Configurational Studies of Complexes of Tea Catechins with Caffeine and Various Cyclodextrins

Table of Contents

Abstract

A suspension of an equimolecular amount of ent-gallocatechin-3-O-gallate (entGCg) and caffeine in water afforded two kinds of crystals, which were 1 : 2 and 2 : 2 complexes of entGCg and caffeine. The stereochemical structures and intermolecular interactions between entGCg and caffeine were determined by X‐ray crystallographic analysis. The crystal structure of entGCg was determined and compared with those of the 1 : 2 and 2 : 2 complexes. Epigallocatechin-3-O-gallate (EGCg) formed a 1 : 1 complex with β-cyclodextrin (CD), in which the aromatic A ring and a part of the heterocyclic C ring were included from the wide secondary hydroxyl group side of the β-CD cavity in aqueous solution, while the B rings and 3-O-gallate groups (B' rings) were left outside the cavity. In contrast, entGCg formed a 1 : 2 complex with β-CD, in which the aromatic A and B rings of entGCg were included by two molecules of β-CD.

Supporting information available online at http://www.thieme-connect.de/ejournals/toc/plantamedica

Introduction

Tea has been consumed worldwide since ancient times to maintain and improve health. Some evidence suggests that tea protects against lifestyle-related diseases such as cancer, high blood pressure, diabetes, obesity, and arteriosclerosis [3]. Tea prepared from leaves of the tea plant Camellia sinensis (Camelliaceae) contains various catechins and caffeine as major ingredients. Catechins are a group of polyphenols that exhibit various pharmacological activities, such as anticarcinogenic [4], [5], anti-metastatic [6], [7], and anti-oxidative effects [8], [9]. The catechins in green tea are commonly classified into two categories, e.g., gallated- and non-gallated catechins, respectively, by the presence and absence of a galloyl group at the C3 position [1], [2], [10]. Generally, gallated catechins show higher activities than their non-gallated analogs [11], [12], [13], [14].

Caffeine is an alkaloid that displays a central nervous system-stimulating effect and that forms complexes with polyphenols, especially in black tea and coffee [15], [16], [17]. Such complexes may possess a unique stereochemical structure. Thus, many researchers have been investigating the structure of such complexes. For example, Maruyama et al. [18] noted that some gallated catechins have a high affinity for caffeine and assumed stacking of caffeine between the aromatic B ring and the 3-O-gallate group (B' ring). These conclusions were based on ¹H NMR chemical shift changes of gallate complexed to caffeine. Cai et al. [19] reported that in non-gallated-type catechins, such as catechin (CA) and epicatechin (EC), the A and C rings provided a general site for caffeine association, but that in gallated-type catechins, such as ent-catechin-3-O-gallate (entCg) and epigallocatechin-3-O-gallate (EGCg), the gallate moiety is the preferred site for complexation ([Fig. 1] ). Furthermore, Hayashi et al. [20] reported the participation of the A ring as well as B ring or 3-O-gallate groups (B' ring) in the complexation with caffeine as concluded from ¹H NMR chemical shift differences, nuclear Overhauser enhancement, and exchange spectroscopy (NOESY) spectra. However, the overall structure of the complex and the detailed intermolecular interactions between catechin and caffeine have not been elucidated sufficiently.

Fig. 1 Ent-gallocatechin-3-O-galate (entGCg), epigallocatechin-3-O-gallate (EGCg), and caffeine.

In this study, the crystal structure of the complex between entGCg and caffeine was determined by X‐ray crystallography, and the intermolecular interactions between entGCg and caffeine moieties were also elucidated [21], [22], [23].

A further study focused on the inclusion complexes comprising cyclodextrins and catechins. Cyclodextrins (CDs) are cyclic oligosaccharides which have six, seven, and eight D-(+)-glucopyranose units for α-, β-, and γ-CDs, respectively ([Fig. 2]). CDs incorporate compounds in their hydrophobic cavities depending on the cavity size. The inclusion complexes alter the physical, chemical, and biological properties of the guest molecule and may yield complexes that have considerable medicinal potential [24].

Fig. 2 Various cyclodextrins.

Special interest has been paid to EGCg, a major green tea component with a broad spectrum of bioactivities. However, catechin powders are bitter, brown and are easily oxidized, making them difficult to use as a medicine or natural food additive. In order to overcome these problems, we investigated the fundamental properties of the inclusion complexes of α-, β-, and γ-CDs with entGCg and EGCg in aqueous solution using a combination of NMR techniques and theoretical approaches [25], [26], [27]. EntGCg is a diastereomer of EGCg, differing in the configuration at C2. The difference between the inclusion complexes is discussed in relation to their conformations in aqueous solution.

The Two Complexes of entGCg and Caffeine [21], [22], [23]

An aqueous suspension containing an equimolecular amount of entGCg and caffeine in water was heated at 90 °C for 30 min. The solution gave a colorless powder ([Fig. 3]), which was recrystallized from water to give colorless needles (crystal A, mp 160–162 °C). The crystals represented a complex of entGCg and caffeine in a molar ratio of 1 : 2 based on the measurement of the integral area of ¹H NMR signals. When the same suspension was heated at 90 °C for 30 s and left at room temperature, a sticky substance was obtained ([Fig. 3]), comprising a complex of entGCg, caffeine, and water in a molar ratio of 1 : 1 : 22 as evident from the integrals of ¹H NMR signals. The sticky material crystallized slowly over a period of ca. three months at room temperature to give colorless needles (crystal B, mp 155–157 °C) which contained complexes of entGCg and caffeine in a molar ratio of 1 : 1 based on measurement of the integral area of ¹H NMR signals. However, it was actually a 2 : 2 complex of entGCg and caffeine based on evidence from X‐ray crystallography [22]. Interestingly, when the sticky substance was heated at 90 °C for 30 min, the product contained entGCg and caffeine in a molar ratio of 1 : 2 and was recrystallized from water to give colorless needles (crystal A). It was concluded that the complex formation energy of the 1 : 2 complex was higher than that of the 2 : 2 complex. Crystallizations of EGCg and caffeine have been also attempted, but they did not yet succeed.

Fig. 3 Preparation of two kinds of crystal of complexes of entGCg and caffeine.

An ORTEP drawing and a one unit cell of the 1 : 2 complex of entGCg and caffeine ([Fig. 4 a]) show that two caffeine molecules were located above the aromatic A ring and the 3-O-gallate group (B' ring) of an entGCg molecule. In one unit cell, four 1 : 2 complex entities and twelve water molecules as the crystal solvent were present. In a unit of the merohedral twinned structure [28] of crystal B with 2 : 2 complexes, the A and C rings of the two entGCg molecules faced each other, and their aromatic B rings and 3-O-gallate groups (B' rings) faced the two caffeine molecules ([Fig. 4 b]). One unit cell contained eight units consisting of the 2 : 2 complex and ninety-six water molecules as the crystal solvent.

Fig. 4 a and b Crystal structure of the 1 : 2 (a) and 2 : 2 (b) complexes of entGCg and caffeine, ORTEP drawing with thermal ellipsoids at a 30 % probability level and one unit cell. Hydrogen atoms and crystal solvent are omitted for clarity.

To compare the crystal structures of the 1 : 2 and 2 : 2 complexes, we carried out X‐ray analysis of entGCg alone, which was crystallized using the different solubility between entGCg and EGCg in water. A solution containing an equimolecular amount of entGCg and EGCg was left at room temperature for a few days to afford a colorless block-shaped single crystal of only entGCg, while EGCg as well as some entGCg were still soluble in the solution. The single crystal of entGCg was determined by X‐ray crystallographic analysis [21]. One unit cell contains two entGCg molecules and two water molecules ([Fig. 5]).

Fig. 5 a and b Crystal structure of entGCg alone (a) ORTEP drawing with thermal ellipsoids at a 30 % probability level. b One unit cell. Hydrogen atoms and crystal solvent are omitted for clarity.

The dihedral angles of C1′-C2-C3-O and H2-C2-C3-H3 of the entGCg moiety of the 1 : 2 complex are 55.93° and 173.18°, respectively, indicating that the B ring and the 3-O-gallate group (B' ring) both adopt equatorial positions ([Fig. 6]; [Table 1]). This also holds true for the 2 : 2 complex, whereas the corresponding aromatic rings of entGCg crystals alone adopt axial and pseudoaxial positions, respectively ([Fig. 6]; [Table 1]). These findings suggested a conformational change of entGCg upon conversion of 1 : 2 to 2 : 2 complexes, facilitated by the conformational flexibility of entGCg molecules owing to puckering of the pyran C ring. On the other hand, the caffeine molecule has a planar and rigid xanthine skeleton.

Fig. 6 Conformation of entGCg moieties in the 1 : 2 and 2 : 2 complexes, and entGCg alone in crystal state.

Table 1 Torsion angle in entGCg in the 1 : 2 and 2 : 2 complexes and entGCg alone.

	Torsion angle (C1′-C2-C3-O)	Torsion angle (H2-C2-C3-H3)
1 : 2 Complex of entGCg and caffeine	55.93°	173.18°
2 : 2 Complex of entGCg and caffeine	61.05° (average)	176.94° (average)
entGCg alone	159.03°	72.80°

For complex formation of compounds with aromatic rings, three kinds of interactions are important, namely face-to-face π–π interaction between the planes of two aromatic rings, offset π–π interaction between the planes of two slightly shifted aromatic rings, and CH–π interaction ([Fig. 7]).

Fig. 7 Intermolecular interactions by non-covalent bonds in the complexes of catechins and caffeine.

A marked difference in the layer structure between the crystal structures of the 1 : 2 and 2 : 2 complexes and entGCg alone was observed ([Figs. 8]–[11]). As shown in [Fig. 8], units of the former piled up in parallel in the same direction as the a-axis. The distances between the aromatic A rings and the 3-O-gallate groups (B' rings) of two entGCg molecules were 6.866 and 6.767 Å, respectively. Two caffeine molecules were located almost in the middle of the A ring and 3-O-gallate group (B' ring) of entGCgs in a sandwich-like manner. This allows for face-to-face π–π interactions between the A ring and the 3-O-gallate group (B' ring) of the upper entGCg and the six-membered ring of caffeine, and offset π–π interactions between the same structural elements involving the lower entGCg. Also, a CH–π interaction was formed between the B ring of the lower entGCg and the methyl group at N7 of caffeine (distance 3.281 Å). As shown in [Table 2], three intermolecular hydrogen bonds between entGCg and caffeine, and entGCgs were observed in the 1 : 2 complex.

Fig. 8 The layer structure and intermolecular interactions of the 1 : 2 complex of entGCg and caffeine.

Fig. 9 Packing of the 2 : 2 complex of entGCg and caffeine in the cell down the a-axis.

Fig. 10 The layer structure and intermolecular interactions of the 2 : 2 complex of entGCg and caffeine.

Fig. 11 The layer structure and intermolecular interaction of entGCg alone.

Table 2 Intermolecular hydrogen bonds in the 1 : 2 complex.

D-H	A	D…A	D-H	H…A	∠ D-H…A
C(4)-OH(2O)	O(14)	2.727	1.108	1.656	160.81
C(6)-OH(3O)	O(7)	2.880	1.045	1.710	157.97
C(20)-OH(10O)	O(5)	2.894	1.059	1.870	161.49

In [Fig. 9], packing of the 2 : 2 complex of entGCg and caffeine in the cell down the a-axis is shown. Each caffeine molecule and the aromatic B ring and 3-O-gallate group (B' ring) of the entGCg molecules were arrayed regularly in the following order: 3-O-gallate group (B' ring), caffeine, 3-O-gallate group (B' ring), caffeine, and B ring. The average distances between each ring were ca. 3.2 Å, ca. 3.3 Å, ca. 3.3 Å, and ca. 3.4 Å, respectively. Furthermore, these caffeine molecules were surrounded on four sides by the aromatic B rings and the 3-O-gallate groups (B' rings) of two entGCg molecules.

In the layer structure, units of the 2 : 2 complex of entGCg and caffeine piled up in parallel to the a-axis, and the A and A rings of entGCgs faced each other by face-to-face π–π interactions ([Fig. 10]). All caffeine molecules were sandwiched between the aromatic B ring and the 3-O-gallate group (B' ring) or the 3-O-gallate groups (B' rings) of entGCg molecules by face-to-face π–π interactions. Also, CH–π interactions occurred between the B rings of entGCg and both the methyl groups at N3 (average distance ca. 3.0 Å) and N7 of caffeine (average distance ca. 2.8 Å). As shown in [Table 3], eight intermolecular hydrogen bonds between entGCgs, entGCg and caffeine were observed in this case.

Table 3 Intermolecular hydrogen bonds in the 2 : 2 complex.

D-H	A	D…A	D-H	H…A	∠ D-H…A
C(36)-OH(16)	N(27)	2.729	0.841	1.891	174.83
C(42)-OH(18)	O(78)	2.660	0.840	1.830	169.96
C(96)-OH(42)	O(13)	2.657	0.839	1.818	177.62
C(104)-OH(46)	O(39)	2.513	0.840	1.705	160.36
C(109)-OH(48)	O(3)	2.816	0.839	2.058	149.88
C(186)-OH(81)	O(52)	2.621	0.840	1.781	177.90
C(194)-OH(85)	O(26)	2.657	0.841	1.820	173.58
C(224)-OH(98)	O(65)	2.570	0.842	1.740	168.46

In the layer structure, entGCg faced in the same direction and accumulated parallel to the a-axis ([Fig. 11]). Offset π–π interactions formed between A and A rings, B and B rings and 3-O-gallate groups (B' rings) of entGCg molecules. However, no face-to-face π–π interaction was observed in the layer of entGCg. Furthermore, five hydrogen bonds were observed between entGCgs, entGCg and water and, as a result, a network of hydrogen bonds was formed in the crystal structure of entGCg ([Table 4]). Generally speaking, the equatorial position of a bulky group is kinetically more stable than the axial position, but it is thought that the cooperative effect of these interactions permits axial and pseudoaxial positions of the B ring and the 3-O-gallate group (B' ring).

Table 4 Intermolecular hydrogen bonds in entGCg alone.

D-H	A	D…A	D-H	H…A	∠ D-H…A
C(7)-OH(7)	O(8)	2.728	1.014	1.750	160.77
C(15)-OH(12)	O(4)	2.801	0.919	1.933	156.53
C(19)-OH(15)	O(11)	2.694	0.972	1.746	164.30
C(20)-OH(16)	O(5)	2.734	0.953	1.810	162.29

Inclusion Complexes of Various Cyclodextrins with EGCg and entGCg [26], [27], [28]

Equation (1) for the formation of the inclusion complex of β-CD with EGCg and entGCg was constructed to calculate the respective n values ([Fig. 12]), which were 1.20 and 1.90 for EGCg and entGCg at 35 °C, respectively. This suggested that the stoichiometry of the formation of the inclusion complex of β-CD with EGCg was mainly 1 : 1, a conclusion that was supported by Job's Plot experiments [29]. In contrast, the stoichiometric composition of the inclusion complex of β-CD with entGCg was mainly 1 : 2.

Fig. 12 Equation for the formation of the inclusion complex of β-CD with EGCg and entGCg. [β-CD]₀: initial concentration of β-CD (mM); [CA]₀: initial concentration of EGCg, entGCg (mM); Kc: binding constant (mM⁻ⁿ). δ _CA, δ _CX and δ _obs represent the chemical shift (ppm) of the H₈ proton signal of EGCg and the H_{2′′,6′′} proton signal of entGCg in a free state, the inclusion complex of β-CD and EGCg, entGCg, and the mixture of β-CD and EGCg, entGCg in ¹H NMR spectra, respectively. Δδ _CX and Δδ _obs represent (δ _CA–δ _obs) and (δ _CA–δ _CX), respectively.

The conformation of EGCg and entGCg in aqueous solution were investigated. In the ¹H NMR spectrum of EGCg, the signal for H₂ appeared as a broad singlet, indicating that the coupling constant J_2,3 was ca. 0 Hz. The dihedral angle ∠H₂-C₂-C₃-H₃ was expected to be approximately 90° as judged from the Karplus equation [30]. [Fig. 13] shows the results of the nuclear Overhauser effect (NOE) differential analysis of EGCg and entGCg in D₂O at 35 °C. NOEs between H₂ and H _4β , H₃ and H_2′,6′, and between H_2′,6′ and H_{2′′,6′′} of EGCg suggested that the aromatic B ring and the 3-O-gallate groups (B' rings) of EGCg adopt equatorial and axial positions, respectively.

Fig. 13 Conformation of EGCg and entGCg in aqueous solution. Measurement temperature of NOE experiments was 35 °C.

In the ¹H NMR spectrum of entGCg, the H₂ signal appeared as a doublet (J_2,3 = 6.5 Hz). As shown in [Fig. 13], NOEs between H₂ and H _4α , H _4α and H_{2′′,6′′}, and between H₃ and H_2′,6′ suggested that the aromatic B ring and 3-O-gallate groups (B' rings) were both in equatorial positions and therefore much more distant when compared with EGCg.

To determine the structure of the 1 : 1 inclusion complex of EGCg and β-CD, rotating frame nuclear Overhauser effect spectroscopy (ROESY) of a solution containing equimolar amounts of β-CD and EGCg in D₂O was measured. Strong intermolecular ROE correlations between the H₈ of EGCg and the H₃, H₅, and H₆ being on the inner surface of β-CD suggested that the A ring of EGCg was included in the β-CD cavity. Furthermore, intermolecular ROE correlations between H_2′,6′ of EGCg and each β-CD proton were detected. Based on the results of the ROESY spectrum and the ¹H NMR chemical shift changes [24], it was concluded that the A ring and a part of the C ring of EGCg were included in the wide secondary hydroxyl group side of the β-CD cavity, and that the aromatic B ring and 3-O-gallate group (B' ring) were left outside the cavity ([Fig. 14]). Also, EGCg intramolecular associations between H₂ and H _4β , H₃ and H_2′,6′, as well as H_2′,6′ and H_{2′′,6′′} indicated that the conformation in which the B ring and 3-O-gallate group (B' ring) of EGCg adopted equatorial and axial positions, respectively, were still maintained upon the formation of the inclusion complex.

Fig. 14 ROESY spectrum of a solution containing an equimolecular amount of β-CD and EGCg in D₂O at 35 °C, and possible structure of 1 : 1 inclusion complex of β-CD and EGCg.

In the ROESY spectrum of a solution containing entGCg and β-CD (ratio 1 : 2) in D₂O, strong intermolecular ROE correlations between the H₈ of entGCg and the H₅ and H₆ of β-CD were observed, suggesting that the A ring of entGCg was included in the β-CD cavity [25]. Upon the formation of the 1 : 2 inclusion complex of entGCg and β-CD, all proton signals of entGCg were shifted upfield due to C‐C bond anisotropy by the two molecules of β-CD. The proton signals of entGCg were broadened, except those for H₆ and H_{2′′,6′′} ([Fig. 15]). A plausible explanation of the observed signal broadening may be due to the restricted motion of these protons by the two molecules of β-CD. Notably, the signal for the H_2′,6′ protons was markedly broadened, while that for the H_{2′′,6′′} protons appeared sharp. These findings suggested that the B ring of entGCg was localized in the cavity of β-CD, while the 3-O-gallate group (B' ring) was outside the cavity. Furthermore, the H₈ signal almost disappeared on formation of the inclusion complex with β-CD, whereas the H₆ signal was still sharp. The disappearance of the H₈ signal may be explained by the close proximity with the hydrogens on the inner surface of β-CD. On the other hand, the motion of the H₆ proton might not be restricted due to its position in the vicinity of the rim of the narrow primary hydroxyl group side of β-CD. It is therefore concluded that entGCg is enclosed in the two β-CD molecules in the manner illustrated in [Fig. 15].

Fig. 15 a and b ¹H NMR spectra of solutions containing (a) entGCg and twice the amount of β-CD, and (b) entGCg alone in D₂O at 35 °C, and possible structure of 1 : 2 inclusion complex of β-CD and entGCg.

To confirm the structure of the 1 : 2 inclusion complex of entGCg and β-CD deduced from NMR experiments, the energies and structure of the 1 : 1 and 1 : 2 inclusion complexes were calculated using the PM5 MO method [31]. The 1 : 2 complex including the aromatic A and B rings of entGCg is ca. 3 kcal/mol more stable than that including the A ring and 3-O-gallate group (B' ring) in the PM5 calculation ([Fig. 16]).

Fig. 16 Structure of the 1 : 1 and 1 : 2 inclusion complexes of entGCg and β-CD by PM5 MO method.

EGCg and entGCg afforded no inclusion complex with α-CD because the cavity of α-CD is not large enough to include these molecules. While EGCg yielded a 1 : 1 inclusion complex with β-CD, entGCg formed a 1 : 2 inclusion complex with β-CD, resulting from the different spacing between the B rings and the 3-O-gallate groups (B' rings) in aqueous solution. EGCg failed to form an inclusion complex with γ-CD, whereas entGCg did form a 1 : 1 inclusion complex with γ-CD due to the large cavity of γ-CD.

The difference in stereochemistry between EGCg and entGCg is only the configuration at the 2 position, but for the two molecules the inclusion modes with various cyclodextrins vary considerably and are summarized in [Fig. 17].

Fig. 17 Inclusion modes of EGCg and entGCg with α, β, and γ-CDs.

Supporting information

X-ray data of the 1 : 2 and 2 : 2 complexes of entGCg and caffeine and entGCg alone, NMR experiments and calculation methods for the inclusion complexes of entGCg and β-CD are available as Supporting Information.

References

• 1 Hemingway R W, Foo L Y, Porter L J. Linkage isomerism in trimeric and polymeric 2,3-cis procyanidins.  J Chem Soc [Perkin I]. 1982;  1209-1216
• 2 Porter L J. Flavans and proanthocyanidins. Harborne JB The flavonoids: advances in research since 1980. London; Chapmann and Hall 1988: 21-62
• 3 Kuroda Y, Hara Y. Health effects of tea and its catechins. New York, Boston, Dordrecht, London, Moscow; Kluwer Academic/Plenum Publishers 2004: 11-60
• 4 Lambert J D, Yang C S. Cancer chemopreventive activity and bioavailability of tea and tea polyphenols.  Mutat Res. 2003;  523–524 201-208
• 5 Ahmad N, Cheng P, Mukhtar H. Cell cycle dysregulation by green tea polyphenol epigallocatechin-3-gallate.  Biochem Biophys Res Commun. 2000;  275 328-334
• 6 Maeda-Yamamoto M, Kawahara H, Tahara N, Tsuji K, Hara Y, Isemura M. Effects of tea polyphenols on the invasion and matrix metalloproteinases activities of human fibrosarcoma HT1080 cells.  J Agric Food Chem. 1999;  47 2350-2354
• 7 Sazuka M, Imazawa H, Shoji Y, Mita T, Hara Y, Isemura M. Inhibition of collagenases from mouse lung carcinoma cells by green tea catechins and black tea theaflavins.  Biosci Biotechnol Biochem. 1997;  61 1504-1506
• 8 Hashimoto F, Ono M, Masuoka C, Ito Y, Sakata Y, Shimizu K, Nonaka G, Nishioka I, Nohara T. Evaluation of the anti-oxidative effect (in vitro) of tea polyphenols.  Biosci Biotechnol Biochem. 2003;  67 396-401
• 9 Kimura M, Umegaki K, Kasuya Y, Sugisawa A, Higuchi M. The relation between single/double or repeated tea catechin ingestions and plasma antioxidant activity in humans.  Eur J Clin Nutr. 2002;  56 1186-1193
• 10 Hayashi N, Ujihara T. ‘Biting effect' stabilizing gallate-type catechin/quaternary ammonium ion complexes.  Tetrahedron. 2007;  63 9802-9809
• 11 Tezuka M, Suzuki H, Suzuki Y, Hara Y, Okada S. Inactivation effect of tea leaf catechins on human type-A influenza virus.  Jpn J Toxicol Environ Health. 1997;  43 311-315
• 12 Okabe S, Suganuma M, Hayashi M, Sueoka E, Komori A, Fujiki H. Mechanisms of growth inhibition of human lung cancer cell line, PC-9, by tea polyphenols.  Jpn J Cancer Res. 1997;  88 639-643
• 13 Miura S, Watanabe J, Tomita T, Sano M, Tomita I. The inhibitory effects of tea polyphenols (flavan-3-ol derivatives) on Cu²⁺ mediated oxidative modification of low density lipoprotein.  Biol Pharm Bull. 1994;  17 1567-1572
• 14 Hara Y, Watanabe M. Antibacterial activity of tea polyphenols against clostridium botulinum.  Nippon Shokuhin Kogyo Gakkaishi. 1989;  36 951-955
• 15 Martin R, Lilley T H, Falshaw C P, Haslam E, Begley M J, Magnolato D. The caffeine-potassium chlorogenate molecular complex.  Phytochemistry. 1986;  26 273-279
• 16 Gaffney S H, Martin R, Lilley T H, Haslam E, Magnolato D. The association of polyphenols with caffeine and α- and β-cyclodextrin in aqueous media.  J Chem Soc Chem Commun. 1986;  2 107-109
• 17 Horman I, Viani R. The nature and conformation of the caffeine-chlorogenate complex of coffee.  J Food Sci. 1972;  37 925-927
• 18 Maruyama N, Suzuki Y, Sakata K, Yagi A, Ina K. NMR spectroscopic and computer graphics studies on the creaming down of tea. Proceedings of the International Symposium on Tea Science, Shizuoka-shi, Japan 1991: 145-149
• 19 Cai Y, Gaffney S H, Lilley T H, Magnolato D, Martin R, Spencer C M, Haslam E. Polyphenol interactions. Part 4. Model studies with caffeine and cyclodextrins.  J Chem Soc [Perkin II]. 1990;  2197-2209
• 20 Hayashi N, Ujihara T, Kohata K. Binding energy of tea catechin/caffeine complexes in water evaluated by titration experiments with ¹H-NMR.  Biosci Biotechnol Biochem. 2004;  68 2512-2518
• 21 Tsutsumi H, Sato T, Ishizu T. Offset π-π interaction in crystal structure of (−)-gallocatechin-3-O-gallate.  Chem Pharm Bull. 2010;  58 572-574
• 22 Ishizu T, Tsutsumi H, Sato T, Yamamoto H, Shiro M. Crystal structure of complex of gallocatechin gallate and caffeine.  Chem Lett. 2009;  38 230-231
• 23 Ishizu T, Tsutsumi H, Sato T. Interaction between gallocatechin gallate and caffeine in crystal structure of 1:2 and 2:2 complexes.  Tetrahedron Lett. 2009;  50 4121-4124
• 24 Wong J W, Yuen K H. Inclusion complexation of artemisinin with α-, β-, and γ-cyclodextrins.  Drug Dev Indian Pharm. 2003;  29 1035-1044
• 25 Ishizu T, Kajitani S, Tsutsumi H, Yamamoto H, Harano K. Diastereomeric difference of inclusion modes between (−)-epicatechin gallate, (−)-epigallocatechin gallate and (+)-gallocatechin gallate, with β-cyclodextrin in aqueous solvent.  Magn Reson Chem. 2007;  46 448-456
• 26 Ishizu T, Hirata C, Yamamoto H, Harano K. Structure and intramolecular flexibility of β-cyclodextrin complex with (−)-epigallocatechin gallate in aqueous solvent.  Magn Reson Chem. 2006;  44 776-783
• 27 Ishizu T, Tsutsumi H, Yamamoto H, Harano K. NMR spectroscopic characterization of inclusion complexes comprising cyclodextrins and gallated catechins in aqueous solution: cavity size dependency.  Magn Reson Chem. 2008;  47 283-287
• 28 Giacovazzo C. Fundamentals of crystallography, 2nd edition. IUCr texts on crystallography 7. Oxford; IUCr/Oxford University Press 2002: 237-243
• 29 Job P. Formation and stability of inorganic complexes in solution.  Ann Chem. 1928;  9 113-203
• 30 Karplus M, Anderson DH. Valence-bond interpretation of electron-coupled nuclear spin interactions; application to methane.  J Chem Phys. 1959;  30 6-10
• 31 WinMOPAC V3.9. Tokyo; Fujitsu Ltd 2004