Formation and Characterization of Polyphenol-Derived Red

Subscriber access provided by University of Florida | Smathers Libraries

Functional Structure/Activity Relationships

Formation and characterization of polyphenol-derived red chromophores: Enhancing the color of processed cocoa powders: Part 1 Daniel Germann, Timo D. Stark, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01049 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

Journal of Agricultural and Food Chemistry

1

Formation and characterization of polyphenol-derived red

2

chromophores: Enhancing the color of processed cocoa

3

powders: Part 1

4 5

Daniel Germann, Timo D. Stark, Thomas Hofmann*

6 7

Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-

8

Meitner-Str. 34, 85354, Freising, Germany

9 10 11 12 13 14

* To whom correspondence should be addressed

15

PHONE

+49-8161/71-2902

16

FAX

+49-8161/71-2949

17

E-MAIL

[email protected]

18

1 ACS Paragon Plus Environment


Page 2 of 35

19

ABSTRACT

20

Although cocoa powder alkalization (Dutching) is a widely used industrial process to

21

improve taste, dispersibility and coloring of the final product, nevertheless knowledge

22

about the compounds causing a change in coloring is fragmentary. By means of alkaline

23

model reactions starting from the major cocoa polyphenol monomers, (+)-catechin or (-)-

24

epicatechin, eight chromophores were derived from the first rearrangement product

25

catechinic acid. LC-MS/MS analysis, 1D- and 2D-NMR, and electron paramagnetic

26

resonance

27

hydroxycatechinic acids (1a, 2a) and their radical states (1b, 2b), which were highlighted

28

as main red chromophores . Six new dehydrocatechinic acid dimers (dehydrocatechinic

29

acid-C-6´B/C-8D-(2R,3S)-catechin (3), dehydrocatechinic acid-C-6´B/C-6D-(2R,3S)-

30

catechin

31

dehydrocatechinic acid-C-6´B/C-6D-(2R,3R)-epicatechin (7,8)) were also characterized

32

as chromophores. 1-8 as well as their precursors were detected and quantified in alkalized

33

cocoa powders via LC-MS/MS. With increasing grade of alkalization a decrease in

34

catechin and epicatechin together with an increase in catechinic acid was observed.

35

Compounds 1b, 2b and 3-8 also showed a decrease in concentration by Dutching, which

36

correlates to the accumulation of/to higher ordered chromophore oligomers and

37

underlined the increase of the high molecular weight fraction. These findings give a first

38

insight into the formation of structures causing the red coloring of cocoa, which offers the

39

opportunity to optimize the alkalization process towards a better color design of cocoa

40

powders.

(EPR)

(4,5),

spectroscopy

dehydrocatechinic

led

to

the

unequivocal

identification

acid-C-6´B/C-8D-(2R,3R)-epicatechin

41


of

(6),

6´-

and

Page 3 of 35


42

KEYWORDS: cocoa powder, Dutching, model reaction, polyphenol, epicatechin,

43

catechinic acid, radical, dehydrocatechinic acid dimer, chromophore

44

INTRODUCTION

45

The alkalization process was invented in 1830 by the Dutch chemist Coenraad

46

Johannes van Houten with the intention to enhance taste, solubility and color of cocoa

47

powders.1 Recently this process was mainly used to change the coloring of cocoa

48

powders, because alkalized cocoa powders have a much darker, and more reddish

49

coloring than natural powders.1–3 This change in coloring induced by alkalization was

50

established as quality standard for cocoa powders.4,5 Today this process is widely used

51

in industrial cocoa processing, and U.S. patents deal with its optimization.4,5 However, up

52

to date the knowledge about the structures of the key chromophores which are generated

53

through the alkalization process is rather rudimentary.

54

Polyphenols are known for their high antioxidant activity and cocoa powders are a rich,

55

natural source of polyphenols.6,7 However, via alkalization these polyphenols were

56

decreased and recent research has strongly focused on the extent of degradation.8–11

57

Former studies have shown that major rearrangements of cocoa natural polyphenols

58

occur in alkaline milieu, e.g. C-glycosylation of flavan-3-ols or the rearrangement of

59

catechin (C) and epicatechin (EC) to catechinic acid (CA) and epicatechinic acid

60

(EPCA).12–15

61

Because of this degradation and modification, we suspected that polyphenols also could

62

play an important role in cocoa color change. Our initial alkalization studies confirmed this

63

assumption as yellow-orange to reddish coloring of aqueous solutions of flavan-3-ols,

64

procyanidins and catechinic acids occurred. 3 ACS Paragon Plus Environment


65

Therefore, the purpose of this study was to generate target chromophores from major

66

single polyphenols by means of model reactions, to isolate and elucidate the chemical

67

structures of compounds showing absorbance at wavelengths in visual range and to

68

detect and quantify the concentrations in alkalized cocoa powder samples.

69 70

MATERIALS AND METHODS

71

Chemicals. The following reagents were obtained commercially in p.A. quality: (+)-

72

C, (-)-EC, ammonium formate (Sigma Aldrich, Steinheim, Germany); potassium

73

hydroxide, potassium carbonate, sodium hydroxide, iron (III) chloride, n-pentane (Merck,

74

Darmstadt, Germany). Catechinic acid (CA) was generated from (+)-C or (-)-EC after the

75

method of Sears et al.15 and purified by means of medium pressure liquid

76

chromatography. Dehydrodicatechin A (dC A) was generated and isolated from (+)-C as

77

described below. Water for chromatographic separations was purified by means of a Milli-

78

Q gradient Integral5 system (Millipore, Schwalbach, Germany), and solvents were used

79

of HPLC-grade (Merck). Deuterated solvents for NMR experiments were obtained from

80

Sigma Aldrich (Steinheim, Germany). Food samples were obtained from a local

81

supermarket.

82

General Experimental Procedure. 1D and 2D NMR spectroscopy 1H, 1H-1H13C

83

gCOSY, gROESY, gHSQC, gHMBC and

were performed on an Avance III 500 MHz

84

spectrometer with a CTCI probe or an Avance III 400 MHz spectrometer with a BBO probe

85

(Bruker, Rheinstetten, Germany). Purity of all standards for external calibration was

86

determined by means of qNMR after the method of Frank et al.16 Electron paramagnetic

87

resonance (EPR) spectroscopy was performed on a JEOL JES-FA 200 spectrometer at 4 ACS Paragon Plus Environment

Page 4 of 35

Page 5 of 35


88

X-band frequency, with a microwave transmitter with Gunn-diode (8.8-9.6 GHz, 0.1-200

89

mW), TM mode cavity (ES-TM1A) and variable temperature control unit (ES-DVT4)

90

operated with liquid nitrogen for cooling. The gauss (g) values were determined by using

91

Mn2+ (nuclear spin I = 5/2) embedded in MgO as standard. The spectral parameters were

92

obtained by spectra simulation using standard software (Isu simu, JEOL). Mass spectra

93

of the compounds were measured on a Waters Synapt G2-S HDMS mass spectrometer

94

(Waters, Manchester, UK) coupled to an Acquity UPLC core system (Waters, Milford, MA,

95

USA). Chromophore screening and quantitation in cocoa powders was performed on a

96

Waters Xevo TQ-S mass spectrometer (Waters) coupled to an Acquity UPLC core system

97

(Waters). HPLC analysis was performed using an analytical HPLC system (PU-2080 Plus;

98

Jasco). HPLC separations were performed using a preparative HPLC system (PU-2087

99

Plus; Jasco, Groß-Umstadt, Germany). Medium pressure liquid chromatography (MPLC)

100

separations were performed on a Büchi Sepacore (Flawil, Switzerland) system using a

101

YMC (YMC Europe, Dinslaken, Germany) DispoPackAT ODS-25 flash cartridge (120g,

102

id. 40mm, l. 150mm). Absorbance maxima and corresponding extinction coefficients of

103

identified chromophores were determined using a UV-VIS 2401PC spectrophotometer

104

(Shimazdu, Kyoto, Japan). Analytes were dissolved in ultrapure water or methanol,

105

transferred into a quartz glass Suprasil (QS, 10 mm, 200-2500 nm) cuvette (Carl Zeiss,

106

Jena, Germany) and measured at UV-VIS range (200-650 nm) in reference to a blank of

107

corresponding solvent.

108

Model-Reactions. For the used strong alkalization model I-I, (+)-catechin or (-)-

109

epicatechin (1 g) was suspended in an aqueous solution of potassium carbonate (50 mL,

110

2.7 g/100 mL, pH 13.1), the reaction vessel was set under oxygen atmosphere and heated

111

for 45 min at 90 °C. After reaction, the model solution was immediately cooled to ambient 5 ACS Paragon Plus Environment


112

temperature in ice and the pH adjusted to 7 with formic acid (10 %, aq) and then the

113

sample was concentrated under reduced pressure to a total volume of 5 mL. An aliquot of

114

this solution (1 mL) was diluted with methanol (1 mL) and acetonitrile (2 mL) and

115

fractionated using preparative HILIC HPLC as described below. For preparative isolation

116

of 1 and 2, CA or EPCA (100 mg, each) were dissolved in an aqueous solution of

117

potassium hydroxide (2 mL, 1 g/100 ml, pH 13.1) and oxygen was bubbled into solution

118

for approximate 15 min at ambient temperature. Dilution and chromatography was

119

performed as described below.

120

Model I-II: for preparative generation of dehydrocatechinic acid dimers (3-8), equal

121

aliquots of catechinic acid (500 mg) and (+)-C or (-)-EC (500 mg) were dissolved in a

122

solution of potassium hydroxide (50 mL, 1 g/100 ml, pH 11). After heating for 2 h at 60 °C,

123

the model solution was cooled to ambient temperature and adjusted to pH 7 with formic

124

acid (10 %, aq), and then, the sample was concentrated under reduced pressure (3 mL).

125

Isolation and Structural Characterization of Target Chromophores. Pre-

126

separation of all model reactions was performed using preparative HPLC and a Luna

127

HILIC column (250 x 21.2 mm, 5 µm; Phenomenex, Aschaffenburg, Germany) as the

128

stationary phase. The effluent (21 mL/min) was monitored at 280 and 392 or 488 nm. The

129

separation started with a mixture (A/B, 0/100, v/v) of aqueous ammonium formate (5

130

mmol/L, pH 5.8) as solvent A and aqueous ammonium formate in acetonitrile (10/90, v/v,

131

5 mmol/L, pH 5.8) as solvent B, increasing the B content up to 90 % within 10 min,

132

following in 10 min to 70 %, and in 2 min to 50 % B. From model I-II, compounds 3-8 were

133

collected as orange colored dehydrocatechinic acid fraction and from model I-I, 1 and 2

134

were collected as reddish colored hydroxycatechinic acid fraction and the last eluting peak

135

was collected as reddish-brown fraction (HMW fraction). Within the first separation step 6 ACS Paragon Plus Environment

Page 6 of 35

Page 7 of 35


136

compounds 1a and 2a were obtained alongside to their radical forms (1b, 2b). The

137

dehydrocatechinic acid fraction, containing compounds 3-8, was further purified by means

138

of semi-preparative HPLC using a RP-column (250 x 10 mm, Phenylhexyl, 5 µm;

139

Phenomenex) as the stationary phase. The effluent (4.2 mL/min) was monitored at 280

140

and 392 nm and the separation started with a mixture (95/05, v/v) of A/B, and the B content

141

was increased up to 20 % within 15 min. Collected fractions were concentrated under

142

reduced pressure and the buffer was removed using the above mentioned MPLC system.

143

Chromatography was performed starting with water for 5 min, increasing the MeOH

144

content up to 100 % within 3 min, holding the conditions for 7 min. The effluent (40 mL/min)

145

was monitored at 280 nm. Ammonium formate free compounds were concentrated under

146

reduced pressure, freeze-dried, and characterized by means of LC-MS and NMR.

147

Catechinic acid was purified using MPLC starting with a mixture (85/15, v/v) of water and

148

methanol, increasing the MeOH content up to 45 % within 15 min, and up to 100 % within

149

5 min, holding the conditions for 7 min. The effluent (40 mL/min) was monitored at 280

150

nm. The isolated compound was concentrated under reduced pressure, freeze-dried, and

151

investigated by means of LC-MS and 1H NMR.

152

(1R,5S,6R,7S)-6´-Hydroxycatechinic acid (1a, 1a´´, Figure 1). Colorless; UV (H2O)

153

λmax = 280 nm; (-) HRESIMS: m/z = 303.0498 [M-H]- (calcd for C15H11O7, 303.0505). 1H

154

NMR (500 MHz, D2O, COSY, ROESY): δ 2.01 [m, 3H, H-C(8α, 8´´α, 8´´β)], 2.50 [m, 1H,

155

J = 5.4, 3.4 Hz, H-C(8β)], 2.55 [m, 2H, H-C(1´´, 5´´)], 3.09 [m, 1H, J = 4.1, 1.9 Hz, H-C(5)],

156

3.14 [m, 1H, J = 4.1, 1.9 Hz, H-C(1)], 3.43 [m, 1H, J = 11.1, 4.1, 0.7 Hz, H-C(6)], 3.52 [m,

157

1H, J = 11.2, 4.3, 0.6 Hz, H-C(6´´)], 4.13 [m, 1H, J = 11.3, 6.3 Hz, H-C(7´´)], 4.40 [m, 1H,

158

J = 11.2, 5.6 Hz, H-C(7)], 6.22 [d, 1H, J = 0.8 Hz, H-C(2´´´)], 6.34 [d, 1H, J = 0.8 Hz, H-

159

C(2´)].

13C

NMR (125 MHz, D2O, HSQC, HMBC): δ 31.5 [C-8´´], 35.3 [C-8], 39.8 [C-6´´], 7 ACS Paragon Plus Environment


160

44.4 [C-6], 51.4 [C-1´´], 56.9 [C-5´´], 57.0 [C-1], 61.5 [C-5], 63.7 [C-7´´], 64.1 [C-7], 95.5

161

[C-2´´], 102.0 [C-3´´], 104.5 [C-3], 129.5 [C-2´´´], 129.9 [C-2´], 147.3 [C-1´], 149.5 [C-1´´´],

162

170.5 [C-4´´´], 170.7 [C-4´], 186.9 [C-6´], 187.7 [C-6´´´], 188.1 [C-3´], 190.0 [C-4], 192.2

163

[C-2], 194.2 [C-4´´], 195.6 [C-9´´], 209.6 [C-9].

164

(1R,5S,6R,7S)-6´-Hydroxycatechinic acid radical (1b, Figure 1). Red; UV (in H2O) λmax =

165

486 nm; ελmax (H2O, 298 K, pH 5.5) = 739 L*mol-1*cm-1; (-) HRESIMS: m/z = 303.0498 [M-

166

H]- (calcd for C15H11O7, 303.0505). The EPR parameter derived from best fit of simulated

167

and experimental spectra are: g =1.990; hyperfine coupling constants ai: a1= 0.115 mT;

168

a2 =0.251 mT; a3 = 0.053 mT.

169

(1S,5R,6S,7R)-6´-Hydroxycatechinic acid (2a, 2a´´, Figure 1). Colorless; UV (H2O) λmax =

170

280 nm; (-) HRESIMS: m/z = 303.0498 [M-H]- (calcd for C15H11O7, 303.0505). 1H NMR

171

(500 MHz, D2O, COSY, ROESY): δ 2.00 [m, 1H, J = 11.3, 7.1, 4.2, 1.6, H-C(8α)], 2.09 [m,

172

2H, H-C(8´´α, 8´´β)], 2.53 [m, 1H, J = 5.4, 3.4 Hz, H-C(8β)], 2.60 [m, 1H, J = 3.5, 2.3 Hz,

173

H-C(5´´)], 2.63 [m, 1H, J = 6.8, 3.5 Hz, H-C(1´´)], 3.11 [m, 1H, J = 4.1, 1.9 Hz, H-C(5)],

174

3.16 [m, 1H, J = 3.6, 1.7 Hz, H-C(1)], 3.46 [m, 1H, J = 11.3, 3.4 Hz, H-C(6)], 3.55 [m, 1H,

175

J = 11.7, 3.4 Hz, H-C(6´´)], 4.15 [m, 1H, J = 11.1, 6.2 Hz, H-C(7´´)], 4.41 [m, 1H, J = 11.3,

176

6.2, 5.5 Hz, H-C(7)], 6.30 [s, 1H, H-C(2´´´)], 6.41 [d, 1H, H-C(2´)], not detectable [H-C(3)],

177

[H-C(5´)].

178

6´´], 44.6 [C-6], 51.5 [C-1´´], 56.8 [C-5´´], 57.2 [C-1], 61.6 [C-5], 63.9 [C-7´´], 64.0 [C-7],

179

95.5 [C-2´´], 102.5 [C-3´´], 105.0 [C-3], 129.8 [C-2´´´], 130.2 [C-2´], 147.3 [C-1´], 149.5 [C-

180

1´´´], 168.0 [C-4´´´], 169.0 [C-4´], 187.3 [C-6´], 188.0 [C-6´´´], 188.8 [C-3´], 189.8 [C-4],

181

191.1 [C-3´´´], 192.5 [C-2], 194.0 [C-4´´], 195.4 [C-9´´], 209.8 [C-9], not detectable [C-5´].

182

(1S,5R,6S,7R)-6´-Hydroxycatechinic acid radical (2b, Figure 1). Red; UV (in H2O) λmax =

183

488 nm; ελmax (H2O, 298 K, pH 5.5) = 1490 L*mol-1*cm-1; (-) HRESIMS: m/z = 303.0498

13C

NMR (125 MHz, D2O, HSQC, HMBC): δ 31.7 [C-8´´], 35.6 [C-8], 40.0 [C-


Page 8 of 35

Page 9 of 35


184

[M-H]- (calcd for C15H11O7, 303.0505). The EPR parameter derived from best fit of

185

simulated and experimental spectra are: g =1.997; hyperfine coupling constants ai: a1=

186

0.180 mT; a2 =0.464 mT; a3 = 0.082 mT, a4 = 0.080 mT.

187

B1´,D7,B3´,C3,B6´,D8-Dehydrocatechinic acid-(2R,3S)-catechin (3, Figure 1). Yellow

188

powder; UV (H2O) λmax = 280, 363, 406 nm; ελmax (H2O, 298 K, pH 7.0) = 1697 (363 nm),

189

1706 (406 nm) L*mol-1*cm-1; (-) HRESIMS: m/z = 575.1189 [M-H]- (calcd for C30H23O12,

190

575.1190). 1H NMR (500 MHz, D2O-d2, COSY): δ 2.04 [m, 1H, J = 8.1, 4.7, 1.0 Hz, H-

191

C(C8α)], 2.45 [m, 1H, J = 6.5, 2.8 Hz, H-C(C8β)], 2.46 [d, 1H, J = 11.4 Hz, H-C(B2´α)],

192

2.56 [dd, 1H, J = 11.1, 3.0 Hz, H-C(C6)], 2.59 [d, 1H, J = 11.3 Hz, H-C(B2´β)], 2.64 [m,

193

1H, J = 16.4, 7.6 Hz, H-C(F4α)], 2.85 [m, 1H, J = 16.2, 4.9 Hz, H-C(F4β)], 3.04 [m, 1H, J

194

= 5.9, 3.8 Hz, H-C(A1)], 3.16 [d, 1H, J = 3.0, H-C(A5)], 4.25 [m, 1H, J = 18.6, 7.4, 5.7, 1.3

195

Hz, H-C(F3)], 4.37 [m, 1H, J = 11.3, 6.0, 5.2, H-C(C7)], 5.10 [d, 1H, J = 7.0 Hz, H-C(F2)],

196

5.19 [s, 1H, H-C(A3)], 6.18 [s, 1H, H-C(D6)], 6.32 [s, 1H, H-C(B5´)], 6.91 [dd, 1H, J = 8.0,

197

1.8 Hz, H-C(E6´)], 6.94 [d, 1H, J = 7.9 Hz, H-C(E5´)], 7.00 [d, 1H, J = 1.8 Hz, H-C(E2´)].

198

1H

199

[m, 1H, J = 15.6, 10.7, 4.4, 1.3 Hz, H-C(B2´α, C8β)], 2.90 [dd, 1H, J = 11.1, 2.8 Hz, H-

200

C(C6)], 3.03 [d, 1H, J = 11.2 Hz, H-C(B2´β)], 3.13 [m, 1H, H-C(F4α)], 3.41 [m, 1H, H-

201

C(F4β)], 3.53 [m, 1H, J = 4.9, 2.4 Hz, H-C(A1)], 3.83 [d, 1H, J = 2.3, H-C(A5)], 4.49 [m,

202

1H, H-C(F3)], 5.00 [m, 1H, H-C(C7)], 5.08 [m, 1H, H-C(F2)], 5.78 [s, 1H, H-C(A3)], 6.55

203

[s, 1H, H-C(D6)], 7.01 [dd, 1H, J = 8.1, 1.6 Hz, H-C(E6´)], 7.04 [s, 1H, H-C(B5´)], 7.16 [d,

204

1H, J = 8.2 Hz, H-C(E5´)], 7.42 [d, 1H, J = 1.9 Hz, H-C(E2´)].

205

d2, HSQC, HMBC): δ 26.4 [C-F4], 33.6 [C-C8], 46.7 [C-B2´], 48.7 [C-C6], 57.0 [C-A1],

206

58.0 [C-A5], 66.1 [C-C7], 66.8 [C-F3], 81.0 [C-F2], 88.8 [C-B1´], 91.2 [C-D6], 94.2 [C-B3´],

207

104.1 [C-D4a], 106.0 [C-D8], 109.0 [C-B5´], 114.6 [C-E2´], 116.4 [C-E5´], 119.3 [C-E6´],

NMR (500 MHz, Pyridine-d5): δ 2.28 [m, 1H, J = 16.2, 11.5, 4.6 Hz, H-C(C8α)], 2.78


13C

NMR (125 MHz, D2O-


Page 10 of 35

208

130.5 [C-E1´], 144.2 [C-E3´,4´], 153.5 [C-D8a], 165.4 [C-B6´], 189.3 [C-A4], 190.5 [C-A2],

209

192.7 [C-B4´], 208.6 [C-9], not detectable: [C-A3], [C-D5], [C-D7].

210

Pyridine-d5, HMBC): δ 28.8 [C-F4], 35.9 [C-C8], 48.9 [C-B2´], 50.6 [C-C6], 56.8 [C-A5],

211

58.9 [C-A1], 66.4 [C-C7], 67.9 [C-F3], 83.7 [C-F2], 89.6 [C-B1´], 95.5 [C-B3´], 104.3 [C-

212

D4a], 107.2 [C-D8], 115.3 [C-E2´], 119.0 [C-E6´], 131.2 [C-E1´], 147.4 [C-E3´], 147.6 [C-

213

E4´], 155.2 [C-D8a], 163.9 [C-B6´], 165.8 [C-D5], 166.0 [C-D7], 181.6 [C-A4], 188.3 [C-

214

A2], 194.6 [C-B4´], 204.9 [C-9], not detectable: [C-D6], [C-B5´], [C-E5´].

215

B1´,D5,B3´,C3,B6´,D6-Dehydrocatechinic

216

B1´,D7,B3´,C3,B6´,D6-Dehydrocatechinic acid-(2R,3S)-catechin (5, Figure 1). Yellow

217

powder; UV (H2O) λmax = 280, 363, 406 nm; ελmax (H2O, 298 K, pH 7.0) = 1697 (363 nm),

218

1706 (406 nm) L*mol-1*cm-1; (-) HRESIMS: m/z = 575.1189 [M-H]- (calcd for C30H23O12,

219

575.1190). 1H NMR (500 MHz, D2O-d2, COSY): δ 2.02 [m, 1H, J = 8.1, 4.7, 1.0 Hz, H-

220

C(C8α)], 2.06 [m, 1H, J = 6.5, 2.8 Hz, H-C(C8β)], 2.43 [d, 1H, J = 11.4 Hz, H-C(B2´α)],

221

2.61 [d, 1H, J = 11.3 Hz, H-C(B2´β)], 2.80 [m, 1H, J = 16.4, 7.6 Hz, H- C(F4α, β)], 2.81

222

[dd, 1H, J = 11.1, 3.0 Hz, H-C(C6)], 3.09 [m, 1H, J = 5.9, 3.8 Hz, H-C(A1)], 3.15 [d, 1H, J

223

= 3.0, H-C(A5)], 4.20 [m, 1H, J = 11.3, 6.0, 5.2, H-C(C7)], 4.27 [m, 1H, J = 18.6, 7.4, 5.7,

224

1.3 Hz, H-C(F3)], 5.14 [d, 1H, J = 7.1 Hz, H-C(F2)], 5.22 [s, 1H, H-C(A3)], 6.20 [s, 1H, H-

225

C(D6)], 6.31 [s, 1H, H-C(B5´)], 6.88 [dd, 1H, J = 8.0, 1.8 Hz, H-C(E6´)], 6.93 [d, 1H, J =

226

7.9 Hz, H-C(E5´)], 6.97 [d, 1H, J = 1.8 Hz, H-C(E2´)]. 13C NMR (125 MHz, D2O-d2, HSQC,

227

HMBC): δ 26.0 [C-F4], 30.5 [C-C8], 45.5 [C-C6], 47.9 [C-B2´], 57.1 [C-A1], 58.2 [C-A5],

228

66.5 [C-C7], 66.8 [C-F3], 80.8 [C-F2], 88.8 [C-B1´], 91.2 [C-D6], 94.2 [C-B3´], 104.1 [C-

229

D4a], 106.0 [C-D6], 109.0 [C-B5´], 114.5 [C-E2´], 116.4 [C-E5´], 119.3 [C-E6´], 129.8 [C-

230

E1´], 144.2 [C-E3´,4´], 153.3 [C-D8a], 165.4 [C-B6´], 189.3 [C-A4], 190.5 [C-A2], 192.7

231

[C-B4´], 208.6 [C-9], not detectable: [C-A3]. 1H NMR of isomers 4/5 (500 MHz, Pyridine-

13C

acid-(2R,3S)-catechin (4,


NMR (125 MHz,

Figure

1);

Page 11 of 35


232

d5): δ 3.16 [m, 1H, H-C(F4α)], 3.44 [m, 1H, H-C(F4β)], 5.09 [d, 1H, J = 1.0 Hz, H-C(F2)],

233

5.79 [s, 1H, H-C(A3)], 6.57 [s, 1H, H-C(D6)]. 13C NMR of isomers 4/5 (125 MHz, Pyridine-

234

d5, HMBC): δ 155.1 [C-D8a], 163.1 [C-D7α], 163.2 [C-B6´], 164.7 [C-D5], 165.0 [C-7β].

235

B1´,D7,B3´,C3,B6´,D8-Dehydrocatechinic acid-(2R,3R)-epicatechin (6, Figure 1). Yellow

236

powder; UV (H2O) λmax = 300, 398 nm; ελmax (H2O, 298 K, pH 4.3) = 6448 (300 nm), 8909

237

(398 nm) L*mol-1*cm-1; (-) HRESIMS: m/z = 575.1189 [M-H]- (calcd for C30H23O12,

238

575.1190). 1H NMR (500 MHz, Pyridine-d5): δ 2.28 [m, 1H, J = 15.6, 12.7, 10.9, 4.8 Hz,

239

H-C(C8α)], 2.68 [d, 1H, J = 11.2 Hz, H-C(B2´α)], 2.74 [m, 1H, J = 12.7, 7.9, 5.4, 2.8 Hz,

240

H-C(C8β)], 2.89 [dd, 1H, J = 11.0, 2.7 Hz, H-C(C6)], 3.00 [d, 1H, J = 11.1 Hz, H-C(B2´β)],

241

3.17 [dd, 1H, J = 16.5, 4.3 Hz, H-C(F4α)], 3.50 [m, 1H, H-C(F4β)], 3.85 [m, 2H, J = 4.5,

242

2.3 Hz, H-C(A1,A5)], 4.55 [m, 1H, H-C(F3)], 4.99 [m, 1H, J = 16.1, 10.9, 5.2 Hz, H-C(C7)],

243

5.09 [d, 1H, J = 1.0 Hz, H-C(F2)], 5.76 [s, 1H, H-C(A3)], 6.53 [s, 1H, H-C(D6)], 7.07 [dd,

244

1H, J = 8.1, 2.0 Hz, H-C(E6´)], 7.10 [s, 1H, H-C(B5´)], 7.28 [d, 1H, J = 8.0 Hz, H-C(E5´)],

245

7.68 [d, 1H, J = 2.0 Hz, H-C(E2´)].

246

F4], 36.9 [C-C8], 49.6 [C-B2´], 51.4 [C-C6], 57.2 [C-A5], 59.7 [C-A1], 67.2 [C-C7], 67.3 [C-

247

F3], 82.2 [C-F2], 90.4 [C-B1´], 90.7 [C-D6], 96.5 [C-B3´], 104.6 [C-4a], 108.3 [C-D8], 113.6

248

[C-B5´], 116.7 [C-E2´], 117.4 [C-E5´], 119.7 [C-E6´], 132.3 [C-E1´], 148.4 [C-E3´,E4´],

249

157.0 [C-D8a], 164.6 [C-B6´], 166.8 [C-D7], 167.3 [C-D5], 181.7 [C-A4], 189.3 [C-A2],

250

195.4 [C-B4´], 205.4 [C-C9].

251

B1´,D5,B3´,C3,B6´,D6-Dehydrocatechinic

252

B1´,D7,B3´,C3,B6´,D6-Dehydrocatechinic acid-(2R,3R)-epicatechin (8, Figure 1). Yellow

253

powder; UV (H2O) λmax = 300, 398 nm; ελmax (H2O, 298 K, pH 4.3) = 6448 (300 nm), 8909

254

(398 nm) L*mol-1*cm-1; (-) HRESIMS: m/z = 575.1189 [M-H]- (calcd for C30H23O12,

255

575.1190). 1H NMR (500 MHz, Pyridine-d5): δ 2.28 [m, 1H, J = 15.6, 12.7, 10.9, 4.8 Hz,

13C

NMR (125 MHz, Pyridine-d5, HMBC): δ 30.8 [C-

acid-(2R,3R)-epicatechin (7,


Figure

1);


256

H-C(C8α)], 2.68 [d, 1H, J = 11.2 Hz, H-C(B2´α)], 2.74 [m, 1H, J = 12.7, 7.9, 5.4, 2.8 Hz,

257

H-C(C8β)], 2.89 [dd, 1H, J = 11.0, 2.7 Hz, H-C(C6)], 3.00 [d, 1H, J = 11.1 Hz, H-C(B2´β)],

258

3.17 [dd, 1H, J = 16.5, 4.3 Hz, H-C(F4α)], 3.50 [m, 1H, H-C(F4β)], 3.51 [m, 1H, H-C(A1)],

259

3.85 [m, 1H, J = 2.3 Hz, H-C(A5)], 4.55 [m, 1H, H-C(F3)], 4.99 [m, 1H, J = 16.1, 10.9, 5.2

260

Hz, H-C(C7)], 5.09 [d, 1H, J = 1.0 Hz, H-C(F2)], 5.76 [s, 1H, H-C(A3)], 6.53 [s, 1H, H-

261

C(D6)], 7.07 [dd, 1H, J = 8.1, 2.0 Hz, H-C(E6´)], 7.10 [s, 1H, H-C(B5´)], 7.28 [d, 1H, J =

262

8.0 Hz, H-C(E5´)], 7.68 [d, 1H, J = 2.0 Hz, H-C(E2´)].

263

HMBC): δ 30.8 [C-F4], 36.9 [C-C8], 49.6 [C-B2´], 51.4 [C-C6], 57.2 [C-A5], 59.7 [C-A1],

264

67.2 [C-C7], 67.3 [C-F3], 82.2 [C-F2], 90.4 [C-B1´], 90.7 [C-D6], 96.5 [C-B3´], 104.6 [C-

265

4a], 108.3 [C-D8], 113.6 [C-B5´], 116.7 [C-E2´], 117.4 [C-E5´], 119.7 [C-E6´], 132.3 [C-

266

E1´], 148.4 [C-E3´,E4´], 157.0 [C-D8a], 164.6 [C-B6´], 166.2 [C-D5α,D7β], 166.8 [C-D7α],

267

167.3 [C-D5β], 181.7 [C-A4], 189.3 [C-A2], 195.4 [C-B4´], 205.4 [C-C9].

13C

NMR (125 MHz, Pyridine-d5,

268

For the estimation of the high molecular weight (HMW) content of the catechin

269

model reactions, the reaction mixture was separated by means of ultrafiltration (UF) into

270

five different MW fractions (100 kDa). For UF

271

a Millipore 5124 Amicon Stirred Cell Model 8400, 400 mL for 76 mm filters or Model 8200,

272

200 mL for 63.5 mm filters with 0.1-0.4 MPa backpressure and Millipore UF membranes

273

(regenerated cellulose) were used. The retentate was flushed with water (3x100 mL) and

274

the low MW fractions were combined. Subsequently all MW fractions were freeze-dried.

275

Quantitation of Compounds 1-8 and their Precursors in Alkalized Cocoa

276

Powders. For the quantitative analysis of the chromophores in cocoa powders, the target

277

compounds were analyzed in raw and 5 days fermented cocoa beans; raw, one and five

278

days fermented as well as low (R) and high roasted (HR) cocoa liquors; three cocoa

279

powders differing in grade of alkalization: cocoa 1 (HPP, light alkalized), cocoa 2 (DP70, 12 ACS Paragon Plus Environment

Page 12 of 35

Page 13 of 35


280

medium alkalized), cocoa 3 (GT78, strong alkalized); one non-alkalized cocoa powder

281

(Amber) and seven cocoa powders obtained from local stores. Each sample was extracted

282

in triplicates after the following procedure: An aliquot of each sample (1 g) was defatted

283

using n-pentane (2x10 mL), shaken for 15 min and centrifuged (10 min, 2500 g)

284

afterwards. The defatted cocoa powder was suspended in a mixture of methanol/water

285

(2x5 mL, 70/30, v/v), treated with ultrasonic for 10 min, shaken for 15 min at ambient

286

temperature and centrifuged (10 min, 2500 g). Aliquots were filtered through a 0.45 µm

287

membrane (Sartorius, Göttingen, Germany) and directly used for LC-MS/MS analysis with

288

the system described above. Chromatography was performed on a BEH C18 column

289

(2.0x150 mm, 1.7 µm) at 45 °C with a flow rate of 0.4 mL/min using a linear gradient from

290

A (water, containing 5 mmol ammonium formate, pH 5.8) to B (acetonitrile/water, 90/10,

291

v/v, containing 5 mmol ammonium formate, pH 5.8), starting with a B content of 20 %,

292

increasing up to 30 % within 5 min, afterwards in 1 min to 50 %, and in 0.5 min to 100 %

293

B. Each sample (1 µL) was analyzed in duplicates using the negative electrospray

294

ionization mode and the following ion source parameters: capillary voltage (2.5 kV),

295

sampling cone voltage (22 V), source offset (50 V), source temperature (150 °C),

296

desolvation temperature (450 °C), cone gas (150 L/h), desolvation gas (850 L/h), collision

297

gas (0.15 mL/min), and nebulizer gas (7.0 bar). The MS/MS parameters of each

298

compound were tuned using IntelliStart of MassLynx v4.1 software, and quantitative

299

analysis was performed by means of the multiple reaction monitoring (MRM) mode with

300

fixed mass transitions as follows: m/z 289.1  245.0 for (+)-catechin, (-)-epicatechin and

301

catechinic acid, m/z 303.05  165.02 for 1-2, and m/z 575.1  395.4 for 3-8. All

302

compounds were quantified by means of external calibration. The chomophore precursors

303

(+)-C and (-)-EC were quantitated via their corresponding calibration curve, CA and EPCA 13 ACS Paragon Plus Environment


304

or hydroxycatechinic and epihydroxycatechinic acid were quantitated in total of catechinic

305

acids or hydroxycatechinic acids according to the calibration curve of CA.

306

Dehydrocatechinic acid-catechin and dehydrocatechinic acid-epicatechin dimers were

307

quantitated in total of their isomers according to the calibration curve of DC A. A defined

308

amount of each compound (~1 mg) was dissolved in water (1 mL, for CA) or methanol (1

309

mL, for DC A) and diluted with water to concentrations ranging from 1 mg/mL to 1 µg/mL.

310

After linear regression of the peak area versus concentration, calibration curves showed

311

linear responses with correlation coefficients of R2 > 0.99. All calibration curves were

312

generated in duplicates.

313

RESULTS AND DISCUSSION

314

Analytical HPLC analysis of the alkaline model approach revealed two main

315

fractions absorbing at wavelengths between 368-488 nm. The first eluting fraction

316

highlighted an absorbance maximum at λ = 392 nm and the second fraction indicated an

317

absorbance maximum at λ = 488 nm (Figure 2). To get a deeper insight into the

318

components of these fractions UPLC-TOF-MSe and NMR spectroscopy was

319

accompanied. However, with the used model I-I it was only possible to obtain sufficient

320

amounts of 1 (5 mg) for structural elucidation. Therefore, compounds 1 and 2 were directly

321

oxidized using CA and EPCA as educts, which were previously generated from (+)-C or

322

(-)-EC (model I-II). The dCA were generated in higher amounts by means of direct reaction

323

of CA with (+)-C or (-)-EC (model I-III), respectively, delivering amounts of ca. 2 mg for

324

dCA and dEPCA as a sum of all isomers.

325

Compound 1a showed the same absorption maximum (280 nm) as catechinic acid.

326

Results from electrospray ionization (ESI) MS indicated, that this compound generated an

327

[M-H]- ion at m/z 303, as well as fragment ions at m/z 179, 165 and 164 (Supporting 14 ACS Paragon Plus Environment

Page 14 of 35

Page 15 of 35


328

Information, Figure S3). High resolution LC-MS analysis confirmed the target compound

329

to have a molecular formula of C15H11O7 and the fingerprint fragments C9H7O4, C8H5O4

330

and C8H4O4. The homonuclear H,H correlation spectroscopy (COSY) spectrum of

331

compound 1a revealed a coupling of proton H-C(1) to H-C(6), of H-C(6) to H-C(7), H-C(7)

332

to H-C(8α,β) as well as a coupling of H-C(8α,β) to H-C(1). By the characteristical shifts of

333

the corresponding carbon atoms determined by HMBC correlations this part of the

334

chemical structure could be confirmed to be identical to the A double-ring moiety of

335

catechinic acid (Figure 3). Moreover, the NMR spectra (Figure S4) revealed a double set

336

of signals, most probably caused by the keto-enol tautomery of the target compound in

337

the used protic solvent. However, attempts to analyze the target compound in aprotic

338

solvents, e.g. dimethylsulfoxide or pyridine, did not result in any clear spectrum.

339

Furthermore, the B-ring of this compound differed to catechinic acid because none

340

aromatic proton signals could be assigned. Instead, two new signals could be observed

341

at 6.22 and 6.34 ppm with characteristical chemical shifts for olefinic protons which were

342

assigned to H-C(2´) and H-C(2´´´) by means of HMBC couplings to C(6) and C(6´´),

343

respectively. The signals of H-C(3) and H-C(5´) could not be detected in the spectrum,

344

due to the acidic nature of these two protons caused by keto-enol tautomery in deuterated

345

protic solvents. Unequivocal assignment of the remaining quarternary carbon atoms and

346

the hydrogen-substituted carbon atoms, respectively, could be successfully achieved by

347

means of HMBC. The newly substituted hydroxyl group at position 6´ was confirmed by

348

the downfield shift of C(6´,6´´´), resonating at 186.9, 187.7 ppm and unequivocally

349

assigned by correlations with protons H-C(6, 6´´) and H-C(2´, 2´´´). In comparison to the

350

CA B-ring, the B-ring of this new compound was in an oxidized state, showing signals with

351

typical chemical shifts for carbonyl groups at 170.5, 170.7 ppm C(4´,4´´´) and 188.1 ppm 15 ACS Paragon Plus Environment


352

C(3´,3´´´), which were assigned by correlations with protons H-C(2´,2´´´). The

353

stereochemistry of 1a was deduced comparing coupling constants and chemical shifts to

354

(+)-C and CA.17 Finally, the target compound was determined as (1R,5S,6R,7S)-6´-

355

hydroxycatechinic acid (HyCA). According to literature,18 in aqueous alkaline solution at a

356

pH above 13 the 6´-HyCA is the main hydroxycatechinic acid isomer which is in line to our

357

findings.

358

During the oxidation of CA to compound 1a the reaction solution changed from

359

colorless to red. This color change was proposed by the formation of HyCA radicals.

360

Relying on the structure of 1a and electron paramagnetic resonance (EPR) spectra of the

361

reddish colored reaction solution the radical structure 1b was postulated (Figure 4A). The

362

reaction solution showed an absorption maximum at 486 nm, typical for red

363

chromophores. The EPR parameter derived from best fit of simulated and experimental

364

spectra of 1b indicated the correlation of three magnetic inequivalent protons with the

365

unpaired electron (Supporting Information, Figure 10). To confirm that the reddish coloring

366

of 6´-HyCA is related to its radical, the colorless precursor CA was measured in direct

367

comparison, showing no radical signal (Figure 4C).

368

Compound 2a was identified as (1S,5R,6S,7R)-6´-HyCA by exactly the same

369

strategy as described above for 1a, just starting from the precursor (-)-EC. Again, the

370

reddish coloring of the reaction solution was explainable by tiny amounts of the radical 2b

371

(Figure 4B). The structure of 2b was also postulated by best fit of simulated to

372

experimental EPR spectra indicating four protons coupling with the unpaired electron and

373

therefore, explaining the higher multiplicity of this spectrum compared to 1b (Figure 1).

374

To get a better understanding of the EPCA reactivity, further EPR spectra of two

375

different alkalization approaches were recorded. On the one hand a soft alkalization using 16 ACS Paragon Plus Environment

Page 16 of 35

Page 17 of 35


376

potassium hydroxide (1 %, KOH) and on the other hand strong alkalization using

377

potassium carbonate (2.7 %, K2CO3) was performed. In the soft alkalization approach a

378

stronger radical signal intensity was detectable (g = 1.997) than in the strong alkalization

379

approach, indicating a decrease in radical concentration with an increasing intensity of

380

alkalization. All EPR spectra showed a second, very broad and intense radical signal with

381

a g-factor of 1.9125 which was slightly higher in the strong alkalization approach (Figure

382

4D). Therefore, it was suspected that this could be the radical signal of the HMW fraction

383

to which the HyCAs presumably polymerize. To confirm this assumption, the most

384

abundant MW fraction (10-30 kDa) was isolated from model I by means of UF and

385

investigated via EPR spectroscopy (Figure 4E). This EPR spectrum showed the same

386

broad unresolved signal (g = 1.9125) as previously observed for the EPCA

387

measurements, and therefore, confirmed the presence of radicals in the HMW fraction as

388

well as the transformation of hydroxycatechinic acid radicals to oligo- or polymers in the

389

model solution.

390

Compounds 3-5 showed typical absorption maxima for yellow chromophores at

391

363 and 406 nm. Results from ESI MS indicated that these type of compounds form the

392

same [M-H]- ion with m/z 575 as dC A as well as a fragment ion with m/z 394

393

characteristical for dehydrodicatechins.19,20 Furthermore an increase in intensity of the

394

fragment ion at m/z 395 was detectable, as well as new fragment ions at m/z 507 and

395

549. High resolution LC-MS analysis confirmed the target compounds to have a molecular

396

formula of C30H23O12 and the fingerprint fragments C21H14O8, C21H15O8, C27H23O10 and

397

C29H25O11 (Figure S7). The 1H NMR measurements of 3-5 were firstly recorded in D2O

398

delivering the clearest signals, however, similar to 1 some signals disappeared resulting

399

from the D/H exchange. Especially the proton signals H-C(D6/8) were affected which are 17 ACS Paragon Plus Environment


400

crucial for confirmation of the C-linkage between both monomers and consequently 3-5

401

were additionally recorded in pyridine-d5. The 1H NMR spectrum in D2O showed signals

402

similar to dC A,20 however, the aromatic proton signals H-C(A6) and H-C(A8) were missing

403

indicating a change of the A-ring catechin moiety. Furthermore, a new olefinic proton

404

singlet H-C(A3) resonating at 5.19 ppm was observed which disappeared over time (D/H

405

exchange). Additionally, changes in chemical shifts and coupling constants of the two

406

aliphatic protons H-C(A1) at 3.04 ppm (J = 5.9, 3.8 Hz) and H-C(A5) at 3.16 ppm (J =

407

3.0 Hz) were observed. Chemical shifts and COSY correlations of these proton signals

408

confirmed the presence of a CA moiety in the dimer which is explainable by the

409

substitution of the dehydrocatechin body by CA. The remaining proton signals were

410

identified by chemical shifts and coupling constants as (+)-C. Unequivocal assignment of

411

CA B ring protons and all other carbon atoms, respectively, could be successfully

412

achieved by means of HSQC and HMBC NMR spectroscopy. The HMBC experiment

413

revealed a correlation between the methylene protons H-C(B2´) resonating at 2.46 and

414

2.59 ppm and one carbonyl group C(B4´) at 192.7 ppm. The only remaining B ring proton

415

H-C(B5´) resonating at 6.32 ppm was clearly assigned by 3J couplings to C(B1´) and

416

C(B3´). C-atom C(B1´) resonating at 88.8 ppm was assigned by correlations to the protons

417

H-C(C6), H-C(B2´) and H-C(B5´). The hemi-acetal structure of the compound was

418

confirmed by the correlations between H-C(B2´), H-C(B5´) and the carbon atom C(B3´) at

419

94.2 ppm, which showed a typical chemical shift for a hydroxy substituted aliphatic carbon

420

atom. All these correlations confirmed the same dehydro-nature of the B ring as for dC

421

A.20 The location of the catechinic acid carbonyl group C(A9) resonating at 208.6 ppm

422

was assigned by the correlation to H-C(A5). H-C(A5) and H-C(C6) showed a second

423

correlation at 189.3 ppm to C(A4). The remaining carbonyl group C(A2) resonating at 18 ACS Paragon Plus Environment

Page 18 of 35

Page 19 of 35


424

190.5 ppm was assigned by the correlation with the methylene protons H-C(C8α,β). Final

425

confirmation of the C-linkage was achieved by repetition of the HMBC experiment with

426

pyridine as aprotic solvent. Similar to dehydrodicatechins in methanol, the main isomer

427

showed a clear and intense correlation between the aromatic D-ring proton resonating at

428

6.55 ppm and carbon atom C(D5) at 165.8 ppm, which was also detectable between

429

methylene protons H-C(F4) resonating at 3.13 and 3.41 ppm and C(D5) (Figure 5). These

430

correlations clearly demonstrated the position of the aromatic proton at D6 and confirming

431

the intermolecular linkage at position D8 for compound 3. The 1H NMR spectra recorded

432

in D2O and pyridine indicated additional proton signals with lower intensity, respectively,

433

which could be assigned to two further constitutional isomers of dCA (5,6) showing an

434

intermolecular linkage at D6. This was confirmed by correlations in the HMBC spectrum

435

between the aromatic D-ring proton, methylene protons H-C(F4α,β) and H-C(F2) to the

436

carbon atom C(D8a) at 155.1 ppm. These correlations clearly confirmed the position of

437

the aromatic proton at H-C(D8). Furthermore a correlation between H-C(F4) and C(D5) at

438

164.7 ppm was detectable. For carbon atom C(D7) two possible correlation signals to H-

439

C(D8) were detectable at 163.1 and 165.0 ppm, indicating the two possible conformers

440

for the D6-linked isomer. According to literature data of similar compounds, the carbon

441

atom C(D7) appears in a more down field range than C(D5), if the oxygen bridge resides

442

between C(B1´) and C(D5), and upfielded, if the oxygen bridge resides between C(B1´)

443

and C(D7).21,22 As two detectable correlation signals for C(D7) could be observed the

444

existence of both conformers is very likely.

445

Compounds 6-8 were identified following the same strategy as described above for

446

3-5. However, in protic solvents the (-)-dEPCA dimers showed rapid epimerization to (-)-

447

dCA, therefore NMR spectroscopy was conducted in pyridine. Again, the 8-linked 19 ACS Paragon Plus Environment


448

constitutional isomer 6 could be confirmed as main isomer, but also the two 6-linked

449

isomers were detectable in lower signal intensity. In conclusion, we assumed that EC

450

generates the same three dEPCA-constitution isomers as identified for dCA.

451

The reaction pathway of dehydrocatechinic acid dimers was postulated according

452

to dC A (Figure 6).20 After rearrangement of C or EC to CA, CA is oxidized to its o-quinone.

453

Afterwards, a second C molecule undergoes a nucleophilic attack from position A8 or A6

454

to the oxidized B ring. After rearrangement and cyclization the main product 3 and the

455

side products 4 and 5 are formed. The EC dimers 6-8 are similarly generated via a

456

nucleophilic attack of EC to the o-quinone of CA (Figure 6).

457

Quantification of Compounds 1-8 and their Corresponding Precursors in

458

Alkalized Cocoa Powder Samples. So far, our studies revealed that all identified

459

chromophores were generated either of C (1a,1b,3-5) or EC (2a,2b,6-8), and the CA

460

appeared as decisive intermediate. Therefore, the concentration of 1b/2b as precursors

461

as well as the chromophores (3-8) were traced in different processed (unfermented,

462

fermented, unroasted, roasted, high roasted, low-, medium- and strong-alkalized) cocoa

463

beans. Compared to unfermented cocoa beans and liquor the five days fermented cocoa

464

beans showed a degradation of EC from 60786-63712 to 39041-50034 µmol/kg. Roasting

465

revealed a decrease from 52670 (0-1d fermented)-36682 (5d fermented) µmol/kg in low

466

to 47506 (0-1d)-33628 (4-5d) µmol/kg in HR samples. However, lowest concentrations

467

were detectable in the alkalized samples ranging from 13065 in low alkalized to 3435

468

µmol/kg in strong alkalized cocoa powders. Also the C content indicated a decrease via

469

fermentation from 3116-3849 (0d) to 1339-2245 (5d) µmol/kg. Inversely, roasting

470

increased the concentration from 3039 (5d)-6695 (0-1d) µmol/kg in R to 3713 (5d)-9734

471

(0-1d) µmol/kg in HR cocoa samples. This could be explained by an increasing 20 ACS Paragon Plus Environment

Page 20 of 35

Page 21 of 35


472

epimerization rate of EC to C as expressed by a decreasing ratio of 17-29 EC/C in U to 8-

473

14 in R and 5-10 in HR cocoa samples and is in line to literature.9–11 Due to the

474

transformation to CA and other possible alkalization products the concentration of EC and

475

C is further degraded in alkalized cocoa samples (Figure 7A). All samples showed slightly

476

higher contents of C (0.59-0.89 mg/g) and EC (0.98-3.89 mg/g) than stated for alkalized

477

cocoa powder samples in literature.10,11 Furthermore, the measured values for the non-

478

alkalized cocoa powder sample indicated average contents of 0.54 mg/g C and 4.34 mg/g

479

EC and literature 0.63-0.88 mg/g C and 1.91-2.23 mg/g C.10,11 These values clearly show,

480

that all commercially obtained cocoa samples were dutched. As expected, the EC

481

concentration showed a constant decrease in the order from low to strong alkalized.9–11,23

482

C however, showed no constant decrease, most probably caused by the increasing

483

epimerization rate of EC towards a distinct equilibrium of both compounds.10 This could

484

be confirmed by the determination of EC to C ratios (Figure 7A).

485

The measured contents of CA (70-151 µmol/kg) were about 14 to 24 times lower

486

than the ones of C. The CAs showed a two fold increase from low to strong alkalized

487

cocoa samples, most obviously according to the catalyzed rearrangement of C and EC to

488

CA. The measured contents of HyCA 1-2 (21-25 µmol/kg) showed about a 3-6 fold lower

489

concentrations than the CAs with a slight decrease detectable from low to strong

490

alkalization (Figure 7B). This could be explained by the previously stated rapid

491

polymerization of the hydroxycatechinic acids to higher ordered polyphenol polymers at

492

strong alkali conditions. The HMW content of the model I-I approach was determined by

493

means of UF delivering three fractions above 1 kDa (

Formation and Characterization of Polyphenol-Derived Red

Recommend Documents