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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
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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).
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Abstract
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Migration of green tea components from an active packaging material containing green
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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
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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
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Introduction
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Green tea leaves contain many characteristic compounds being catechins and caffeine
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the major ingredients of tea. Catechins are a group of polyphenols that show beneficial
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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
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acts as a central nervous system stimulant.10 However, the excessive consumption of
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caffeine can produce negative effects in the organism such as anxiety disorders.11
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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
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coffee.13-15 Many researchers have been investigating the structure of the complexes
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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
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catechin and epicatechin, the A and C rings provided a general site for caffeine
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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
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shift change and Nuclear Overhauser Effect Spectroscopy (NOESY) spectra in
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catechins and caffeine solution showed the participation of A rings of catechins in
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complexes, as well as B or Bʼ rings. All of these works mentioned16-18 were performed
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in solution using NMR techniques, but their overall structures were still unclear and the
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detailed interactions between caffeine and catechins have not been sufficiently
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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.
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gallate,
and
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gallocatechin
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intermolecular interactions by X-ray crystallographic analysis. Subsequently, they have
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investigated the crystal stereochemical structures of caffeine complexes and the detailed
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non-covalent
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Furthermore, they were focused on the inclusion complexes comprising cyclodextrins
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and catechins.23 All the crystal structures were prepared in water solution at 90 ºC and
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left at room temperature to crystallize.
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Green tea extract is generally considered a potent antioxidant that can be either used as
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direct soft drink in water, applied to the food surface or incorporated as active agent into
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polymeric packaging materials to protect the food against the oxidation process and
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extend the shelf life of packaged food.24-30 Therefore, the behavior of the green tea
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extract incorporated into an active plastic packaging has to be studied taking into
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account the formation of complexes by intermolecular interactions between the main
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components of green tea16-23, catechins and caffeine, and their behavior in different
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media.
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For this, the aims of this work were: (1) to evaluate the influence of two different food
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simulants in the migration tests from an active packaging material containing green tea
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extract, (2) to investigate in depth by liquid chromatography the variations observed in
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the concentration values of green tea components in both neutral and acidic media, (3)
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to demonstrate the formation of complexes between catechins and caffeine through
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intermolecular interactions and to confirm the formation of these complexes by the
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continuous variation method (Job’s method), which was applied to know the
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stoichiometry of the complexes. Finally, (4) to confirm the experimental results
interaction
investigated
with
their
galloylated
and
stereochemical
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non-galloylated
structures
and
catechins.21,22
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obtained in both liquid chromatography and spectroscopy studies by a computer
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simulation program.
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Materials and methods
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Reagents and solutions
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Caffeine (58-08-2); (+)-Catechin (>99.0% (HPLC), CAS 154-23-4) (C); (-)-Epicatechin
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(>95.0% (HPLC), CAS 490-46-0) (EC); (-)-Epicatechin Gallate (>98% (HPLC), CAS
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1257-08-5) (ECG); (-)-Catechin Gallate (>98% (HPLC), CAS 130405-40-2) (CG); (-)-
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Epigallocatechin (>95.0% (HPLC), CAS 970-74-1) (EGC); (-)-Gallocatechin (>98%
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(HPLC), CAS 3371-27-5) (GC); (-)-Gallocatechin Gallate (>98% (HPLC), CAS 4233-
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96-9) (GCG); (-)-Epigallocatechin Gallate (>95.0% (HPLC), CAS 989-51-5) (EGCG);
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formic acid (>98%, CAS 64-18-6) and acetic acid (>99%, CAS 64-19-7) were all
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supplied by Sigma-Aldrich Química S.A. Methanol (high-performance liquid
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chromatography (HPLC) grade) CAS 67-56-1 was provided by Scharlab (Mollet del
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Vallés, Spain). Ultrapure water was obtained from a Millipore Milli-QPLUS 185 system
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(Madrid, Spain).
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An individual solution of each catechin and caffeine standards of 50 µg/g each in
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methanol was used for the study. For building the calibration curve, a mixture of
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standards from caffeine and eight catechins with concentrations ranging between 1 ng/g
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and 75 µg/g in methanol was prepared. A 50 µg/g solution of GTE was also prepared in
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methanol. The solution was filtered through a syringe filter of 0.22 µm pore size (KX
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Syringe Filter, 25mm, 0.22 µm Nylon, Kinesis, UK) prior to injection.
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Green tea extract and polymeric active films
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Green tea extract Sunphenon 90 MB (GTE) was supplied by TAIYO Europe
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(Filderstadt, Germany) and it contained around 75% total catechins (w/w), according to
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the HPLC determination provided by the supplier company.
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The active packaging was manufactured and supplied by the Spanish company
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ARTIBAL S.A. (Sabiñánigo, Spain). It consisted of a solvent base coating layer
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(varnish) with a constant concentration of GTE (1% of green tea extract in the coating
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solution) applied on a plastic film of polyethylene terephthalate (PET). The system was
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under the EU patent EP1477519-A1.31 The coating varnish is approved for food contact.
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Active films contained the active substance expressed as percentage of active
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agent/weight active layer and the grammage of the material was 3.0 g/m2. The PET film
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was 23µm thick with a density of 18.73 ± 0.02 g/m2. Coated films without GTE were
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used as blanks.
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UPLC-MS/Q-TOF for the analysis of catechins and caffeine standards
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Chromatography was carried out in an Acquity TM system using an Acquity UPLC
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BEH C18 column of 1.7µm particle size (2.1 mm x 100 mm), both from Waters
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(Milford, MA, USA). Chromatography was carried out at 0.3 mL/min column flow and
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the column temperature was 35 ºC. The solvents used as mobile phase were water with
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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
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sample injected was 5 µL.
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Eluting compounds were detected by a time-of-flight mass spectrometer (TOF) LCT
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Premier XE from Waters (Milford, MA, USA) with an electrospray probe in positive
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mode (ESI+) and in negative mode (ESI-) in W mode. Cone voltages were optimized 6
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between 20 and 50 V. Finally, 30 V was selected for the analysis because all catechins
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peaks were detected. Other MS parameters were as follows: the desolvation gas flow
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600 L/h, the desolvation gas temperature 450 ºC and the source temperature was 120 ºC.
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The MS range acquired was 50-1200 Da.
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MassLynx (v. 4.1) software (Waters, Milford, MA, USA) was used to acquire and
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process the chromatographic and MS data.
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UPLC-MS/TQ for the analysis of GTE and for the migration tests
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A system consisting of an Acquity TM Ultra Performance LC TQ detector (triple
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quadrupole; Waters, Milford, MA, USA) was used for the analysis. An electrospray
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(ESI) probe was used in positive (ESI+) and in negative (ESI-) as the ionization source,
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and MassLynx (v. 4.1) software (Waters, Milford, MA, USA) was used to acquire and
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process the chromatographic and MS data.
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Chromatography was carried out in the Acquity system using an Acquity UPLC BEH
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C18 column of 1.7µm particle size (100 mm x 2.1 mm) from Waters (Milford, MA,
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USA). Catechins and caffeine were separated under the following conditions: the flow
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rate was 0.3 mL/min; the injection volume was 10 µL; the column temperature was 35
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ºC; the solvents used as mobile phase were water with 0.1% formic acid (eluent A) and
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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.
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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;
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the source temperature was 120 ºC; the desolvation gas temperature was 450 ºC; the
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cone gas flow was 40 L/h and the desolvation gas flow was 450 L/h. The cone voltage 7
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selected was 30 V. The compounds were detected in SIR mode and the m/z ratios
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selected were: 289.07 (C and EC, ESI-); 305.07 (EGC and GC, ESI-); 441.08 (ECG and
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CG, ESI-); 457.08 (EGCG and GCG, ESI-) and 195.08 (Caffeine, ESI+).
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Migration tests
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For migration experiments, a 6 cm x 12 cm piece of active plastic film was immersed in
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a 20 mL simulant solution. The simulants used for the migration test were: Milli-Q
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water (simulant A from Directive 2002/72
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(simulant B). The solutions were kept in an oven at 70 ºC for 2h. Finally, the solutions
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were analyzed by UPLC-MS/TQ using the same chromatographic method previously
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described. The concentration of GTE in the active plastic films was 1% in the coating
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solution. Plastic films without GTE were used as blanks. All these samples were
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prepared in triplicate. The migration values were expressed as µg compound per kg
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food simulant. The migration values were corrected taking into account the proportion
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of laminate/food simulant used in these experiments (72 cm2 laminate/20 mL simulant)
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and those established in the EU Regulation 10/2011 on plastic materials (6 dm2
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laminate/1 kg food simulant).33
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Spectroscopic analysis of catechins and caffeine standards. The continuous
155
variations method (Job’s method)
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A 0.09 µM solution of catechin standard and 0.09 µM solution of epicatechin standard
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were prepared in methanol. A 0.13 µM solution of caffeine was also prepared in
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methanol. Five solutions were prepared and mixed to give solutions of mole fraction (X)
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of catechins solution varying from 0 to 1. Specifically, the different molar fraction
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solutions prepared were 0, 0.25, 0.5, 0.75 and 1. The measurements were carried out in
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) and 3% acetic acid in purified water
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a 1 cm quartz cell and the volume of the final mixture was 3 mL. The absorbance of the
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mixtures was measured at 279 nm with a UV-1700 PharmaSpec UV-Vis
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spectrophotometer (Shimadzu, Japan). All the measurements were performed in
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triplicate.
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Computer simulation of complexes between caffeine and catechins
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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.
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This software is a powerful tool for producing a nearly unlimited variety of biological
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and chemical drawings and can generate, operate, calculate and predict realistic
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molecular structures and associated properties such as energies involved. Using Chem
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3D Pro energy calculations with MM2 force field can be carried out.34 MM2 methods
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include: (1) Energy Minimization for locating stable conformation (global minimum);
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(2) Molecular Dynamics for studying molecular motion of atoms and (3) Compute
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Properties for reporting the total steric energy (TSE) in a current conformation of a
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model.35
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To optimize the model measurements, the Minimum RMS Gradient was fixed as 0.100,
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which was a reasonable compromise between accuracy and speed of calculations and
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afforded results close to a global minimum value of energy. The step interval, which
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determines the time between molecular dynamics steps, was fixed as 2 fs and the frame
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interval value, which determines the interval at which frames and statistics are collected,
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was 10 fs. These values provided short periods of analysis. The Heating/Cooling Rate
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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
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Results and discussion
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Migration test
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Catechins and caffeine are the major ingredients in GTE. These compounds are known
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for their antioxidant properties and can be incorporated into packaging materials in
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order to protect foodstuff. In this work, active plastic films containing 1% green tea
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extract in the coating formula were evaluated. The migration values of catechins and
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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
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terms of limit of detection (LOD), limit of quantification (LOQ) and reproducibility.
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The LOD values were between 0.02 µg/kg (caffeine) and 2.90 µg/kg (GC). In fact, the
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stereoisomers GC and EGC were the catechins with the lowest LOD. The RSD values
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were below 4%. The linear ranges obtained were calculated with at least five calibration
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points and the results varied from 0.09 µg/kg to 52.34 µg/kg for catechins. For caffeine,
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the linear range obtained was from 0.07 µg/kg to 24.56 µg/kg.
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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
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and caffeine except for GC and EGC, which values were not detected in any simulant.
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Caffeine exhibited the highest migration value for simulant A and simulant B,
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548.27±4.33 µg/kg and 1107.51±5.65 µg/kg, respectively. Approximately, the
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migration value of caffeine in simulant B was twice the migration value in simulant A.
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Catechins showed migration values from 3.94±0.16 to 18.76±0.04 µg/kg for simulant A
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and from 13.30±2.34 to 213.80±6.76 µg/kg for simulant B. It can be seen that the
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migration values for all green tea components took place in a major extension for
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simulant B. This fact can be related with the solubility of the catechins and caffeine in 10
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the different simulants. For simulant B, which was 3% acetic acid in purified water, the
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catechins and caffeine can be easily protonated and therefore the solubility of these
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compounds from the plastic material is increased. However, for simulant A, which was
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Milli-Q water, the solubility was not so favorable, as the molecules are not protonated
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in water and therefore the migration values were lower than in simulant B.
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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
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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.
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Migration values of catechins and caffeine were higher for the acidic food simulant
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(simulant B) than for water. To understand the behavior of green tea components,
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several experiments based on the influence of pH on the catechins and caffeine
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standards as well as on the green tea extract were investigated.
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Influence of acidic media in catechins and caffeine standards and in GTE
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The high increase of caffeine is of concern, as this compound would be incorporated
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into the food. The migration tests showed a considerable influence of pH in the specific
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migration values of catechins and caffeine. To understand better the release of free
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caffeine and some catechins a study in depth was carried out. Firstly, an acidic media
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(formic acid) was added to several standards solutions of tea catechins and caffeine to
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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
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mixture. Specifically, the mixtures prepared in methanol were as follows: C, EC and
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caffeine; GC, EGC and caffeine; ECG, CG and caffeine; EGCG, GCG and caffeine. All
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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
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the differences observed in the different mixtures were expressed as % of area increase.
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Figure 1 shows the results obtained for these experiments. The combination of C, EC
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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
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of EC increased 13.41% and the area of caffeine increased 14.45%. The proportion of
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C, EC and caffeine increase was about the same and it can be related to the
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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
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was as follows: GC (1.89%), EGC (0,58%) and caffeine (0,41%); ECG (0,56%), CG
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(0,30%) and caffeine (0,06%); EGCG (0,89%), GCG (0,16)% and caffeine (0,04%).
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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
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showed an initial concentration value of 4.10 µg/g. As can be seen, the proportion of 12
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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
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the GTE. The trans isomers showed initial concentration values below 2.18 µg/g (GC).
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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%.
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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
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increase for C and EC was approximately the same percentage value obtained for
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caffeine. These results suggest that for each molecule of caffeine liberated, one
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molecule of C or one molecule of EC will be liberated after the breakdown of the C-
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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.
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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
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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
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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
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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
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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
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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
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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
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References
421
1.
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major green tea polyphenol, (-)-epigallocatechin-3-gallate, inhibits obesity, metabolic
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efficiency of major catechins and caffeine. Food Chem. 2006, 96, 597-605.
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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
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Hayashi, N.; Ujihara, T.; Kohata, K. Binding energy of tea catechin/caffeine
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complexes in water evaluated by titration experiments with H-1-NMR. Biosci.
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caffeine in crystal structure of 1:2 and 2:2 complexes. Tetrahedron Lett. 2009, 50, 4121-
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complex of gallocatechin gallate and caffeine. Chem. Lett. 2009, 38, 230-231.
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determination of caffeine complexes with galloylated and non-galloylated catechins.
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Chem. Lett. 2010, 39, 607-609.
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complexes of various tea catechins and caffeine in crystal state. Chem. Pharm. Bull.
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2011, 59, 1008-1015.
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Configurational studies of complexes of tea catechins with caffeine and various
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cyclodextrins. Planta Med. 2011, 77, 1099-1109.
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Munoz, P. Development of new antioxidant active packaging films based on ethylene
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vinyl alcohol copolymer (EVOH) and green tea extract. J. Agric. Food Chem. 2011, 59,
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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
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Vargas, M.; Pastor, C.; Chiralt, A.; McClements, D. J.; Gonzalez-Martinez, C.
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Recent advances in edible coatings for fresh and minimally processed fruits. Crit. Rev.
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Food Sci. Nutr. 2008, 48, 496-511.
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film containing green tea, green coffee, and grapefruit extracts. J. Agric. Food Chem.
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2012, 60, 9842-9849.
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films for active packaging materials. Int. J. Biol. Macromol. 2013, 59, 282-289.
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Natural additives in bioactive edible films and coating: Funcionality and applications in
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foods. Food Eng. Rev. 2013, 5, 200-216.
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30.
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analysis of non-volatile migrants from new active packaging materials. Anal. Bioanal.
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Chem. 2012, 404, 1945-1957.
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Garces, O.; Nerin C.; Beltran, J. A.; Roncales, P. EU patent EP1477519-A1.
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32.
Commission Directive (EU) No 72/2002 of 6 August 2002 relating to plastic
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materials and articles intended to come into contact with foodstuffs.
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33.
508
materials and article intended to come into contact with food.
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34.
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York 2002, 14, 285-314.
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512
MA, USA 2005.
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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.
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37.
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theoretical and experimental study. Chem. Phys. 2013, 415, 222-227.
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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
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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
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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
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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
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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
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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
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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
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FIGURES
Figure 1
31
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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
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Figure 3
33
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Figure 4
Non-protonated C-caffeine complex
Non-protonated EC-caffeine complex
Protonated C-caffeine complex
Protonated EC-caffeine complex
34
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TOC GRAPHIC
LIBERATED
molecules
NON-PROTONATED complex
PROTONATED complex
35
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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
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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).
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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References
421
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major green tea polyphenol, (-)-epigallocatechin-3-gallate, inhibits obesity, metabolic
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Yuan, J. M.; Sun, C.; Butler, L. M. Tea and cancer prevention: Epidemiological
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Gaffney, S. H.; Martin, R.; Lilley, T. H.; Haslam, E.; Magnolato, D. The
Horman, I.; Viani, R. Nature and conformation of caffeine-chlorogenate
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Maruyama, N.; Suzuki, Y.; Sakata, K.; Yagi, A.; Ina, K.; Duke, C. C. NMR
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determination of caffeine complexes with galloylated and non-galloylated catechins.
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complexes of various tea catechins and caffeine in crystal state. Chem. Pharm. Bull.
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Configurational studies of complexes of tea catechins with caffeine and various
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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-
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film containing green tea, green coffee, and grapefruit extracts. J. Agric. Food Chem.
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films for active packaging materials. Int. J. Biol. Macromol. 2013, 59, 282-289.
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Natural additives in bioactive edible films and coating: Funcionality and applications in
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foods. Food Eng. Rev. 2013, 5, 200-216.
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analysis of non-volatile migrants from new active packaging materials. Anal. Bioanal.
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Chem. 2012, 404, 1945-1957.
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Garces, O.; Nerin C.; Beltran, J. A.; Roncales, P. EU patent EP1477519-A1.
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materials and articles intended to come into contact with foodstuffs.
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materials and article intended to come into contact with food.
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York 2002, 14, 285-314.
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MA, USA 2005.
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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
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Stan, T. C. An introduction to computational biochemistry. Wiley-Liss, Inc., New
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Job, P. Formation and stability of inorganic complexes in solution. Ann. Chim.
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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
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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
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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
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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
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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
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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°
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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
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FIGURES
Figure 1
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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
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Figure 3
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Figure 4
Non-protonated C-caffeine complex
Non-protonated EC-caffeine complex
Protonated C-caffeine complex
Protonated EC-caffeine complex
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TOC GRAPHIC
LIBERATED
molecules
NON-PROTONATED complex
PROTONATED complex
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