Structural Changes in Mulberry (Morus Microphylla. Buckl) and Chokeberry (Aronia melanocarpa) Anthocyanins during Simulated In Vitro Human Digestion

Journal Pre-proofs Structural Changes in Mulberry (Morus Microphylla. Buckl) and Chokeberry (Aronia melanocarpa) Anthocyanins during Simulated In Vitro Human Di‐ gestion Inhwan Kim, Joon Kwan Moon, Sun Jin Hur, Jihyun Lee PII: DOI: Reference:

S0308-8146(20)30311-3 https://doi.org/10.1016/j.foodchem.2020.126449 FOCH 126449

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Food Chemistry

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Please cite this article as: Kim, I., Moon, J.K., Hur, S.J., Lee, J., Structural Changes in Mulberry (Morus Microphylla. Buckl) and Chokeberry (Aronia melanocarpa) Anthocyanins during Simulated In Vitro Human Digestion, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.2020.126449

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Structural Changes in Mulberry (Morus Microphylla. Buckl) and Chokeberry (Aronia melanocarpa) Anthocyanins during Simulated In Vitro Human Digestion

Inhwan KIMa, Joon Kwan MOONb, Sun Jin HURc, and Jihyun LEEa* a Department

of Food Science and Technology, Chung-Ang University, Anseong, 17546,

Republic of Korea b Department

of Plant Life and Environmental Science, Hankyong National University,

Anseong, 17580, Republic of Korea c Department

of Animal Science and Technology, Chung-Ang University, Anseong, 17546,

Republic of Korea

Corresponding Author *(J.L.)

Phone: +82-31-670-3266. Fax: +82-31-675-3108. E-mail: [email protected]

1

1

ABSTRACT

2

Mulberry and chokeberry are rich sources of anthocyanins. In this study, the effect of the

3

anthocyanin composition on the anthocyanin profile changes during in vitro digestion

4

(mimicking the physiological conditions) was investigated by UHPLC-(ESI)-qTOF and

5

UHPLC-(ESI)-QqQ. The antioxidant activity before and after in vitro digestion was elucidated.

6

Cyanidin-3-O-glucoside and cyanidin-3-O-galactoside were dominant in mulberry and

7

chokeberry, respectively. Moreover, the loss of cyanidin-3-O-galactoside in the chokeberry

8

extract after digestion was greater than that of cyanidin-3-O-glucoside in the mulberry extract.

9

After digestion, phenolic acids including protocatechuic acid and various cyanidin conjugates

10

were newly formed because of decomposition and changes in the cyanidin-glycosides. The

11

phenolic acid and cyanidin conjugate levels varied depending on the cyanidin glycoside

12

sources in the colonic fraction. Finally, antioxidant activity before and after digestion was

13

higher in the chokeberry extract than in the mulberry extract. Moreover, this activity

14

continuously decreased until intestinal digestion but increased in the colonic fraction.

15 16 17

Keywords: cyanidin glycoside, cyanidin, anthocyanin, metabolite, in vitro digestion model,

18

phenolic acid, UHPLC-(ESI)-qTOF, UHPLC-(ESI)-QqQ

2

20

1. Introduction

21

Mulberry (e.g., Morus Alba, Morus Lhou (Ser.), Morus Microphylla, and Morus rubra) is

22

a member of the Morus species belonging to the Moraceae family. It has a long history of

23

cultivation as a feed for silkworms in China and India. Furthermore, mulberry fruits have been

24

used in China to lower fever and for protection against liver diseases (Bae & Suh, 2007). Thus,

25

the increasing interest in health worldwide has resulted in an increased consumption of

26

mulberry fruits. For example, in Korea, the yield of mulberry fruits increased twofold from

27

2008 to 2011. On the other hand, chokeberry (Aronia melanocarpa), which belongs to the

28

Rosaceae family, is used as both a natural food colorant and in herbal medicine in Russia

29

because of its high anthocyanin contents (Kokotkiewicz, Jaremicz, & Luczkiewicz, 2010).

30

Anthocyanins are natural pigments found in various berries in the glycoside form, which

31

varies in different berries. For example, anthocyanins occur mostly as perlagonidin-3-O-

32

glucoside in strawberries (Veberic, Slatnar, Bizjak, Stampar, & Mikulic-Petkovsek, 2015). On

33

the other hand, bilberry contains various cyanidin glycosides, such as cyanidin-3-O-

34

galactoside, cyanidin-3-O-glucoside, and cyanidin-3-O-arabinoside (Veberic, Slatnar, Bizjak,

35

Stampar, & Mikulic-Petkovsek, 2015), whereas the main anthocyanin in mulberry fruits is

36

cyanidin-3-O-glucoside (Kim & Lee, 2017; Veberic, Slatnar, Bizjak, Stampar, & Mikulic-

37

Petkovsek, 2015).

38

Epidemiological studies have revealed that the consumption of anthocyanins is associated

39

with protective effects against cancer as well as cardiovascular and age-related diseases.

40

Moreover, beneficial effects of anthocyanins on health, such as their anti-inflammatory, anti-

41

cancer, and antioxidant effects, have been reported both in in vivo and in vitro studies

42

(Edirisinghe, Banaszewski, Cappozzo, Sandhya, Ellis, Tadapaneni, et al., 2011; Hu, Deng,

43

Chen, Zhou, Liu, Fu, et al., 2016; Zhao, Giusti, Malik, Moyer, & Magnuson, 2004). These

44

effects may result from anthocyanins and their metabolites formed in the body. Interestingly, 3

45

the bioavailability of anthocyanins was reported to be in the range of 0.26–1.8%, (Borges,

46

Roowi, Rouanet, Duthie, Lean, & Crozier, 2007; Edirisinghe, et al., 2011; Marczylo, Cooke,

47

Brown, Steward, & Gescher, 2009). An in vivo study has reported that anthocyanins are

48

metabolized to anthocyanidin by the removal of sugars in the intestine, followed by

49

degradation to smaller compounds such as protocatechuic acid and ferulic acid (Fang, 2014).

50

An in vitro study have revealed that the gut microbiota in the colon metabolize the

51

anthocyanins

52

glucoside into phenolic acids such as protocatechuic acid, gallic acid, and syringic acid

53

(Fernandes, Faria, de Freitas, Calhau, & Mateus, 2015).

cyanidin-3-O-glucoside,

delphinidin-3-O-glucoside,

and

malvidin-3-O-

54

In a previous study, the bioavailability levels of quercetin-3-O-glucoside and quercetin-3-

55

O-rutinoside were reported to be 148% and 23%, respectively, of that of quercetin aglycone

56

(Cermak, Landgraf, & Wolffram, 2003). In humans, the plasma metabolites reached a

57

maximum level less than one hour after the intake of quercetin-4′-O-glucoside or onion,

58

whereas after the ingestion of rutin, the maximum level of metabolites in the plasma was

59

observed after 6–9 h (Graefe, Wittig, Mueller, Riethling, Uehleke, Drewelow, et al., 2001;

60

Hollman, Bijsman, van Gameren, Cnossen, de Vries, & Katan, 1999; Hollman, Van Trijp,

61

Buysman, vd Gaag, Mengelers, De Vries, et al., 1997). These results indicate that quercetin-

62

4′-O-glucoside was absorbed in the upper small intestine, whereas rutin was absorbed in the

63

terminal ileum or large intestine (Cermak, Landgraf, & Wolffram, 2003). Metabolite profiles

64

differ depending on the metabolism site because of factors such as the differences in the

65

enzymes and microbiota present in the colon (McGhie & Walton, 2007). Thus, glycoside forms

66

attached to anthocyanins may also affect the absorption site, thereby affecting the

67

bioavailability and metabolism of anthocyanins.

68

Information on the changes in the anthocyanin structures is essential for understanding

69

their bioactivities. This is because the anthocyanin structures are altered after consumption, 4

70

which in turn leads to changes in their bioactivities. Various factors such as the pH, temperature,

71

and gut microbiota in the colon may induce changes in or degradation of the anthocyanin

72

structure (Avila, Hidalgo, Sanchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009;

73

Barbagallo, Palmeri, Fabiano, Rapisarda, & Spagna, 2007; Fossen, Cabrita, & Andersen, 1998).

74

For example, the flavylium cation form of anthocyanin is the most abundant form in acidic

75

environments such as the stomach (McGhie & Walton, 2007). However, when the pH increases,

76

the blue quinonoidal structure is generated by the loss of a proton (McGhie & Walton, 2007).

77

The much slower hydration of the flavylium cation results in the production of a colorless

78

hemiketal form by opening of the C-ring to yield the chalcone (cis and trans) forms (McGhie

79

& Walton, 2007). Moreover, it was reported that the degradation rate of anthocyanins is higher

80

at a pH of 6.0 than at a pH of 2.2 (Sui, Dong, & Zhou, 2014). However, few studies have

81

examined whether the differences in the glycosyl moiety bonded to anthocyanin affects

82

anthocyanin metabolism. For example, Corre-Betanazo et al. studied the changes in the

83

anthocyanin glycosides from blueberry extract using an in vitro model. However, the blueberry

84

extract comprised various anthocyanins, including delphinidin-3-O-galactoside, delphinidin-

85

3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-galactoside, peonidin-3-O-galactoside,

86

and malvidin-3-O-arabinoside (Correa-Betanzo, Allen-Vercoe, McDonald, Schroeter,

87

Corredig, & Paliyath, 2014). In another study, the changes in various phenolic compounds

88

including anthocyanins (e.g., pelargonidin arabinoside, pelargonidin glucoside, and cyanidin

89

glucoside), flavan-3-ols (e.g., gallocatechin, catechin, and epicatechin gallate), and phenolic

90

acids (gallic acid, galloylquinic acid, and gallic acid hexoside) from Arbutus unedo were

91

investigated during in vitro digestion (Mosele, Macia, Romero, & Motilva, 2016). In both

92

studies, however, it is not clear which anthocyanin glycosides were used to form the

93

metabolites (e.g., protocatechuic acid, caffeic acid, and syringic acid).

5

94

Anthocyanin metabolites have been examined in both in vitro and in vivo models. In vitro

95

digestion models are useful alternatives to in vivo models because they provide results in a

96

shorter time (Lee, Lee, & Hur, 2015). In vitro digestion models have been used to investigate

97

the survival of bioactive compounds and drugs through the gastrointestinal (GI) tract (Jantratid,

98

Janssen, Reppas, & Dressman, 2008; Korhonen & Pihlanto, 2006). In previous studies, in vitro

99

anthocyanin digestion models included the oral, gastric, and intestinal fractions, whereas the

100

colonic fraction was not simulated (Liang, Wu, Zhao, Zhao, Li, Zou, et al., 2012; McDougall,

101

Fyffe, Dobson, & Stewart, 2007). The recoveries of anthocyanin after the simulated intestinal

102

phase were 0.34% (serum-available material) (Liang, et al., 2012) and 25% (McDougall, Fyffe,

103

Dobson, & Stewart, 2007). Furthermore, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside

104

were metabolized in vitro into cyanidin aglycone, ferulic acid, and caffeic acid among others

105

(Liang, et al., 2012). However, the colon may be an important site of anthocyanin metabolism

106

in the GI tract (Zhang, Yang, Wu, & Weng, 2016). Therefore, it is necessary to design a colonic

107

fraction to understand anthocyanin degradation during in vitro digestion. Additionally, to the

108

best of our knowledge, no in vitro studies have evaluated the changes in cyanidin-3-O-

109

galactoside, which is the most abundant form of anthocyanins in chokeberry.

110

Anthocyanins and their metabolites found in vitro and in vivo are typically analyzed by

111

high-performance liquid chromatography (HPLC) and/or ultra-high-performance liquid

112

chromatography (UHPLC) equipped with a triple quadrupole mass spectrometer (Bae & Suh,

113

2007; Chen, Xin, Yuan, Su, & Liu, 2014; Natic, Dabic, Papetti, Aksic, Ognjanov, Ljubojevic,

114

et al., 2015). A triple quadrupole (QqQ) mass spectrometer provides the unit masses of the

115

target pseudomolecular ions. Furthermore, LC-QqQ is suitable for quantifying the target

116

pseudomolecular ions in multiple reaction monitoring (MRM) mode. However, LC-QqQ

117

cannot identify unknown pseudomolecular ions because it provides the unit mass but not an

118

accurate mass. On the other hand, quadrupole time-of-flight (qTOF) mass spectrometry gives 6

119

an accurate mass of the pseudomolecular ions and structure information based on the accurate

120

mass of fragmental ions (Wang, Wang, & Cai, 2013). Therefore, UHPLC-(ESI)-qTOF is more

121

suitable than LC-QqQ for identifying unknown pseudomolecular ions.

122

To date, the phenolic profiles of mulberry and chokeberry have not been evaluated in detail.

123

The major anthocyanins in mulberry and chokeberry fruits are cyanidin-3-O-glucoside and

124

cyanidin-3-O-galactoside, respectively. However, it remains unknown how the different

125

glycoside forms attached to cyanidin affect the changes in cyanidin glycosides during digestion.

126

In this study, the phenolic profiles of chokeberry and mulberry were determined, and the

127

effects of different anthocyanin compositions on the changes in the phenolic profiles during

128

simulated in vitro human digestion were elucidated by UHPLC-(ESI)-qTOF and UHPLC-

129

(ESI)-QqQ.

7

130

2. Materials and methods

131

2.1. Chemicals and reagents

132

Cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, cyanidin-3-O-galactoside, cyanidin-3-

133

O-arabinoside, cyanidin aglycone, pelargonidin-3-O-glucoside, and quercetin-3-O-glucoside

134

were purchased from Extrasynthese (Genay, France). Protocatechuic acid, chlorogenic acid,

135

rutin, trolox, trifluoroacetic acid, formic acid, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were

136

purchased from Sigma-Aldrich (St. Louis, MO, USA), and HPLC-grade water, methanol and

137

acetonitrile were obtained from J.T. Baker (Phillipsburg, NJ, USA).

138 139

2.2. Mulberry and chokeberry samples

140

Mulberry (Morus Microphylla Buckl. Shimgang) and chokeberry (Aronia melanocarpa

141

Viking) were purchased from Yangpyeong Yangjam Youngnong Co., Ltd. (Yangpyeong,

142

Korea) and Sacheol Aronia Co., Ltd. (Jeongeup, Korea), respectively, in 2017. The mulberry

143

and chokeberry fruits were harvested at commercial maturity. Fresh mulberry and chokeberry

144

fruits, weighing 15–20 kg each, were purchased and used to prepare the composite samples.

145

After harvesting, the mulberry and chokeberry samples were immediately frozen, lyophilized,

146

and stored at -80°C until anthocyanin analysis. For the in vitro digestion experiments, the

147

anthocyanins were extracted from the mulberry and chokeberry fruits and purified.

148 149

2.3. Quantification of the phenolic compounds in the mulberry and chokeberry samples by

150

HPLC

151

For anthocyanin analysis, 300 mg of mulberry or chokeberry lyophilized powders were

152

extracted with 30 mL 0.1% HCl methanol as described in a previous study (Kim & Lee, 2017).

153

The mixtures were sonicated for 30 min and centrifuged at 10,621 ×g for 15 min. For the

154

analysis of the other phenolic compounds, 100% methanol was used in the extraction process 8

155

instead of 0.1% HCl methanol. Next, the supernatants were filtered through a 0.22 µm

156

polyvinylidene fluoride syringe filter, and the collected filtrates were analyzed by HPLC (1260

157

Infinity II LC Systems, Agilent Technologies, Santa Clara, CA, USA). The injection volume

158

was 20 μL, and the phenolic compounds were separated on a Zorbax Eclipse XDB-C18 column

159

(4.6 × 250 mm, 5 µm, Agilent) at 40°C. The mobile phases comprised 1% trifluoroacetic acid

160

in water (A) and 1% trifluoroacetic acid in acetonitrile (B). The flow rate of the mobile phase

161

was 1 mL/min. The gradient was as follows: 0–6.5 min, 10–12% (B); 6.5–10.5 min, 12–13%

162

(B); 10.5–33 min, 13–17% (B); 33–60 min, 17–65% (B); 60–70 min, 65–95% (B). The

163

phenolic compounds were monitored at 320 nm for phenolic acid (chlorogenic acid), 360 nm

164

for the flavonols (rutin and quercetin-3-O-glucoside), and 520 nm for the anthocyanins

165

(cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, cyanidin-3-O-galactoside, cyanidin-3-O-

166

arabinoside, pelargonidin-3-O-glucoside, and cyanidin aglycone). The retention times and

167

spectra of the identified phenolic compounds were then compared with the authentic standards.

168

The compounds were quantified using external calibration curves of the authentic standards.

169

The samples were analyzed in triplicate (n = 3).

170 171

2.4. Purification of the anthocyanins from the mulberry and chokeberry fruits for the in vitro

172

digestion model

173

Anthocyanins from the mulberry and chokeberry fruits were isolated using a previously

174

described method with some modifications (Liu, Zhang, Wu, Wang, Wei, Wu, et al., 2014).

175

Briefly, lyophilized mulberry and chokeberry fruit powders were mixed with methanol (20

176

mL/g sample), and the extracts were centrifuged at 4000 ×g for 15 min. The supernatants were

177

then filtered through filter paper (Whatman No. 41, Maidstone, UK). The residues were re-

178

extracted using the same procedure. The two extracts were then mixed and subsequently

179

concentrated in a rotary evaporator (n-1300, EYELA, Tokyo, Japan) at 40°C. The resultant 9

180

concentrates were passed through a Sephadex LH20 column, and the anthocyanins were eluted

181

with 0.1% HCl 20% methanol solution (v/v). Next, the eluents were concentrated using a rotary

182

evaporator and dehydrated by lyophilization. The dried eluents were re-dissolved with 0.1%

183

HCl solution and then purified using Sep-Pak C18 SPE cartridges (Waters, Milford, MA, USA).

184

Each cartridge was conditioned with water and methanol. After loading the sample, each

185

cartridge was first washed with 0.1% HCl and subsequently with ethyl acetate. Anthocyanin

186

was eluted with 10% formic acid in methanol (v/v). Finally, the pH values of the purified

187

anthocyanin extracts were adjusted to 6.8 with 2 mol/L NaOH for in vitro digestion.

188 189

2.5. Static in vitro digestion

190

The simulated in vitro digestion model was prepared using a modified method described

191

in our previous study (Lee, Lee, & Hur, 2015). The digestion model was composed of oral,

192

gastric, intestinal, and colonic fractions. The composition of each fraction is listed in Table 1.

193

The purified anthocyanin extracts (equivalent to ~2.3 mg anthocyanins/g fruit DW) from

194

the mulberry and chokeberry fruits were digested sequentially at 37°C using a water bath as

195

follows: oral fraction (addition of saliva juice and mixing for 5 min)—gastric fraction (addition

196

of gastric juice and mixing for 2 h)—intestinal fraction (addition of an intestinal juice mixture

197

of duodenal and bile juices and mixing for 2 h)—colonic fraction (addition of the

198

microorganism and mixing for 4 h). Aliquots (500 µL) of each fraction were collected,

199

centrifuged, filtered through a 0.22-µm filter, and stored at -20°C until UHPLC-(ESI)-qTOF

200

and UHPLC-(ESI)-QqQ analyses. The samples were prepared in triplicate (n = 3).

201 202

2.6. Determination of DPPH radical scavenging activity in each digestion fraction

203

The antioxidant activity before and after in vitro digestion was evaluated by 2,2-diphenyl-

204

1-picrylhydrazyl (DPPH) radical scavenging activity according to a previously described 10

205

method with some modifications (Vieira, Borges, Copetti, Di Pietro, Nunes, & Fett, 2011).

206

The mulberry and chokeberry extracts and the digestion fractions were each mixed with 200

207

µL of 0.15 mM DPPH dissolved in ethanol and allowed to stand for 15 min. The absorbance

208

was measured by spectrophotometry (Multiskan go, Thermo Scientific, Waltham, USA) at 517

209

nm. The results were reported in µg Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-

210

carboxylic acid) equivalents (te)/g DW.

211 212

2.7. Analysis of the anthocyanin metabolites in each digestion fraction by UHPLC-(ESI)-qTOF

213

and UHPLC-(ESI)-QqQ

214

The anthocyanin metabolites were identified by UHPLC-(ESI)-qTOF (Acquity UPLC

215

system coupled with SYNAPT G2-Si HDMS, Waters) with an injection volume of 5 μL. The

216

anthocyanin metabolites were separated on an Acquity BEH C18 column (2.1 × 100 mm, 1.7

217

μm, Waters) at 40°C. The mobile phases comprised 0.1% formic acid in water (A) and 0.1%

218

formic acid in acetonitrile (B). The gradient was as follows: 0–8 min, 5–40% (B); 8–9 min,

219

40–80% (B); 9–9.2 min, 80–100% (B); 9.2–10.2 min, 100% (B); 10.2–10.7 min, 100–5% (B);

220

10.7–12 min, 5% (B); a flow rate of 0.4 mL/min was employed. The anthocyanin metabolites

221

were analyzed as pseudomolecular ions in ESI-positive and -negative modes. The capillary

222

and cone voltages were 1.5 kV and 30 V, respectively, and the source and devolution

223

temperatures were 120 and 500°C, respectively. Mass scanning was conducted in the m/z range

224

of 60–1400.

225

Each anthocyanin metabolite was quantified in multiple reaction monitoring (MRM) mode

226

of the UHPLC-(ESI)-QqQ (Nexera x2 coupled to LCMS-8050, Shimadzu, Kyoto, Japan). The

227

metabolites were separated on an Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters)

228

with a flow rate of 0.40 mL/min. The mobile phase consisted of 0.1% formic acid in water (A)

229

and 0.1% formic acid in acetonitrile (B). The anthocyanin metabolites were quantified in ESI11

230

positive mode for the anthocyanins and ESI-negative mode for the phenolic acids. The

231

nebulizing and drying gas flow rates were 3 and 10 L/min, respectively, and the collision

232

energy was 35 V in negative and positive modes. Mass scanning was conducted in the m/z

233

range of 50–1000. The absolute quantification of cyanidin-3-O-glucoside, cyanidin-3-O-

234

rutinoside, cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, pelargonidin-3-O-glucoside,

235

cyanidin aglycone, pelargonidin aglycone, protocatechuic acid, and p-coumaric acid was

236

performed with authentic standards. When authentic standards were unavailable, external

237

calibration curves of cyanidin-3-O-glucoside and protocatechuic acid were used to respectively

238

quantify the anthocyanins and phenolic acids.

239

The cyanidin conjugates (i.e., cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, cyanidin-

240

3-O-galactoside, and cyanidin-3-O-arabinoside) were quantified using external calibration

241

curves by measuring the area of the m/z of the precursor ions to m/z 287 (product ion, cyanidin

242

aglycone ion). Pelargonidin-3-O-glucoside was quantified using the area of m/z 433 (precursor

243

ion) to m/z 271 (product ion, pelargonidin aglycone). Cyanidin aglycone and pelargonidin

244

aglycone were quantified using the area of the m/z of the precursor ions to their characteristic

245

fragments (product ions) (de Rosso, Hillebrand, Montilla, Bobbio, Winterhalter, Mercadante,

246

et al., 2008). Protocatechuic acid hexoside was quantified by measuring the area of m/z 315

247

(precursor ion) to m/z 153 (product ion, protocatechuic acid). p-Hydroxybenzoyl hexoside was

248

quantified by measuring the area of m/z 298 (precursor ion) to m/z 137 (product ion, hydroxy-

249

benzoate). Protocatechuic acid was quantified by measuring the area of m/z 153 (precursor

250

ion) to m/z 109 (product ion, a fragment ion corresponding to the loss of CO2 from the

251

carboxylic acid moiety). Coumarylquinic acid was quantified by measuring the area of m/z

252

337 (precursor ion) to m/z 163 (product ion, p-coumaric acid). Feruloylquinic acid was

253

quantified by measuring the area of m/z 367 (precursor ion) to m/z 191 (product ion, quinic

254

acid). p-Coumaric acid was quantified by measuring the area of m/z 163 (precursor ion) to m/z 12

255

119 (product ion, a fragment ion that corresponds to the loss of CO2). Finally, caffeoyl quinic

256

acid was quantified by measuring the area of m/z 353 (precursor ion) to m/z 173 (product ion,

257

quinic acid-H2O).

258 259

2.8. Statistical analysis

260

Statistical analysis was performed using SPSS statistics 23 (SPSS, Inc., Chicago, IL, USA)

261

and XLSTAT (ver. 2017.03, Microsoft Excel Add-in software, New York, USA) software.

262

Significant differences in the anthocyanin metabolites in the in vitro digestion fractions were

263

determined by one-way analysis of variance followed by Duncan′s post-hoc test at p < 0.05.

264

Principal component analysis (PCA) and hierarchical cluster analysis were conducted based

265

on the anthocyanins, and their metabolites were identified and quantified by UHPLC-(ESI)-

266

qTOF and UHPLC-(ESI)-QqQ using XLSTAT. To visualize the data discrimination, PCA

267

plots mapped variables (16 metabolites) together with samples (n = 10) through loading, and

268

scores in dimensional spaces were determined.

13

269

3. Results and Discussion

270 271

3.1. Determination of the phenolic compounds in the fruit and purified extracts of mulberry

272

and chokeberry

273

Table 2 details the phenolic contents in the mulberry and chokeberry fruits and their

274

isolated extracts analyzed by HPLC. The major anthocyanin in mulberry was cyanidin-3-O-

275

glucoside (17.22 mg/g DW). Mulberry also contained cyanidin-3-O-rutinoside (5.91 mg/g DW)

276

and pelargonidin-3-O-glucoside (0.31 mg/g DW), as well as other phenolic compounds such

277

as rutin (i.e., quercetin-3-O-rutinoside) and chlorogenic acid. The total polyphenol content in

278

mulberry was 24.01 mg/g DW.

279

Chokeberry contained cyanidin-3-O-galactoside (20.41 mg/g DW) as the major

280

anthocyanin. It also contained cyanidin-3-O-arabinoside (3.92 mg/g DW), cyanidin-3-O-

281

glucoside (0.30 mg/g DW), and cyanidin aglycone (0.41 mg/g DW), as well as other phenolic

282

compounds such as chlorogenic acid and quercetin-3-O-glucoside. The total polyphenol

283

content in chokeberry was 28.92 mg/g DW.

284

Purified anthocyanin extracts of the mulberry and chokeberry fruits were used for in vitro

285

studies. Notably, all the flavonoids and phenolic acids were removed from the two extracts

286

during the purification process. Thus, the purified mulberry and chokeberry extracts only

287

contained anthocyanins. Moreover, among all the anthocyanins, cyanidin-3-O-glucoside and

288

cyanidin-3-O-galactoside presented the highest levels in the purified (anthocyanin) extracts of

289

mulberry and chokeberry, respectively.

290 291

3.2. Identification of anthocyanin metabolites in the in vitro digestion model using UHPLC-

292

(ESI)-qTOF

14

293

The compositions of mulberry and chokeberry anthocyanin metabolites in the simulated in

294

vitro digestion model is illustrated in Table 3. The anthocyanin metabolites were identified by

295

UHPLC-(ESI)-qTOF and quantified in ESI-positive mode because the signal was much higher

296

in this mode. Other phenolic compounds were identified and quantified in ESI-negative mode

297

because their response was higher in negative mode.

298

The theoretical mass of each metabolite is listed in Table 3, with mass errors of the

299

measured masses of the identified metabolite compounds of < 4.7 ppm when analyzed by

300

UHPLC-(ESI)-qTOF. Seven anthocyanins (cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside,

301

cyanidin hexosylhexoside, pelargonidin-3-O-glucoside, cyanidin dioxaloylhexoside, cyanidin

302

aglycone, and pelargonidin aglycone) and five phenolic acids (protocatechuic acid hexoside,

303

p-hydroxybenzoyl hexoside, protocatechuic acid, coumaroylquinic acid, and feruloylquinic

304

acid) were identified in the purified mulberry extract and its digestive juices in the oral, gastric,

305

intestinal, and colonic fractions. On the other hand, six anthocyanins (cyanidin-3-O-

306

galactoside, cyanidin-3-O-glucoside, cyanidin hexosylhexoside, cyanidin-3-O-arabinoside,

307

cyanidin aglycone, and pelargonidin aglycone) and six phenolic acids (p-coumaric acid,

308

protocatechuic acid hexoside, protocatechuic acid, caffeoylquinic acid, coumaroylquinic acid,

309

and feruloylquinic acid) were identified in the purified chokeberry extract and its digestive

310

juices. Among them, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, cyanidin-3-O-

311

arabinoside, cyanidin-3-O-galactoside, pelargonidin-3-O-glucoside, cyanidin aglycone, and

312

protocatechuic acid were identified by comparison of their retention times and accurate masses

313

with the authentic standards.

314

Cyanidin dioxaloylhexoside was absent from the purified mulberry extract but present in

315

the digestive juices of purified mulberry extract. Most phenolic acids were not present in the

316

mulberry and chokeberry extracts because they were removed during the purification process;

317

however, they were newly found in the digestive juices. Thus, protocatechuic acid hexoside, 15

318

p-hydroxybenzoyl hexoside, coumaroylquinic acid, and feruloylquinic acid were absent from

319

the mulberry extract but found in the digestive juices. Additionally, p-coumaric acid,

320

protocatechuic acid hexoside, caffeoylquinic acid, coumaroylquinic acid, and feruloylquinic

321

acid were newly formed during the in vitro digestion process of the chokeberry extract.

322

Following digestion, p-hydroxybenzoyl hexoside was found in the digestive juices of the

323

mulberry extract but not in those of the chokeberry extract, whereas p-coumaric acid and

324

caffeoylquinic acid were only found in the chokeberry juices. These results indicated that

325

different anthocyanin profiles (particularly different glycoside profiles of cyanidin: cyanidin-

326

3-O-glucoside for the mulberry extract and cyanidin-3-O-galactoside for the chokeberry

327

extract) result in the formation of different metabolites (particularly catabolites) after digestion.

328 329

3.3. Quantification of the anthocyanin metabolites in the in vitro digestion model using

330

UHPLC-(ESI)-QqQ

331

The purified anthocyanin extracts (equivalent to ~2.3 mg anthocyanins/g fruit DW) from

332

mulberry and chokeberry were digested sequentially in the following order: oral fraction,

333

gastric fraction, intestinal fraction (mixture of duodenal juice and bile juice), and colonic

334

fraction. The content of each metabolite in the different fractions was quantified in MRM mode

335

by UHPLC-(ESI)-QqQ.

336

In the oral fraction, the cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside levels

337

decreased to 84% and 48%, respectively, of the levels found in the mulberry anthocyanin

338

extract. The pelargonidin-3-O-glucoside content in the oral fraction also decreased to 44% of

339

the level found in the mulberry extract. In contrast, the cyanidin aglycone and pelargonidin

340

aglycone levels increased by 3.8 and 4.1 times after simulated oral digestion. The increased

341

contents of cyanidin aglycone and pelargonidin aglycone may have been derived from their

342

glycoside forms (i.e., cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, and pelargonidin-316

343

O-glucoside). The total anthocyanin level in the mulberry extract decreased to 76% of the

344

original value after simulated oral digestion. On the other hand, the phenolic acid levels in the

345

mulberry extract presented up to a 17.4 times increase from the purified mulberry extract (138

346

μg/g fruit DW) to the oral fraction (2400 μg/g fruit DW). Particularly, the protocatechuic acid

347

level increased 14.9 times after simulated oral digestion. These results indicate that the

348

anthocyanins in the purified mulberry extract were degraded and that smaller molecular

349

phenolic acids were formed during oral digestion.

350

Compared to the mulberry anthocyanins, larger amounts of chokeberry anthocyanins were

351

recovered after simulated oral digestion. Specifically, 93% of the cyanidin-3-O-galactoside

352

content in chokeberry extract was recovered after simulated oral digestion. After oral digestion,

353

the cyanidin-3-O-arabinoside level in the chokeberry extract decreased to 78% compared to

354

that in the chokeberry extract. However, the pelargonidin aglycone content increased from 129

355

to 314 μg/g fruit DW after simulated oral digestion; this compound was not derived from

356

pelargonidin glycosides as they were not present in our chokeberry extract. Interestingly, this

357

indicates that cyanidin was converted to perlagonidin after losing a 4-hydroxyl group. The

358

phenolic acid composition of the chokeberry extract after simulated oral digestion differed

359

from that of the mulberry extract. Feruloylquinic acid and caffeoylquinic acid were only

360

detected in the chokeberry extract after simulated oral digestion. However, coumaroylquinic

361

acid and p-hydroxybenzoyl hexoside, which were found in the mulberry extract, were also

362

identified in the chokeberry extract after simulated oral digestion. These results suggested that

363

the sugar moiety may have affected the metabolite profile of cyanidin glycoside after oral

364

digestion.

365

After gastric digestion of the mulberry extract, 73% of the total anthocyanins was

366

recovered. The total anthocyanins in the extract, oral fraction, and gastric fraction displayed a

367

significant decrease (p < 0.05), suggesting that the anthocyanins may have been converted to 17

368

other metabolites during in vitro digestion. After simulated gastric digestion, the levels of

369

cyanidin-3-O-glucoside derived from the mulberry extract and cyanidin-3-O-galactoside

370

derived from the chokeberry extract were not significantly different from those observed after

371

simulated oral digestion (p > 0.05). The acidic conditions of the simulated gastric fluid

372

contributed to the stability of cyanidin-3-O-glucoside (McGhie & Walton, 2007). A previous

373

study reported that anthocyanin was absorbed via the stomach in rat (Talavera, Felgines, Texier,

374

Besson, Lamaison, & Rémésy, 2003). Therefore, the high stability of the anthocyanins after

375

gastric digestion may be very important because it suggests that in vivo, circulating metabolites

376

may be present as the anthocyanin metabolites found in the gastric fluid. Notably, the cyanidin

377

aglycone and pelargonidin aglycone contents of mulberry and chokeberry extracts after

378

simulated gastric digestion were higher than those observed after simulated oral digestion.

379

After simulated intestinal digestion, the total anthocyanin content of the mulberry extract

380

was considerably decreased to 56% compared to that observed after simulated gastric digestion.

381

The cyanidin-3-O-glucoside content also decreased to 56% compared to that observed after

382

simulated gastric digestion of the mulberry extract. Cyanidin dioxaloylhexoside was only

383

detected in the mulberry extract after gastric and intestinal digestion. After simulated intestinal

384

digestion, 69% cyanidin aglycone and 12% pelargonidin aglycone were recovered compared

385

to the amounts recovered after simulated gastric digestion. These results suggest that cyanidin

386

aglycone was more stable than pelargonidin aglycone at the alkaline pH of simulated intestinal

387

digestion. The total phenolic acid level of the mulberry extract after simulated intestinal

388

digestion increased by 1.5 times compared to that observed after simulated gastric digestion.

389

Protocatechuic acid hexoside was the predominant phenolic acid after intestinal digestion of

390

the mulberry extract.

391

The cyanidin-3-O-galactoside level of the chokeberry extract after simulated intestinal

392

digestion decreased to 37% compared to that recovered after simulated gastric digestion. 18

393

Cyanidin-3-O-galactoside exhibited greater degradation than cyanidin-3-O-glucoside in the

394

intestinal environment. Protocatechuic acid hexoside in the digested chokeberry extract after

395

simulated intestinal digestion increased by 5.1 times, compared to that observed after simulated

396

gastric digestion. Moreover, p-Coumaric acid was newly detected in the intestinal fraction of

397

the chokeberry extract.

398

After simulated colonic digestion, the cyanidin-3-O-glucoside level in the mulberry extract

399

decreased to 64% compared to that recovered after simulated intestinal digestion. The

400

cyanidin-3-O-rutinoside, cyanidin hexosylhexoside, pelargonidin-3-O-glucoside, cyanidin

401

dioxaloylhexoside, and cyanidin aglycone levels also decreased to 83, 71, 53, 48, and 23% of

402

the levels recovered after simulated intestinal digestion, respectively. The protocatechuic acid

403

hexoside and p-hydroxybenzoyl hexoside levels of the mulberry extract after simulated colonic

404

digestion decreased to 3–4% compared to those observed after simulated intestinal digestion.

405

However, the protocatechuic acid contents significantly increased (by 2.3 times) in the colonic

406

fraction compared to those observed in the intestinal fraction (p < 0.05). It has also been

407

reported that anthocyanins are degraded to protocatechuic acid by Lactobacillus casei (Marin,

408

Miguelez, Villar, & Lombo, 2015). However, the exact mechanism of protocatechuic acid

409

production has not been reported (Stevens & Maier, 2016).

410

The cyanidin-3-O-galactoside level in the chokeberry extract after simulated colonic

411

digestion decreased to 37% compared to that observed after simulated intestinal digestion. This

412

indicates that, compared to cyanidin-3-O-glucoside, cyanidin-3-O-galactoside was recovered

413

at a higher rate after oral and gastric digestions but significantly degraded after intestinal and

414

colonic digestions. After colonic digestion, the cyanidin-3-O-arabinoside, cyanidin aglycone,

415

and pelargonidin aglycone contents decreased to 43, 44, and 18% of the values recovered after

416

intestinal digestion, respectively. Cyanidin hexosylhexoside was not detected after colonic

417

digestion. The protocatechuic acid hexoside level of the chokeberry extract after simulated 19

418

colonic digestion decreased to 58% compared to that observed after simulated intestinal

419

digestion. The protocatechuic acid level of the chokeberry extract after colonic digestion

420

increased by 2.0 times compared to that observed after simulated intestinal digestion.

421

Coumaroylquinic acid and feruloylquinic acid were newly detected after simulated colonic

422

digestion of the chokeberry extract. Additionally, caffeoylquinic acid was only detected after

423

simulated colonic digestion of the chokeberry extract. Caffeoylquinic acid was identified as a

424

metabolite of cyanidin-O-xylosyl glucoside in a previous study (Pinto, Spinola, Llorent-

425

Martinez, Fernandez-de Cordova, Molina-Garcia, & Castilho, 2017). However, in our study,

426

the cyanidin-3-O-glucoside from the mulberry extract did not degrade into caffeoylquinic acid.

427

Therefore, we supposed that the anthocyanin metabolite composition was affected by the sugar

428

moiety in anthocyanin.

429

After simulated digestion, the levels of cyanidin-3-O-glucoside and cyanidin-3-O-

430

galactoside decreased significantly. It has been reported that anthocyanins are metabolized by

431

the opening of the intramolecular heterocyclic flavylium ring under alkaline conditions in the

432

intestinal fraction (Stevens & Maier, 2016). Anthocyanins are typically stable at an acidic pH

433

but unstable at an alkaline pH. Moreover, the pH stability of anthocyanins depends on their

434

chemical structures. The methoxyl groups on the B-ring of anthocyanins seems to enhance the

435

stability of anothocyanins at an alkaline pH. For example, it has been reported that malvidin-

436

3-O-glucoside, which has methoxyl groups on the B-ring, exhibited higher stability than

437

cyanidin-3-O-glucoside across the alkaline pH range (Loypimai, Moongngarm, & Chottanom,

438

2016).

439

In our study, cyanidin-3-O-galactoside degradation was greater during in vitro digestion

440

compared to that of cyanidin-3-O-glucoside. No studies have compared the digestion stability

441

of the hexoside moiety of cyanidin; however, one study has evaluated the digestion stability

442

of the hexoside moiety of peonidin (Jiao, Li, Zhang, Gao, Zhang, Meng, et al., 2018). Jiao et 20

443

al. reported that peonidin-3-O-glucoside was recovered at a higher rate (46.7%) than peonidin-

444

3-O-galactoside (10.8%) after in vitro intestinal digestion (Jiao, et al., 2018). Therefore, the

445

hexoside moiety of anthocyanin affects the stability of anthocyanins in the in vitro digestion

446

model, and the glucoside moiety may be more stable than the galactoside moiety of

447

anthocyanin during digestion.

448

During in vitro digestion, the cyanidin aglycone levels increased in the oral fraction and

449

finally decreased after further digestion of the mulberry and chokeberry extracts. Cyanidin

450

aglycone has been reported to form by degradation of the glycoside moiety (Kay, Kroon, &

451

Cassidy, 2009). Thus, the decrease in the cyanidin aglycone levels might have occurred

452

because this compound was used to form other anthocyanins (e.g., cyanidin dioxaloylhexoside)

453

or it might have degraded into phenolic acids, such as protocatechuic acid, during digestion.

454

Previous studies have also revealed that cyanidin-3-O-glucoside, delphinidin-3-O-glucoside,

455

and malvidin-3-O-glucoside are degraded to smaller phenolic compounds such as

456

protocatechuic acid, gallic acid, and syringic acid by in vitro digestion (Fernandes, Faria, de

457

Freitas, Calhau, & Mateus, 2015). Blood orange juice contains cyanidin-3-O-glucoside as the

458

major anthocyanin (Mondello, Cotroneo, Errante, Dugo, & Dugo, 2000). After consumption

459

of blood orange juice, protocatechuic acid was formed in the human plasma, which may have

460

been derived from the anthocyanins present in the orange juice (Vitaglione, Donnarumma,

461

Napolitano, Galvano, Gallo, Scalfi, et al., 2007). However, the metabolites produced from

462

cyanidin-3-O-galactoside have not been investigated to date. Thus, we identified the

463

metabolites produced from cyanidin-3-O-glucoside and cyanidin-3-O-galactoside during in

464

vitro digestion.

465

The composition of the anthocyanin metabolites (i.e., anthocyanin and phenolic acid) of

466

the mulberry and chokeberry extracts were altered during in vitro digestion. For example,

467

cyanidin dioxaloylhexoside, p-coumaric acid, protocatechuic acid, p-hydroxylbenzoylhexose, 21

468

caffeoylquinic acid, coumaroylquinic acid, and feruloylquinic acid were newly formed in the

469

gastric, intestinal, and colonic fractions of the mulberry and chokeberry extracts. The

470

anthocyanin metabolite composition after in vitro digestion of the mulberry extract differed

471

from that observed in the chokeberry extract. For example, cyanidin dioxaloylhexoside and p-

472

hydroxybenzoylhexose were only detected after in vitro digestion of the mulberry extract,

473

whereas p-coumaric acid and caffeoylquinic acid were only detected after in vitro digestion of

474

the chokeberry extract. Interestingly, protocatechuic acid hexoside (44% of the total

475

metabolites) was the predominant metabolite after digestion of the chokeberry extract, whereas

476

cyanidin-3-O-glucoside (66% of the total metabolites) was the predominant metabolite after

477

digestion of the mulberry extract.

478 479

3.4. Antioxidant activity in the in vitro digestion model

480

The DPPH radical scavenging activity results of the mulberry and chokeberry anthocyanin

481

extracts before and after the simulated in vitro digestion model are presented in Table 3. The

482

anthocyanin extracts of mulberry and chokeberry presented 12,718 and 18,109 mg trolox

483

equivalent/g fruit DW, respectively. The DPPH radical scavenging activity decreased from the

484

oral to the intestinal fraction, regardless of the anthocyanin sources. Particularly, in the

485

intestinal fraction of mulberry extract, the antioxidant activity decreased to 40% of that of the

486

oral fraction of the mulberry extract. This decrease in antioxidant activity was attributed to the

487

loss of anthocyanins from the gastric to the intestinal fraction. Interestingly, the antioxidant

488

activities in the mulberry and chokeberry extracts increased by up to 2.1 and 1.3 times,

489

respectively, from the intestinal to the colonic fractions. These results may be explained by the

490

newly formed degradation products of the anthocyanins (e.g., phenolic acids) during colonic

491

digestion. An increase in the antioxidant activity of pomegranate juices during simulated

22

492

digestion by lactic acid bacteria has also been reported (Valero-Cases, Nuncio-Jauregui, &

493

Frutos, 2017).

494 495

3.5. PCA and hierarchical cluster analysis

496

To visualize the samples (mulberry/chokeberry extracts and different simulated digestion

497

fractions) and anthocyanin metabolite clustering, PCA and hierarchical cluster analysis were

498

performed. Figure 1 presents the PCA results of the anthocyanin metabolites in the in vitro

499

digestion model. The first two principal components (F1 and F2) explained 65.47% of the total

500

variables, with values of 42.93% and 22.54% for F1 and F2, respectively. The simulated

501

digestion fractions containing mulberry and chokeberry extracts were clearly grouped to the

502

left and right sides, respectively. Cyanidin-3-O-glucoside was correlated with the simulated

503

digestion fractions containing the mulberry extract, whereas cyanidin-3-O-galactoside was

504

correlated with the simulated digestion fractions containing the chokeberry extract.

505

Furthermore, the intestinal and colonic fractions were located in the lower part of the PCA

506

plot, whereas p-coumaric acid, protocatechuic acid hexoside, and protocatechuic acid were

507

correlated with the intestinal and colonic fractions of both extracts.

508

Figure 2 presents a heatmap of the mulberry and chokeberry anthocyanin metabolites in

509

the in vitro digestion model. The data reveal that the cyanidin-3-O-glucoside and cyanidin-3-

510

O-rutinoside levels of the mulberry extract decreased during in vitro digestion. The cyanidin

511

aglycone and pelargonidin aglycone levels were high in the gastric fraction containing the

512

mulberry extract. On the other hand, the protocatechuic acid hexoside, p-hydroxybenzoyl

513

hexoside, cyanidin dioxaloylhexoside, feruloylquinic acid, and coumaroylquinic acid levels

514

were high in the intestinal and colonic fractions of the mulberry extract.

515

The cyanidin-3-O-galactoside levels of the chokeberry extract decreased during in vitro

516

digestion. The levels of feruloylquinic acid, p-coumaric acid, protocatechuic acid hexoside, 23

517

and coumaroylquinic acid were high in the intestinal and colonic fractions containing the

518

chokeberry extract. Interestingly, protocatechuic acid and protocatechuic acid hexoside

519

displayed opposite patterns. Therefore, protocatechuic acid in the oral and gastric fractions

520

may be converted to protocatechuic acid hexoside in the intestinal and colonic fractions. The

521

caffeoylquinic acid levels increased during in vitro digestion, whereas the cyanidin-3-O-

522

arabinoside levels increased after gastric digestion of the chokeberry extract.

523

In previous studies, whole berry fruits have often been used to understand the anthocyanin

524

changes in in vitro digestion (Quatrin, Rampelotto, Pauletto, Maurer, Nichelle, Klein, et al.,

525

2019). We used purified anthocyanin extracts for the in vitro digestion study instead of whole

526

fruit because the purpose of this study was to investigate how the different glycoside forms

527

attached to cyanidin affect the changes in the cyanidin glycosides during digestion. If whole

528

fruits were to be used, the presence of other flavonoids and phenolic acids, which would remain

529

with the anthocyanins during digestion, would make it difficult to establish the origin of the

530

metabolites that were newly formed during digestion.

531 532

4. Conclusion

533

After in vitro digestion, the recovered contents of cyanidin-3-O-glucoside and cyanidin-3-

534

O-galactoside were 29% and 12% of the levels detected in the mulberry and chokeberry

535

extracts, respectively. The anthocyanin sugar moiety affected the anthocyanin stability in in

536

vitro digestion. Cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside in the mulberry extract

537

were metabolized to cyanidin aglycone, cyanidin hexosyl hexoside, and pelargonidin-3-O-

538

glucoside in the gastric fraction. Metabolites in the gastric fraction were converted to

539

protocatechuic acid hexoside, p-hydroxybenzoyl hexoside, cyanidin dioxaloylhexoside,

540

feruloylquinic acid, and coumaroylquinic acid in the intestinal and colonic fractions. The

541

chemical changes in different anthocyanin glycoside moieties (i.e., cyanidin-3-O-glucoside 24

542

from the mulberry extract and cyanidin-3-O-galactoside from the chokeberry extract) varied

543

after in vitro digestion. For example, cyanidin-3-O-galactoside was degraded into

544

caffeoylquinic acid, which was not found after in vitro digestion of cyanidin-3-O-glucoside

545

from the mulberry extract. The bioactivity (DPPH radical scavenging activity) of the

546

anthocyanin metabolites decreased in the intestinal fraction. However, the bioactivity

547

increased after simulated colonic digestion, possibly because of the newly formed anthocyanin

548

metabolites during colonic digestion. Furthermore, anthocyanin metabolites from the

549

chokeberry extract exhibited higher DPPH radical activities than those from the mulberry

550

extract. This research provides basic information of the chemical changes of cyanidin

551

glycosides during in vitro gastrointestinal digestion. We concluded that berries with high

552

cyanidin-3-O-galactoside contents (e.g., chokeberry) may be better anthocyanin sources than

553

berries with cyanidin-3-O-glucoside contents (e.g., mulberry) owing to the high bioactivities

554

of the anthocyanin metabolites although cyanidin-3-O-glucoside is more stable than cyanidin-

555

3-O-galactoside during digestion.

556 557

Acknowledgements

558

This work was supported by the National Research Foundation of Korea (NRF) funded by

559

the Korea government (MSIP) [grant numbers NRF-2016R1C1B1014851 and NRF-

560

2019R1F1A1062634].

561 562

Conflict of interest

563

The authors declare no conflict of interest.

564

25

565

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Figure Legends

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Figure 1. Principal component analysis (PCA) of anthocyanin and its metabolites in the in

708

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709

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710

Figure 2. Heatmap visualization and hierarchical clustering of changes in the (a) mulberry and

711

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712

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713

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714

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32

Figure 1.

33

Figure 2.

34

Table 1. Composition of the oral, gastric, intestinal, and colonic fractions in the simulated digestion model for the purified mulberry and chokeberry extracts

Oral fraction

Gastric fraction

Intestinal fraction

Colonic fraction

Saliva

Gastric juice

Duodenal juice

Bile juice

Microorganism in colon

Compounds

1.7 mL NaCl 8 mL urea 15 mg uric acid

6.5 mL HCl 18 mL CaCl2·2H2O 1 g BSA

6.3 mL KCl 9 mL CaCl2·2H2O 1 g BSA

68.3 mL NaHCO3 10 mL CaCl2·2H2O 1.8 g BSA 30 g bile

E. coil (log108–1010) L. casei (log108–1010)

Enzymes

290 mg α-amylase 25 mg mucin

2.5 g pepsin 3 g mucin

9 g pancreatin 1.5 g lipase

pH

6.8 ± 0.2

1.50 ± 0.02

8.0 ± 0.2

7.0 ± 0.2

35

Table 2. Phenolic composition of the mulberry and chokeberry fruits and purified mulberry and chokeberry extracts Mulberry fruit (mg/g)

Chokeberry fruit (mg/g)

Purified mulberry extract (mg/mL)

Purified chokeberry extract (mg/mL)

Cyanidin-3-O-glucoside

17.22 ± 0.13

0.30 ± 0.01

0.99 ± 0.11

0.05 ± 0.00

Cyanidin-3-O-rutinoside

5.91 ± 0.02

n.d.

0.19 ± 0.00

n.d.

Cyanidin-3-O-galactoside

n.d.

20.41 ± 1.91

n.d.

1.75 ± 0.13

Pelargonidin-3-O-glucoside

0.31 ± 0.02

n.d.

0.04 ± 0.00

n.d.

Cyanidin-3-O-arabinoside

n.d.

3.92 ± 0.30

n.d.

0.14 ± 0.02

Cyanidin aglycone

n.d.

0.41 ± 0.00

0.12 ± 0.01

0.13 ± 0.01

Rutin

0.61 ± 0.00

n.d.

n.d.

n.d.

Quercetin-3-O-glucoside

n.d.

0.04 ± 0.01

n.d.

n.d.

Chlorogenic acid

0.03 ± 0.00

3.91 ± 0.22

n.d.

n.d.

Sum

24.01 ± 0.11

28.92 ± 2.52

1.35 ± 0.15

2.07 ± 0.18

All compounds were identified and quantified using authentic standards. 36

Table 3. Mulberry and chokeberry anthocyanin metabolite contents (μg/g fruit DW) and DPPH radical scavenging activity (mg trolox equivalent/g fruit DW) in the simulated digestion model

　

Mulberry

Theoretical mass (m/z) 　

Error (ppm)

MRM transition

Anthocyanin extracts

　

Oral fraction

Gastric fraction

Intestinal fraction

Colonic fraction

　

Anthocyanins Cyanidin-3-O-glucoside+*

449.1078

4.7

449 → 287

17323 a

14492 b

13969 b

7754 c

4954 d

Cyanidin-3-O-rutinoside+*

595.1663

4.2

595 → 287

3600 a

1723 b

1600 b

1292 b

1077 c

Cyanidin hexosylhexoside+

611.1612

-0.5

611 → 287

492 a

431 b

415 b

108 c

77 d

37

Pelargonidin-3-O-glucoside +*

433.1135

1.6

433 → 271

1338 a

585 b

538 b

292 c

154 d

Cyanidin dioxaloylhexoside+

593.0779

2.2

593 → 287

n.d.

n.d.

n.d.

208 a

100 b

Cyanidin aglycone+*

287.0556

3.5

287 → 189

62 c

238 b

292 a

200 b

46 c

Pelargonidin aglycone+

271.0606

4.1

271 → 173

92 c

377 a

331 b

38 d

69 cd

24062 a

18238 b

17477 c

9862 d

6485 e

Sum of anthocyanins Phenolic acids Protocatechuic acid hexoside

315.0722

3.5

315 → 153

n.d.

77 c

169 b

862 a

31 c

p-Hydroxybenzoyl hexoside

299.0772

0.7

298 → 161

n.d.

46 c

185 b

554 a

15 c

Protocatechuic acid*

153.0193

-2.6

153 → 109

138 c

2062 a

508 b

292 c

662 b

Coumaroylquinic acid

337.0929

1.5

337 → 163

n.d.

215 c

231 c

354 a

308 b

Feruloylquinic acid

367.1035

-2.7

367 → 191

n.d.

n.d.

46 b

77 a

31 c

Sum of phenolic acids

138 d

2400 a

1131 c

1746 b

1038 c

DPPH radical scavenging activity

12718 a

5640 b

4543 c

2218 d

4643 c 38

Chokeberry

Anthocyanins Cyanidin-3-O-galactoside+*

449.1078

3.1

449 → 287

20529 a

19000 b

17543 b

6543 c

2400 d

Cyanidin-3-O-glucoside+*

449.1078

4.7

449 → 287

414 a

200 b

57 c

n.d.

n.d.

Cyanidin hexosyl hexoside+

611.1612

1.3

611 → 287

329 a

171 b

171 b

64 c

n.d.

Cyanidin-3-O-arabinoside+*

419.0978

0.2

419 → 287

1143 a

886 b

950 b

650 c

279 d

Cyanidin aglycone+*

287.0556

3.5

287 → 189

350 d

743 b

871 a

586 c

257 d

Pelargonidin aglycone+

271.0606

3

271 → 173

129 b

314 a

271 a

157 b

29 c

22893 a

21307 b

19864 b

8007 c

2979 d

n.d.

n.d.

n.d.

43 a

29 b

Sum of anthocyanins Phenolic acids p-Coumaric acid

163.0401

-1.2

163 → 119

39

+

Protocatechuic acid hexoside

315.0722

3.5

315 → 153

n.d.

471 d

1671 c

4686 a

2714 b

Protocatechuic acid*

153.0193

-0.7

153 → 109

143 d

1629 a

1014 b

186 d

371 c

Caffeoylquinic acid

353.0878

4.5

353 → 173

n.d.

157 a

29 bc

14 c

29 b

Coumaroylquinic acid

337.0929

-1.5

337 → 163

n.d.

n.d.

n.d.

n.d.

71 a

Feruloylquinic acid

367.1035

0.5

367 → 191

n.d.

86 b

100 b

157 a

n.d.

Sum of phenolic acids

143 d

2336 c

2807 c

5086 a

3221 b

DPPH radical scavenging activity

18109 a

6156 b

5293 d

4324 e

5750 c

Metabolites were identified and quantified in ESI-positive mode; other metabolites were analyzed in ESI-negative mode. *Metabolites were

identified and quantified using authentic standards. Highlights 

Anthocyanin composition changes during the digestion of chokeberry and mulberry



Cyanidin-3-O-glucoside and cyanidin-3-O-galactoside were dominant anthocyanins



Digestion decreases anthocyanin levels at different rates



Anthocyanin metabolites varied depending on the cyanidin glycosides 40



Decomposition of anthocyanins afforded phenolic acids during digestion



Antioxidant activity increased from the intestinal to the colonic fractions

41