Molecular Interactions between Caffeine and Catechins in Green Tea

Jul 1, 2014 - Catechin, epicatechin, and caffeine from the green tea extract exhibited ... known that caffeine forms complexes with catechins in black...
0 downloads 0 Views 548KB Size
Subscriber access provided by UNIV MUNICH

Article

MOLECULAR INTERACTIONS BETWEEN CAFFEIN AND CATECHINS IN GREEN TEA. Marta Colon, and Cristina Nerin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5011287 • Publication Date (Web): 01 Jul 2014 Downloaded from http://pubs.acs.org on July 6, 2014

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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

MOLECULAR INTERACTIONS BETWEEN CAFFEIN AND CATECHINS IN GREEN TEA. M. Colon, C. Nerin*. Department of Analytical Chemistry, Aragon Institute of Engineering Research I3A, CPS-University of Zaragoza, Torres Quevedo Building, María de Luna St. 3, E-50018 Zaragoza, Spain. * Corresponding author, Tel.: +34 976 761873; fax: +34 9762388 E-mail address: [email protected] (C.Nerin).

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 35

1

Abstract

2

Migration of green tea components from an active packaging material containing green

3

tea extract was performed in water and 3% acetic acid in water. The migration values

4

for acid simulant were much higher than the values obtained in water. The influence of

5

the acidic media in solutions of catechins standards and green tea extract was evaluated

6

by liquid chromatography. Catechin, Epicatechin and Caffeine from the green tea

7

extract exhibited the major variation in their concentrations values, with an increase of

8

29.90%, 20.75% and 15.95% respectively in acidic medium. The results suggested that

9

catechins and caffeine form complexes through intermolecular interactions in neutral

10

media and these interactions are broken in acidic media. The continuous variation

11

method was also performed to confirm the stoichiometry of the complexes between

12

catechins and caffeine. Finally, a computer simulation was applied by Chem Pro 12.0

13

and the energies involved were calculated to confirm the experimental results obtained.

14

15

16

17

18

caffeine,

(+)-catechin,

19

Keywords:

20

simulation, migration, active packaging.

(-)-epicatechin,

intermolecular

interactions,

21

22 2

ACS Paragon Plus Environment

Page 3 of 35

Journal of Agricultural and Food Chemistry

23

Introduction

24

Green tea leaves contain many characteristic compounds being catechins and caffeine

25

the major ingredients of tea. Catechins are a group of polyphenols that show beneficial

26

effects in human health such as anti-hypercholesterolemic,1,2 anti-bacterial,3,4 anti-

27

oxidative,5,

28

Caffeine, which is the principal member of methylated xanthines, is a naturally

29

occurring alkaloid found in tea, coffee, mate, guarana and kola nuts. In humans, caffeine

30

acts as a central nervous system stimulant.10 However, the excessive consumption of

31

caffeine can produce negative effects in the organism such as anxiety disorders.11

32

Generally, green tea leaves contain high levels of caffeine, which can be as high as 10%

33

(w/w).12 It is known that caffeine forms complexes with catechins in black tea and

34

coffee.13-15 Many researchers have been investigating the structure of the complexes

35

between caffeine and catechins. Maruyama et al.16 described that some gallated

36

catechins have a high affinity for caffeine and this conclusion was based on 1H NMR

37

chemical shift change of gallate complexed to caffeine. Cai et al.17 noted that in

38

catechin and epicatechin, the A and C rings provided a general site for caffeine

39

association but in gallated catechins, the galloyl ester is the preferred site for

40

complexation. Hayashi et al.18 reported that an investigation of the 1H NMR chemical

41

shift change and Nuclear Overhauser Effect Spectroscopy (NOESY) spectra in

42

catechins and caffeine solution showed the participation of A rings of catechins in

43

complexes, as well as B or Bʼ rings. All of these works mentioned16-18 were performed

44

in solution using NMR techniques, but their overall structures were still unclear and the

45

detailed interactions between caffeine and catechins have not been sufficiently

46

elucidated. In 2009, Ishizu et al.19,20 prepared crystals of complexes of caffeine and

6

and anti-cancereffects,7-9 mainly because of their antioxidant properties.

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

gallate,

and

47

gallocatechin

48

intermolecular interactions by X-ray crystallographic analysis. Subsequently, they have

49

investigated the crystal stereochemical structures of caffeine complexes and the detailed

50

non-covalent

51

Furthermore, they were focused on the inclusion complexes comprising cyclodextrins

52

and catechins.23 All the crystal structures were prepared in water solution at 90 ºC and

53

left at room temperature to crystallize.

54

Green tea extract is generally considered a potent antioxidant that can be either used as

55

direct soft drink in water, applied to the food surface or incorporated as active agent into

56

polymeric packaging materials to protect the food against the oxidation process and

57

extend the shelf life of packaged food.24-30 Therefore, the behavior of the green tea

58

extract incorporated into an active plastic packaging has to be studied taking into

59

account the formation of complexes by intermolecular interactions between the main

60

components of green tea16-23, catechins and caffeine, and their behavior in different

61

media.

62

For this, the aims of this work were: (1) to evaluate the influence of two different food

63

simulants in the migration tests from an active packaging material containing green tea

64

extract, (2) to investigate in depth by liquid chromatography the variations observed in

65

the concentration values of green tea components in both neutral and acidic media, (3)

66

to demonstrate the formation of complexes between catechins and caffeine through

67

intermolecular interactions and to confirm the formation of these complexes by the

68

continuous variation method (Job’s method), which was applied to know the

69

stoichiometry of the complexes. Finally, (4) to confirm the experimental results

interaction

investigated

with

their

galloylated

and

stereochemical

Page 4 of 35

non-galloylated

structures

and

catechins.21,22

4

ACS Paragon Plus Environment

Page 5 of 35

Journal of Agricultural and Food Chemistry

70

obtained in both liquid chromatography and spectroscopy studies by a computer

71

simulation program.

72

Materials and methods

73

Reagents and solutions

74

Caffeine (58-08-2); (+)-Catechin (>99.0% (HPLC), CAS 154-23-4) (C); (-)-Epicatechin

75

(>95.0% (HPLC), CAS 490-46-0) (EC); (-)-Epicatechin Gallate (>98% (HPLC), CAS

76

1257-08-5) (ECG); (-)-Catechin Gallate (>98% (HPLC), CAS 130405-40-2) (CG); (-)-

77

Epigallocatechin (>95.0% (HPLC), CAS 970-74-1) (EGC); (-)-Gallocatechin (>98%

78

(HPLC), CAS 3371-27-5) (GC); (-)-Gallocatechin Gallate (>98% (HPLC), CAS 4233-

79

96-9) (GCG); (-)-Epigallocatechin Gallate (>95.0% (HPLC), CAS 989-51-5) (EGCG);

80

formic acid (>98%, CAS 64-18-6) and acetic acid (>99%, CAS 64-19-7) were all

81

supplied by Sigma-Aldrich Química S.A. Methanol (high-performance liquid

82

chromatography (HPLC) grade) CAS 67-56-1 was provided by Scharlab (Mollet del

83

Vallés, Spain). Ultrapure water was obtained from a Millipore Milli-QPLUS 185 system

84

(Madrid, Spain).

85

An individual solution of each catechin and caffeine standards of 50 µg/g each in

86

methanol was used for the study. For building the calibration curve, a mixture of

87

standards from caffeine and eight catechins with concentrations ranging between 1 ng/g

88

and 75 µg/g in methanol was prepared. A 50 µg/g solution of GTE was also prepared in

89

methanol. The solution was filtered through a syringe filter of 0.22 µm pore size (KX

90

Syringe Filter, 25mm, 0.22 µm Nylon, Kinesis, UK) prior to injection.

91

Green tea extract and polymeric active films

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 35

92

Green tea extract Sunphenon 90 MB (GTE) was supplied by TAIYO Europe

93

(Filderstadt, Germany) and it contained around 75% total catechins (w/w), according to

94

the HPLC determination provided by the supplier company.

95

The active packaging was manufactured and supplied by the Spanish company

96

ARTIBAL S.A. (Sabiñánigo, Spain). It consisted of a solvent base coating layer

97

(varnish) with a constant concentration of GTE (1% of green tea extract in the coating

98

solution) applied on a plastic film of polyethylene terephthalate (PET). The system was

99

under the EU patent EP1477519-A1.31 The coating varnish is approved for food contact.

100

Active films contained the active substance expressed as percentage of active

101

agent/weight active layer and the grammage of the material was 3.0 g/m2. The PET film

102

was 23µm thick with a density of 18.73 ± 0.02 g/m2. Coated films without GTE were

103

used as blanks.

104

UPLC-MS/Q-TOF for the analysis of catechins and caffeine standards

105

Chromatography was carried out in an Acquity TM system using an Acquity UPLC

106

BEH C18 column of 1.7µm particle size (2.1 mm x 100 mm), both from Waters

107

(Milford, MA, USA). Chromatography was carried out at 0.3 mL/min column flow and

108

the column temperature was 35 ºC. The solvents used as mobile phase were water with

109

0.1% formic acid (eluent A) and methanol with 0.1% formic acid (eluent B). The

110

gradient used was 0-6 min, 5% B; 6-8 min, 95% B; 8-10 min, 5% B. The volume of

111

sample injected was 5 µL.

112

Eluting compounds were detected by a time-of-flight mass spectrometer (TOF) LCT

113

Premier XE from Waters (Milford, MA, USA) with an electrospray probe in positive

114

mode (ESI+) and in negative mode (ESI-) in W mode. Cone voltages were optimized 6

ACS Paragon Plus Environment

Page 7 of 35

Journal of Agricultural and Food Chemistry

115

between 20 and 50 V. Finally, 30 V was selected for the analysis because all catechins

116

peaks were detected. Other MS parameters were as follows: the desolvation gas flow

117

600 L/h, the desolvation gas temperature 450 ºC and the source temperature was 120 ºC.

118

The MS range acquired was 50-1200 Da.

119

MassLynx (v. 4.1) software (Waters, Milford, MA, USA) was used to acquire and

120

process the chromatographic and MS data.

121

UPLC-MS/TQ for the analysis of GTE and for the migration tests

122

A system consisting of an Acquity TM Ultra Performance LC TQ detector (triple

123

quadrupole; Waters, Milford, MA, USA) was used for the analysis. An electrospray

124

(ESI) probe was used in positive (ESI+) and in negative (ESI-) as the ionization source,

125

and MassLynx (v. 4.1) software (Waters, Milford, MA, USA) was used to acquire and

126

process the chromatographic and MS data.

127

Chromatography was carried out in the Acquity system using an Acquity UPLC BEH

128

C18 column of 1.7µm particle size (100 mm x 2.1 mm) from Waters (Milford, MA,

129

USA). Catechins and caffeine were separated under the following conditions: the flow

130

rate was 0.3 mL/min; the injection volume was 10 µL; the column temperature was 35

131

ºC; the solvents used as mobile phase were water with 0.1% formic acid (eluent A) and

132

methanol with 0.1% formic acid (eluent B) and the gradient used was 0-6 min, 5% B; 6-

133

8 min, 95% B; 8-10 min, 5% B.

134

Eluting compounds were detected and quantified by MS in both positive and negative

135

modes under the following ionization conditions: the capillary voltage was ±3.50 kV;

136

the source temperature was 120 ºC; the desolvation gas temperature was 450 ºC; the

137

cone gas flow was 40 L/h and the desolvation gas flow was 450 L/h. The cone voltage 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 35

138

selected was 30 V. The compounds were detected in SIR mode and the m/z ratios

139

selected were: 289.07 (C and EC, ESI-); 305.07 (EGC and GC, ESI-); 441.08 (ECG and

140

CG, ESI-); 457.08 (EGCG and GCG, ESI-) and 195.08 (Caffeine, ESI+).

141

Migration tests

142

For migration experiments, a 6 cm x 12 cm piece of active plastic film was immersed in

143

a 20 mL simulant solution. The simulants used for the migration test were: Milli-Q

144

water (simulant A from Directive 2002/72

145

(simulant B). The solutions were kept in an oven at 70 ºC for 2h. Finally, the solutions

146

were analyzed by UPLC-MS/TQ using the same chromatographic method previously

147

described. The concentration of GTE in the active plastic films was 1% in the coating

148

solution. Plastic films without GTE were used as blanks. All these samples were

149

prepared in triplicate. The migration values were expressed as µg compound per kg

150

food simulant. The migration values were corrected taking into account the proportion

151

of laminate/food simulant used in these experiments (72 cm2 laminate/20 mL simulant)

152

and those established in the EU Regulation 10/2011 on plastic materials (6 dm2

153

laminate/1 kg food simulant).33

154

Spectroscopic analysis of catechins and caffeine standards. The continuous

155

variations method (Job’s method)

156

A 0.09 µM solution of catechin standard and 0.09 µM solution of epicatechin standard

157

were prepared in methanol. A 0.13 µM solution of caffeine was also prepared in

158

methanol. Five solutions were prepared and mixed to give solutions of mole fraction (X)

159

of catechins solution varying from 0 to 1. Specifically, the different molar fraction

160

solutions prepared were 0, 0.25, 0.5, 0.75 and 1. The measurements were carried out in

32

) and 3% acetic acid in purified water

8

ACS Paragon Plus Environment

Page 9 of 35

Journal of Agricultural and Food Chemistry

161

a 1 cm quartz cell and the volume of the final mixture was 3 mL. The absorbance of the

162

mixtures was measured at 279 nm with a UV-1700 PharmaSpec UV-Vis

163

spectrophotometer (Shimadzu, Japan). All the measurements were performed in

164

triplicate.

165

Computer simulation of complexes between caffeine and catechins

166

Chem Draw & Chem 3D Pro 12.0 (Cambridge Soft Corporation, Cambridge, MA,

167

USA) was the software used to simulate the complexes between caffeine and catechins.

168

This software is a powerful tool for producing a nearly unlimited variety of biological

169

and chemical drawings and can generate, operate, calculate and predict realistic

170

molecular structures and associated properties such as energies involved. Using Chem

171

3D Pro energy calculations with MM2 force field can be carried out.34 MM2 methods

172

include: (1) Energy Minimization for locating stable conformation (global minimum);

173

(2) Molecular Dynamics for studying molecular motion of atoms and (3) Compute

174

Properties for reporting the total steric energy (TSE) in a current conformation of a

175

model.35

176

To optimize the model measurements, the Minimum RMS Gradient was fixed as 0.100,

177

which was a reasonable compromise between accuracy and speed of calculations and

178

afforded results close to a global minimum value of energy. The step interval, which

179

determines the time between molecular dynamics steps, was fixed as 2 fs and the frame

180

interval value, which determines the interval at which frames and statistics are collected,

181

was 10 fs. These values provided short periods of analysis. The Heating/Cooling Rate

182

was approximately 1.0 kcal/atom/picoseconds because minimally disturbed the

183

trajectory of atoms. Finally, the Target Temperature was 300 Kelvin. The simulation is

184

terminated when the target temperature is reached. 9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 35

185

Results and discussion

186

Migration test

187

Catechins and caffeine are the major ingredients in GTE. These compounds are known

188

for their antioxidant properties and can be incorporated into packaging materials in

189

order to protect foodstuff. In this work, active plastic films containing 1% green tea

190

extract in the coating formula were evaluated. The migration values of catechins and

191

caffeine were studied in two different food simulants. The most relevant analytical

192

parameters for UPLC-MS/TQ are shown in Table 1. Good results were obtained in

193

terms of limit of detection (LOD), limit of quantification (LOQ) and reproducibility.

194

The LOD values were between 0.02 µg/kg (caffeine) and 2.90 µg/kg (GC). In fact, the

195

stereoisomers GC and EGC were the catechins with the lowest LOD. The RSD values

196

were below 4%. The linear ranges obtained were calculated with at least five calibration

197

points and the results varied from 0.09 µg/kg to 52.34 µg/kg for catechins. For caffeine,

198

the linear range obtained was from 0.07 µg/kg to 24.56 µg/kg.

199

Table 2 shows the migration values of catechins and caffeine found in the migration

200

experiments for both simulants. The migration values were calculated for all catechins

201

and caffeine except for GC and EGC, which values were not detected in any simulant.

202

Caffeine exhibited the highest migration value for simulant A and simulant B,

203

548.27±4.33 µg/kg and 1107.51±5.65 µg/kg, respectively. Approximately, the

204

migration value of caffeine in simulant B was twice the migration value in simulant A.

205

Catechins showed migration values from 3.94±0.16 to 18.76±0.04 µg/kg for simulant A

206

and from 13.30±2.34 to 213.80±6.76 µg/kg for simulant B. It can be seen that the

207

migration values for all green tea components took place in a major extension for

208

simulant B. This fact can be related with the solubility of the catechins and caffeine in 10

ACS Paragon Plus Environment

Page 11 of 35

Journal of Agricultural and Food Chemistry

209

the different simulants. For simulant B, which was 3% acetic acid in purified water, the

210

catechins and caffeine can be easily protonated and therefore the solubility of these

211

compounds from the plastic material is increased. However, for simulant A, which was

212

Milli-Q water, the solubility was not so favorable, as the molecules are not protonated

213

in water and therefore the migration values were lower than in simulant B.

214

On the other hand, the cis isomers (EC, ECG, and EGCG) exhibited higher migration

215

values than the trans isomers (C, CG and GCG). This fact can be related with the initial

216

concentration of each catechin in the GTE (Table 3, Column 2). For all cis isomers, the

217

initial concentration was higher than the initial concentration of the trans isomers. As a

218

consequence, the migration of the cis isomers such as EC, ECG and EGCG from the

219

material was higher than that for the trans isomers.

220

Migration values of catechins and caffeine were higher for the acidic food simulant

221

(simulant B) than for water. To understand the behavior of green tea components,

222

several experiments based on the influence of pH on the catechins and caffeine

223

standards as well as on the green tea extract were investigated.

224

Influence of acidic media in catechins and caffeine standards and in GTE

225

The high increase of caffeine is of concern, as this compound would be incorporated

226

into the food. The migration tests showed a considerable influence of pH in the specific

227

migration values of catechins and caffeine. To understand better the release of free

228

caffeine and some catechins a study in depth was carried out. Firstly, an acidic media

229

(formic acid) was added to several standards solutions of tea catechins and caffeine to

230

evaluate the influence of the media in the tea components. Different stereoisomer

231

mixtures of pure catechins were prepared and caffeine was added to each catechin 11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 35

232

mixture. Specifically, the mixtures prepared in methanol were as follows: C, EC and

233

caffeine; GC, EGC and caffeine; ECG, CG and caffeine; EGCG, GCG and caffeine. All

234

these mixtures were prepared in equimolecular concentrations of 50 µg/g each one

235

(1:1:1). The samples were analyzed by UPLC-MS/Q-TOF in absence of formic acid and

236

after the addition of 5% formic acid. The area values were calculated in both cases and

237

the differences observed in the different mixtures were expressed as % of area increase.

238

Figure 1 shows the results obtained for these experiments. The combination of C, EC

239

and caffeine (1:1:1) was the mixture that experimented the major change in the % of

240

area increase. After the addition of formic acid, the area of C increased 15.83%, the area

241

of EC increased 13.41% and the area of caffeine increased 14.45%. The proportion of

242

C, EC and caffeine increase was about the same and it can be related to the

243

concentration of each standard in the mixture. The % area increase for the rest of

244

mixtures was almost unchanged. In fact, the % area increase for the different mixtures

245

was as follows: GC (1.89%), EGC (0,58%) and caffeine (0,41%); ECG (0,56%), CG

246

(0,30%) and caffeine (0,06%); EGCG (0,89%), GCG (0,16)% and caffeine (0,04%).

247

After the analysis of the different standard mixtures, the influence of the acidic media

248

was evaluated in the GTE. GTE contains the eight catechins and caffeine previously

249

studied as standards, but not as equimolecular proportions. A 50µg/g solution of GTE in

250

methanol was prepared in triplicate. The chromatographic analysis was carried out by

251

UPLC-MS/TQ before and after the addition of 5% formic acid. This technique allowed

252

us to quantify the variation of the different catechins and caffeine when formic acid was

253

added. The results are shown in Table 3, where the second column lists the initial

254

concentration of each green tea component before the addition of formic acid. Caffeine

255

showed an initial concentration value of 4.10 µg/g. As can be seen, the proportion of 12

ACS Paragon Plus Environment

Page 13 of 35

Journal of Agricultural and Food Chemistry

256

catechins was not equimolecular, being the cis isomers EC (3.13 µg/g), EGC (3.10

257

µg/g), ECG (2.35 µg/g) and EGCG (29.62 µg/g) the most concentrated compounds in

258

the GTE. The trans isomers showed initial concentration values below 2.18 µg/g (GC).

259

The last column in Table 3 shows the concentration increase of the different catechins

260

and caffeine after addition of formic acid, expressed as percentage. Again, C (29.90%),

261

EC (20.75%) and caffeine (44.93%) exhibited the major change. The other catechins

262

exhibited a variation below 2.39% (CG). In all cases the percentage of relative standard

263

deviation was below 5%.

264

From the results obtained for the standards by UPLC-MS/Q-TOF and for GTE by

265

UPLC-MS/TQ, it can be concluded that caffeine, C and EC increased their

266

concentration as a consequence of the addition of formic acid. According to the

267

literature, C forms a 1:1 complex with caffeine by intermolecular hydrogen bonds

268

well as EC, which forms also a 1:1 complex.21 It is known that caffeine behaves as a

269

very feeble base and reacts with acids. Experimental methods have explored the most

270

stable protonated structure corresponding to the most basic site in the molecule and the

271

structure protonated from the N7 site (Fig. 2), MH+ (N7) was the most stable one among

272

the ions studied.36 From this bibliography, it can be concluded that in absence of formic

273

acid, C and EC can exist as C-caffeine complex and EC-caffeine complex, which

274

present intermolecular interactions between molecules. However, in presence of formic

275

acid, the molecules of caffeine can be protonated as quaternary ammonium salts and

276

consequently, the intermolecular interactions present in the complexes between C and

277

caffeine and the complexes of EC and caffeine can be broken. As a result of this

278

breakdown, molecules of C, EC and caffeine can be released as individual free

279

molecules and their concentrations increase. Furthermore, the sum of % concentration

22

as

13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 35

280

increase for C and EC was approximately the same percentage value obtained for

281

caffeine. These results suggest that for each molecule of caffeine liberated, one

282

molecule of C or one molecule of EC will be liberated after the breakdown of the C-

283

caffeine complex or EC-caffeine complex. This conclusion can be supported by the

284

stoichiometry of the C-caffeine (1:1) or EC–caffeine (1:1) complexes proposed by

285

Ishizu et al 21,22 This fact is interesting and opens new ways to trap active compounds by

286

chemical interaction with catechins.

287

Spectroscopic analysis of catechins and caffeine standards. The continuous

288

variations method (Job’s method)

289

To confirm the formation of complexes between catechin and epicatechin with caffeine

290

and their stoichiometry, a spectroscopic analysis based on the continuous variations

291

method has been carried out. This method is often referred to as Job’s method

292

is an easy and common method for the determination of the reactant stoichiometry of

293

chemical equilibrium. In this method, the measured concentration of the complex

294

between catechin or epicatechin with caffeine (or a parameter that is proportional to its

295

concentration such as its UV/vis maximum absorbance) is plotted against the mole

296

fraction of the catechin or epicatechin solution reactant while the sum of the reactants

297

concentrations (catechin solution plus caffeine solution) is kept constant. This plot is

298

named to as Job’s plot. Figure 3 shows the Job’s plots of the C-caffeine system (dashed

299

line) and the EC-caffeine system (continuous line). Five solutions of catechin derivate

300

and caffeine were measured at different mole fractions, from 0 to 1 with a constant

301

concentration of 0.03 µM. The maximum point of the curve, which corresponds to the

302

maximum concentration of the catechin caffeine complex, determined the stoichiometry

303

of equilibrium reaction. From this curve, the stoichiometry obtained for the C-caffeine

36

and it

14

ACS Paragon Plus Environment

Page 15 of 35

Journal of Agricultural and Food Chemistry

304

complex was 1:1 and the absorbance value at this point was 0.518. In the case of EC-

305

caffeine complex, the stoichiometry of the complex was also 1:1 and the absorbance

306

value was 0.556. Error bars showed the relative standard deviation expressed as

307

percentage (% RSD) and % RSD was below to 2% for all measures.

308

The continuous variations method demonstrated that the complex between catechin and

309

epicatechin with caffeine is formed in equimolecular proportions (1:1). These results

310

confirm the conclusions given after the UPLC analysis of the green tea standards and

311

the green tea extract. UPLC and spectrophotometric analysis confirm that catechin and

312

epicatechin form complexes with caffeine by intermolecular interactions and these

313

complexes are formed in 1:1 complex association.

314

An approach: Simulation of complexes of tea catechins with caffeine

315

According to the results presented above, C and EC formed a complex with caffeine by

316

intermolecular interactions and this fact agrees with the previous bibliography.21,22

317

Spectroscopic analysis also demonstrated that these complexes are formed in 1:1

318

complex association, catechin-caffeine complex and epicatechin-caffeine complex. The

319

increase of the concentration value of free caffeine in presence of formic acid means

320

that caffeine was protonated as a quaternary ammonium salt. Consequently, the

321

complexes between C and EC with caffeine can be broken and the different molecules

322

can be liberated, thus increasing their concentrations. An approach of a molecular

323

modeling was proposed to confirm the results experimentally obtained. Chem 3D Pro

324

software was selected as the molecular modeling to perform this study.

325

The structures of C, EC, caffeine, protonated caffeine (Fig. 2) and the complexes of C

326

and EC with caffeine based on the structures determined by Ishazu et al.21,22 (Fig.4) 15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 35

327

were drawn by Chem Draw. On the other hand, the protonated complexes of C and EC

328

with caffeine were also drawn by Chem Draw software (Fig.4). The protonated site of

329

caffeine was the N7 site according to literature 37. The hydrogen bond is represented by

330

a dashed line in all structures (Fig.4).

331

Several dihedral angles of the catechin-caffeine complexes were calculated by Chem 3D

332

Pro and the values obtained are shown in Table 4. The dihedral angle values calculated

333

to protonated and non-protonated structures were the same. Therefore, Table 4 shows

334

the dihedral angle values of non-protonanted structures. The dihedral angle values

335

obtained by the software were compared with the values determined by Ishazu et al.21,22

336

and they exhibited high similarity. Therefore, the results afforded by the software could

337

be considered sufficiently reliable.

338

Table 5 shows the computed properties of C, EC, caffeine, protonated caffeine and the

339

different complexes, non-protonated and protonated, between C and EC with caffeine

340

after the geometry optimization. For this, MM2 calculations were carried out by Chem

341

3D Pro software. The protocol of the procedure was as follows: (1) To calculate the

342

computed properties of the structures proposed before the geometry optimization.

343

Different energy values were given such as stretch, bend, torsion, van der Waals,

344

dipole-dipole and charge-dipole. The energy values calculated for C, EC, caffeine and

345

protonated caffeine were the best energy values obtained after the MM2 job. However,

346

the energy values obtained for all complexes were high TSE for the current

347

conformation and these results indicated that the geometry of these complexes was not

348

optimized. The TSE values of complexes were approximately 2000 kcal/mol for all

349

cases. (2) To obtain the geometry optimization, which corresponds to a minimum

350

energy point, an energy minimization job was performed for each complex. For all 16

ACS Paragon Plus Environment

Page 17 of 35

Journal of Agricultural and Food Chemistry

351

structures evaluated, the TSE of the structures was much lower after geometry

352

optimization. (3) A molecular dynamic job was performed to simulate the motion of the

353

forces acting on the atoms. (4) Finally, a second computed properties job was performed

354

after the energy minimization and molecular dynamic jobs. At this point, the total steric

355

energy value was the best value obtained by running the MM2 methodology, which

356

corresponded to the minimum energy value for the complex studied, and therefore the

357

conformation that was more likely to exist.

358

These TSE values as well as the energy values of stretch, bend, torsion, van der Waals,

359

dipole-dipole and charge-dipole, are shown in Table 5 for the different structures. The

360

aim of this simulation was to compare the main differences between non-protonated and

361

protonated complexes. For this purpose, the conclusions of this study were based on the

362

total steric energy values. First, the sum of individual TSE values of C and caffeine

363

(14.6584 kcal/mol) was compared to the TSE value of non-protonated C-caffeine

364

complex (13.9091 kcal/mol). Similarly, the sum of individual TSE values of EC and

365

caffeine (16.5743 kcal/mol) was compared to the TSE value of the non-protonated EC-

366

caffeine complex (14.4597 kcal/mol). In both cases, the TSE values were lower for the

367

C and EC complexes and it was concluded that both C and EC were more likely to form

368

caffeine complexes. With respect to the protonation of caffeine, the sum of the

369

individual TSE values of C and protonated caffeine (21.5857 kcal/mol) was compared

370

to the TSE value of protonated C-caffeine complex (22.5772 kcal/mol) and the sum of

371

the individual TSE values of EC and protonated caffeine (23.5016 kcal/mol) was

372

compared to the TSE value of protonated EC-caffeine complex (23.8679kcal/mol).

373

These differences showed that the protonated complexes of C and EC were less likely to

374

exist than the molecules not arranged. 17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 35

375

The differences of energy between non-protonated and protonated complexes were also

376

compared. The TSE obtained for C-caffeine (13.9091 kcal/mol) and EC-caffeine

377

(14.4597 kcal/mol) non-protonated complexes was lower than the values obtained for

378

C-caffeine (22.5772 kcal/mol) and EC-caffeine (23.8679 kcal/mol) protonated

379

complexes. Once again, these results suggested that the non-protonated complexes were

380

more likely to exist compared to the protonated complexes.

381

The simulation data showed that protonated complexes were less stable than non-

382

protonated complexes and these results consolidated the idea that the intermolecular

383

hydrogen bond among catechins-caffeine complexes can be broken giving individual

384

molecules of C, EC and the ammonium salt of caffeine, in order to establish the most

385

stable conformation. Both, the experimental work and the simulation studio concluded

386

that C and EC form equimolecular complexes with caffeine in neutral media. However,

387

the presence of an acidic media involves the breakdown of the intermolecular

388

interactions between molecules giving C, EC and the ammonium salt of caffeine as free

389

molecules.

390

This work recovers important information about the green tea components and their

391

behavior depending on the pH media. The starting migration analysis of active

392

packaging films containing green tea extract showed unexpected migration values for

393

3% acetic acid simulant. Specifically, the migration values in acidic simulant were more

394

than double of those migration values for aqueous simulant. To explain these results, a

395

detailed UPLC analysis of green tea standards as well as green tea extract was

396

performed. From the chromatographic results obtained and the previous reported

397

literature, the formation of equimolecular complexes between catechins and caffeine

398

through intermolecular interactions is proposed. The complexes can exist in neutral 18

ACS Paragon Plus Environment

Page 19 of 35

Journal of Agricultural and Food Chemistry

399

media but can be broken in acidic media as a consequence of the protonation of

400

caffeine. A spectroscopic analysis based on the continuous variations method, also

401

named as Job’s method, was carried out to verify the stoichiometry of the C-caffeine

402

complex and EC-caffeine complex proposed. The results obtained for the Job’s method

403

confirm that these complexes are formed in 1:1 complex association. Finally, a

404

simulation studio based on the stabilization energies of the protonated and non-

405

protonated catechin complexes was performed to confirm the conclusions achieved

406

from the experimental work. The simulation showed that the total steric energies of the

407

non-protonated complexes are higher than the protonated complexes and therefore more

408

stable to exist. The conclusions reached from the simulation studio support the

409

information given by the experimental analysis.

410

411

412

413

414

415

416

417

418

419 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 35

420

References

421

1.

422

major green tea polyphenol, (-)-epigallocatechin-3-gallate, inhibits obesity, metabolic

423

syndrome, and fatty liver disease in high-fat-fed mice. J. Nutr. 2008, 138, 1677-1683.

424

2.

425

induced obesity by a combination of tea-catechin intake and regular swimming. Int. J.

426

Obes. 2006, 30, 561-568.

427

3.

428

Possible mechanism and antibacterial activity on skin pathogens. Food Chem. 2012,

429

135, 672-675.

430

4.

431

Tsuchiya, T. Marked reduction in the minimum inhibitory concentration (MIC) of beta-

432

lactams in methicillin-resistant Staphylococcus aureus produced by epicatechin gallate,

433

an ingredient of green tea (Camellia sinensis). Biol. Pharm. Bull. 1999, 22, 1388-1390.

434

5.

435

from animal studies. J. Nutr. 2003, 133, 3275-3284.

436

6.

437

ORAC and DPPH assay comparison to assess antioxidant capacity of tea infusions:

438

Relationship between total polyphenol and individual catechin content. Int. J. Food Sci.

439

Nutr. 2010, 61, 109-124.

440

7.

441

931-937.

Bose, M.; Lambert, J. D.; Ju, J.; Reuhl, K. R.; Shapses, S. A.; Yang, C. S. The

Murase, T.; Haramizu, S.; Shimotoyodome, A.; Tokimitsu, I. Reduction of diet-

Sharma, A.; Gupta, S.; Sarethy, I. P.; Dang, S.; Gabrani, R. Green tea extract:

Shiota, S.; Shimizu, M.; Mizushima, T.; Ito, H.; Hatano, T.; Yoshida, T.;

Frei, B.; Higdon, J. V. Antioxidant activity of tea polyphenols in vivo: Evidence

Roy, M. K.; Koide, M.; Rao, T. P.; Okubo, T.; Ogasawara, Y.; Juneja, L. R.

Chung, S. Y.; Xin, W. Green tea and cancer prevention. Nutr. Cancer 2010, 62,

20

ACS Paragon Plus Environment

Page 21 of 35

Journal of Agricultural and Food Chemistry

442

8.

Singh, B. N.; Shankar, S.; Srivastava, R. K. Green tea catechin,

443

epigallocatechin-3-gallate (EGCG): Mechanisms, perspectives and clinical applications.

444

Biochem. Pharm. 2011, 82, 1807-1821.

445

9.

446

studies. Pharm. Res. 2011, 64, 123-135.

447

10.

448

occurring pesticides. Science 1984, 226, 184-187.

449

11.

Glade, M. J. Caffeine-Not just a stimulant. Nutrition 2010, 26, 932-938.

450

12.

Perva-Uzunalic, A.; Skerget, M.; Knez, Z.; Weinreich, B.; Otto, F.; Gruner, S.

451

Extraction of active ingredients from green tea (Camellia sinensis): Extraction

452

efficiency of major catechins and caffeine. Food Chem. 2006, 96, 597-605.

453

13.

454

Association of polyphenols with caffeine and alpha-cyclodextrin and beta-cyclodextrin

455

in aqueous-media. Chem. Commun. 1986, 2, 107-109.

456

14.

457

complex of coffee. J. Food Sci. 1972, 37, 925-927.

458

15.

459

D. The caffeine potassium chlorogenate molecular-complex. Phytochemistry 1987, 26,

460

273-279.

461

16.

462

spectroscopic and computer-graphics studies on the creaming down of tea. Int. Symp.

463

Tea Sci. 1991, 145-149.

464

17.

465

M.; Haslam, E. Polyphenol Interactions. 4. Model studies with caffeine and

466

cyclodextrins. J. Chem. Soc., Perkin Trans. 2 1990, 12, 2197-2209.

Yuan, J. M.; Sun, C.; Butler, L. M. Tea and cancer prevention: Epidemiological

Nathanson, J. A. Caffeine and related methylxanthines -possible naturally-

Gaffney, S. H.; Martin, R.; Lilley, T. H.; Haslam, E.; Magnolato, D. The

Horman, I.; Viani, R. Nature and conformation of caffeine-chlorogenate

Martin, R.; Lilley, T. H.; Falshaw, C. P.; Haslam, E.; Begley, M. J.; Magnolato,

Maruyama, N.; Suzuki, Y.; Sakata, K.; Yagi, A.; Ina, K.; Duke, C. C. NMR

Cai, Y.; Gaffney, S. H.; Lilley, T. H.; Magnolato, D.; Martin, R.; Spencer, C.

21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 22 of 35

467

18.

Hayashi, N.; Ujihara, T.; Kohata, K. Binding energy of tea catechin/caffeine

468

complexes in water evaluated by titration experiments with H-1-NMR. Biosci.

469

Biotechnol. Biochem. 2004, 68, 2512-2518.

470

19.

471

caffeine in crystal structure of 1:2 and 2:2 complexes. Tetrahedron Lett. 2009, 50, 4121-

472

4124.

473

20.

474

complex of gallocatechin gallate and caffeine. Chem. Lett. 2009, 38, 230-231.

475

21.

476

determination of caffeine complexes with galloylated and non-galloylated catechins.

477

Chem. Lett. 2010, 39, 607-609.

478

22.

479

complexes of various tea catechins and caffeine in crystal state. Chem. Pharm. Bull.

480

2011, 59, 1008-1015.

481

23.

482

Configurational studies of complexes of tea catechins with caffeine and various

483

cyclodextrins. Planta Med. 2011, 77, 1099-1109.

484

24.

485

Munoz, P. Development of new antioxidant active packaging films based on ethylene

486

vinyl alcohol copolymer (EVOH) and green tea extract. J. Agric. Food Chem. 2011, 59,

487

7832-7840.

488

25.

489

inhibition of lipid oxidation. Food Sci. Biotechnol. 2003, 12, 737-746.

Ishizu, T.; Tsutsumi, H.; Sato, T. Interaction between gallocatechin gallate and

Ishizu, T.; Tsutsumi, H.; Sato, T.; Yamamoto, H.; Shiro, M. Crystal structure of

Ishizu, T.; Sato, T.; Tsutsumi, H.; Yamamoto, H. Stereochemical structure

Tsutsumi, H.; Kinoshita, Y.; Sato, T.; Ishizu, T. Configurational studies of

Ishizu, T.; Kajitani, S.; Tsutsumi, H.; Sato, T.; Yamamoto, H.; Hirata, C.

De Dicastillo, C. L.; Nerin, C.; Alfaro, P.; Catala, R.; Gavara, R.; Hernandez-

Shin, H. S.; Lee, Y. Antioxidant-impregnated food packaging materials for

22

ACS Paragon Plus Environment

Page 23 of 35

Journal of Agricultural and Food Chemistry

490

26.

Vargas, M.; Pastor, C.; Chiralt, A.; McClements, D. J.; Gonzalez-Martinez, C.

491

Recent advances in edible coatings for fresh and minimally processed fruits. Crit. Rev.

492

Food Sci. Nutr. 2008, 48, 496-511.

493

27.

494

film containing green tea, green coffee, and grapefruit extracts. J. Agric. Food Chem.

495

2012, 60, 9842-9849.

496

28.

497

films for active packaging materials. Int. J. Biol. Macromol. 2013, 59, 282-289.

498

29.

499

Natural additives in bioactive edible films and coating: Funcionality and applications in

500

foods. Food Eng. Rev. 2013, 5, 200-216.

501

30.

502

analysis of non-volatile migrants from new active packaging materials. Anal. Bioanal.

503

Chem. 2012, 404, 1945-1957.

504

31.

Garces, O.; Nerin C.; Beltran, J. A.; Roncales, P. EU patent EP1477519-A1.

505

32.

Commission Directive (EU) No 72/2002 of 6 August 2002 relating to plastic

506

materials and articles intended to come into contact with foodstuffs.

507

33.

508

materials and article intended to come into contact with food.

509

34.

510

York 2002, 14, 285-314.

511

35.

512

MA, USA 2005.

513

36.

514

1928, 9, 113-203.

Colon, M.; Nerin, C. Role of Catechins in the antioxidant capacity of an active

Peng, Y.; Wu, Y.; Li, Y. Development of tea extracts and chitosan composite

Silva-Weiss, A.; Ihl, M.; Sobral, P. J. A.; Gomez-Guillen, M. C.; Bifani, V.

Aznar, M.; Rodriguez-Lafuente,A.;Alfaro, P.; Nerin, C. UPLC-Q-TOF-MS

Commission Regulation (EU) No 10/2011 of 14 January 2011 on plastic

Stan, T. C. An introduction to computational biochemistry. Wiley-Liss, Inc., New

Office, C. Chem 3D User´s Manual. Cambridge Soft Corporation, Cambridge,

Job, P. Formation and stability of inorganic complexes in solution. Ann. Chim.

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

515

37.

516

theoretical and experimental study. Chem. Phys. 2013, 415, 222-227.

Page 24 of 35

Bahrami, H.; Tabrizchi, M.; Farrokhpour, H. Protonation of caffeine: A

517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533

This research has been financed by the Project INNPACTO 2010/0486 from the

534

MICINN, Ministerio de Ciencia e Innovación, Spain. The authors also thank the Project

535

Gobierno de Aragón and European Social Funds for financing the research group

536

GUIA-T-10.

537

24

ACS Paragon Plus Environment

Page 25 of 35

Journal of Agricultural and Food Chemistry

FIGURE CAPTIONS

Figure 1. Percentage of Area Increase of Different Equimolecular Mixtures of Stereoisomers of Catechins and Caffeine (1:1:1) After Addition of 5% Formic Acid.

Figure 2. Structure of Caffeine, (+)-Catechin and (-)-Epicatechin.

Figure 3. The Job’s Plots of C-Caffeine System (Dashed Line) and EC-Caffeine System (Continuous Line).

Figure 4. Structure of Non-Protonated and Protonated C-Caffeine and EC-Caffeine Complexes.

25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 26 of 35

TABLES

Table 1. Analytical Features of the UPLC-MC/TQ Method.

Linear range

LOD

LOQ

RSD

(µg/kg)

(µg/kg)

(µg/kg)

(%, n=3)

(+)-C

0.22-52.34

0.07

0.22

2.60

(-)-EC

0.09-52.34

0.03

0.09

3.37

(-)-GC

9.66-52.34

2.90

9.66

1.41

(-)-EGC

4.70-52.34

1.41

4.70

1.14

(-)-ECG

0.95-52.34

0.29

0.95

2.90

(-)-CG

0.44-52.34

0.13

0.44

1.25

(-)-EGCG

0.29-52.34

0.09

0.29

3.29

(-)-GCG

0.47-52.34

0.14

0.47

2.27

Caffeine

0.07-24.56

0.02

0.07

1.05

Name

26

ACS Paragon Plus Environment

Page 27 of 35

Journal of Agricultural and Food Chemistry

Table 2. Migration Values of Catechins and Caffeine in Two Aqueous Simulants by UPLC-MS/TQ.

Simulant Milli-Q

Simulant 3% acetic

water

acid in water

(µg/kg)

(µg/kg)

(+)-C

10.44±0.76

22.52±0.98

(-)-EC

18.76±0.04

82.12±0.87

(-)-GC

NDa

NDa

(-)-EGC

NDa

NDa

(-)-ECG

15.46±1.02

160.52±4.51

(-)-CG

5.79±0.45

13.30±2.34

(-)-EGCG

4.20±1.08

213.80±6.76

(-)-GCG

3.94±0.16

16.17±0.43

Caffeine

548.27±4.33

1107.51±5.65

Name

a

ND: not detected

27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 28 of 35

Table 3. Concentration Values of Catechins and Caffeine of Green Tea Extract in Absence (without H+) and Presence (5% H+) of Formic Acid.

[ ] µg/g,

%RSD (n=3)

[ ] µg/g,

%RSD (n=3)

Concentration

Name +

+

without H

without H

5% H

(+)-C

0.18

1.31

(-)-EC

3.13

(-)-GC

+

+

5% H

increase, (%)

0.25

3.23

29.90

0.95

3.95

1.23

20.75

2.18

1.05

2.21

1.45

1.68

(-)-EGC

3.10

3.47

3.12

0.43

0.66

(-)-ECG

2.35

4.82

2.35

2.01

0.01

(-)-CG

0.98

1.47

1.01

0.23

2.39

(-)-EGCG

29.58

0.37

29.62

1.56

0.14

(-)-GCG

1.92

1.64

1.96

4.56

1.70

Caffeine

4.10

4.74

4.88

4.87

15.95

28

ACS Paragon Plus Environment

Page 29 of 35

Journal of Agricultural and Food Chemistry

Table 4. Comparison of Chem 3D Pro Values and Ishazu et al.21,22 Values of Several Dihedral Angles of the Complexes Between C and EC With Caffeine.

Dihedral angle

Chem 3D Pro value

Ishazu et al. value

H2-C2-C3-H3 (C-caffeine)

173.1°

169.0°

C1’-C2-C3-O (C-caffeine)

52.4°

48.7°

H2-C2-C3-H3 (EC-caffeine)

66.1°

60.23°

O1-C2-C3-H3 (EC-caffeine)

176.0 °

179.6°

29

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 30 of 35

Table 5. Energy Values of Several Parameters Calculated by Chem Pro 12.0 of Catechin, Epicatechin, Caffeine, Protonated Caffeine and Non-Protonated and Protonated C-Caffeine and EC-Caffeine Complexes.

Energy (kcal/mol)

C

EC

Protonated

Non-protonated

Protonated

Non-protonated

Protonated

Caffeine

C-Caffeine

C-Caffeine

EC-Caffeine

EC-Caffeine

Caffeine

Strech

1.0824

1.1529

0.8574

0.8763

2.2376

2.2405

2.2845

2.2357

Bend

5.4820

6.6179

22.3571

21.5206

16.2340

29.4483

16.9875

29.8070

Strech-Bend

-0.0838

-0.0048

-0.0961

-0.0124

-0.0097

-0.0664

0.0271

-0.0321

Torsion

-14.6109

-13.6778

1.9123

1.9651

-8.4744

-10.8780

-9.3759

-11.2062

Non-1,4 VDW

-12.4550

-12.3518

0.3622

1.0650

-13.6243

-5.4612

-13.7413

-5.8387

1,4-VDW

12.6950

12.2932

9.2112

9.2406

25.3927

25.5631

25.2569

25.3394

Charge/Dipole

-

-

-

6.6489

-

-5.2226

-

-4.3414

Dipole/Dipole

0.0655

0.0616

-12.1210

-11.8936

-7.8468

-13.0465

-6.9793

-12.0958

Total Steric Energy

-7.8248

-5.9089

22.4832

29.4105

13.9091

22.5772

14.4597

23.8679

30

ACS Paragon Plus Environment

Page 31 of 35

Journal of Agricultural and Food Chemistry

FIGURES

Figure 1

31

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 32 of 35

Figure 2

5` 6` B

8

OH 7

8a O

2 1`

C

A

6 5

4a

3 4

OH 4`

2`

3` OH

OH

OH

Caffeine

(+)-Catechin

(-)-Epicatechin

32

ACS Paragon Plus Environment

Page 33 of 35

Journal of Agricultural and Food Chemistry

Figure 3

33

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 34 of 35

Figure 4

Non-protonated C-caffeine complex

Non-protonated EC-caffeine complex

Protonated C-caffeine complex

Protonated EC-caffeine complex

34

ACS Paragon Plus Environment

Page 35 of 35

Journal of Agricultural and Food Chemistry

TOC GRAPHIC

LIBERATED

molecules

NON-PROTONATED complex

PROTONATED complex

35

ACS Paragon Plus Environment