.82 153 875 % %

1.98 8.65 22.89 23.34 0 Time -0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 1.98 8.65 22.89 23.34 0 (b) Time -0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 () 509.2 100 (b) 68

509.2 100 68 97.0 % 91.0 510.2 347.1 97.0 856.3 113.0137.0 355.1 531.2 % 186.9 219.0 607.2 629.1 69.0 260.9 329.1 567.1 577.2 651.1 953.2 510.2 0 91.0 m/z 347.1 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850856.3 900 950 1000 113.0137.0 186.9 219.0 355.1 531.2 607.2 629.1 69.0 260.9 329.1 567.1 577.2 651.1 953.2 0 (c) m/z 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 Figure A2. Mass spectrum of (a,b) syringic acid and(c) ((c), negative electrospray ionization mode) malvidin 3-O-glucoside-chalcone during the simulated digestion process. FigureFigure A2. A2.Mass Mass spectrum spectrum of of (a ,(ba,)b syringic) syringic acidacid andand (((cc, ),negative negative electrospray electrospray ionization ionization mode) mode) malvidin 3‐O‐glucoside‐chalcone during the simulated digestion process. malvidinReferences 3-O -glucoside-chalcone during the simulated digestion process. References1. Han, F.L.; Li, Z.; Xu, Y. Contribution of monomeric anthocyanins to the color of young red wine: Statistical Referencesand experimental approaches. J. Food Sci. 2015, 80, C2751–C2758. 1. Han, F.L.; Li, Z.; Xu, Y. Contribution of monomeric anthocyanins to the color of young red wine: Statistical 2. Han, F.L.; Zhang, W.N.; Pan, Q.H.; Zheng, C.R.; Chen, H.Y.; Duan, C.Q. Principal component regression and experimental approaches. J. Food Sci. 2015, 80, C2751–C2758. 1. Han,analysis F.L.; Li, of Z.; the Xu, relation Y. Contribution between CIELAB of monomeric color and monomeric anthocyanins anthocyanins to the color in young of young cabernet red wine:sauvignon Statistical 2. Han, F.L.; Zhang, W.N.; Pan, Q.H.; Zheng, C.R.; Chen, H.Y.; Duan, C.Q. Principal component regression and experimentalwines. Molecules approaches. 2008, 13, 2859–2870.J. Food Sci. 2015, 80, C2751–C2758. [PubMed] analysis of the relation between CIELAB color and monomeric anthocyanins in young cabernet sauvignon 2. Han,3. Baiano, F.L.; Zhang, A.; De W.N.; Gianni, Pan, A.; Q.H.; Mentana, Zheng, A.; C.R.;Quinto, Chen, M.; H.Y.;Centonze, Duan, D.; C.Q. Del PrincipalNobile, M.A. component Colour-related regression wines. Molecules 2008, 13, 2859–2870. analysisphenolics, of the volatile relation composition, between CIELAB and sensory color profile and monomeric of Nero di anthocyaninsTroia wines treated in young with oak cabernet chips sauvignonor by wines.micro-oxygenation.Molecules 2008, 13 Eur., 2859–2870. Food Res. Technol [CrossRef. 2016][, 242PubMed, 1631–1646.] 4. Sáenz-Navajas, M.P.; Ferreira, V.; Dizy, M.; Fernández-Zurbano, P. Characterization of taste-active 3. Baiano, A.; De Gianni, A.; Mentana, A.; Quinto, M.; Centonze, D.; Del Nobile, M.A. Colour-related phenolics,fractions volatile in red composition,wine combining and HPLC sensory fractionatio profilen, of sensory Nero dianalysis Troia winesand ultra treated performance with oak liquid chips or chromatography coupled with mass spectrometry detection. Anal. Chim. Acta 2010, 673, 151–159. by micro-oxygenation. Eur. Food Res. Technol. 2016, 242, 1631–1646. [CrossRef] 5. Ma, W.; Guo, A.Q.; Zhang, Y.L.; Wang, H.; Liu, Y.; Li, H. A review on astringency and bitterness perception 4. Sáenz-Navajas, M.P.; Ferreira, V.; Dizy, M.; Fernández-Zurbano, P. Characterization of taste-active fractions of tannins in wine. Trends Food Sci. Technol. 2014, 40, 6–19. in6. redPojer, wine E.; combining Mattivi, F.; HPLCDan, J.; fractionation,Stockley, C.S. The sensory case for analysis anthocyanin and ultraconsumptio performancen to promote liquid human chromatography health: coupledA review. with massCompr. spectrometry Rev. Food Sci. detection.Food Saf. 2013Anal., 12, Chim.483–508. Acta 2010, 673, 151–159. [CrossRef][PubMed] 5. Ma, W.; Guo, A.Q.; Zhang, Y.L.; Wang, H.; Liu, Y.; Li, H. A review on astringency and bitterness perception of tannins in wine. Trends Food Sci. Technol. 2014, 40, 6–19. [CrossRef] Molecules 2018, 23, 354 17 of 20

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Sample Availability: Samples of the compounds are not available from the authors.

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