Tea has been drunk in many countries throughout the world since ancient times for its taste, and to maintain and improve health [1]. Tea is commonly prepared by pouring hot or boiling water over leaves of the tea plant, Camellia sinensis, Theaceae, which includes caffeine, catechins, vitamins, theanine, etc.

The main eight tea catechins are classified into four categories by the existence of a galloyl group on the oxygen atom at the C3 position and the relative stereochemistry between the C2 and C3 positions: 2,3-cis gallate-type, 2,3-trans gallate-type, 2,3-cis-non gallate-type, and 2,3-trans non-gallate-type (Figure 1) [2]. Generally speaking, gallate-type catechins show higher physiological activities than non-gallate-type catechins [3-6].

When a hot tea beverage cools down, brown-white turbidity and precipitate occurs in the tea. This phenomenon is called “creaming down (reaction)”. Since the creaming down is a trigger that reduces the original taste and flavor of tea, it is one of the most serious problems in making a tea beverage. It has been suggested that caffeine formed precipitates of a cream down with tea polyphenols such as catechins, theaflavins and thearubigins [7].

Ina et al. reported that all the main signals were assigned to tea catechins such as 2,3-cis gallated catechins (-)-epigallocatechin-3-O-gallate (EGCg) and (-)-epicatechin-3-O- gallate (ECg) and caffeine in the ¹³C NMR spectrum of a hot water solution of a precipitate formed by the creaming down of a tea infusion [8]. Ina et al. also reported that creaming down eventually occurs when an aqueous caffeine solution is poured into an aqueous solution of EGCg, which is most abundant in tea catechins [8].

However, creaming down in tea beverages has not been chemically elucidated sufficiently. Thus, we investigated what kind of catechins predominantly precipitated using a catechin mixture and caffeine by the quantitative ¹H NMR. The catechin mixture (purchased from Nagara Science Co., Ltd) is a fraction of extracted catechins included in green tea. We attempted crystallization of a precipitate formed by the creaming down of EGCg and caffeine, the stereochemical structure of the complex of EGCg and caffeine was investigated using X-ray crystallographic analysis. Furthermore, mechanism of creaming down was investigated using molecular interactions forming between tea catechins and caffeine.

Based on the mechanism of the creaming down found in this way, molecular capture of heterocyclic compouds by EGCg from an aqueous solution was investigated.

Precipitates occurred when aqueous solution of a variety of heterocyclic compounds poured into aqueous solution of EGCg. The molecular capture abilities of the heterocyclic compounds were evaluated with the ratio of the amount of the heterocyclic compounds included in the precipitates to that of the total heterocyclic compounds used.

Figure 1: The Eight Major Tea Catechins and Caffeine.

Analysis of the Precipitate of Creaming Down Made From Catechin Mixture and Caffeine

The content of tea catechins contained in the catechin mixture was investigated by the integrated value of the proton signal for H2 of each tea catechin in the quantitative ¹H NMR spectrum [9]. No overlap of the methine H2 proton signal of each tea catechin in deuterated acetone (acetone-d₆) was observed in the ¹H NMR spectrum [10]. Then amount of each tea catechin included in the catechin mixture was measured with the integrated value of the H2 proton signal using the methyl proton signal (singlet) of Boc-glycine as an internal standard (Figure 2).

As shown in Figure 2, the H2 proton signal of 2,3-cis catechins of ECg, EGCg, EC, and EGC was observed as singlet-like, whereas that of 2,3-trans catechins of (+)-catechin (CA) and (+)-gallocatechin (GC) was observed as a doublet in the ¹H NMR spectrum. Such a difference in coupling patterns resulted from differences in configuration. Judging from the coupling constant J_2,3 of ECg, EGCg, EC, and EGC being ca. 0 Hz, it was thought that their dihedral angles ∠H₂-C₂-C₃-H₃ were approximately 90°, judging from the Kurplus equation [12]. On the other hand, since J_2,3 of CA and GC were 7.8 Hz and 7.4 Hz, respectively, their dihedral angles ∠H₂-C₂-C₃-H₃ were expected to be considerably larger than 90°[12,13].

Figure 2: The methine proton H₂ signal of each catechin of the catechin mixture in1H NMR Spectrum.

The content of the catechin mixture is shown in Table 1, indicating that it included a large amount of 2,3-cis-type catechins, such as EGCg, ECG, EC, and ECg [9]. Thus, a simple and easy method for quantitative analysis of a sample containing many kinds of catechins using the ¹H NMR spectrum was developed. The reported contents of catechins in green tea are listed in Table 1 [11].

Table 1: The content of tea catechins in the catechin mixture.

We investigated molecular capture using EGCg, which formed a strong hydrophobic space in water. Aqueous solution of various heterocyclic compounds (Table 3) was poured into an aqueous solution of an equimolar amount of EGCg, and it afforded sticky precipitates containing EGCg and the heterocyclic compounds at a molar ratio of 1:1, based on measurement of the integrated value of 1H NMR signals.
Analysis of the Complex of Egcg and 2-Chloropyrimidine by X-Ray Crystallography
To confirm that the precipitates made from an aqueous solution of equimolar of EGCg and the heterocyclic compounds were 2:2 complexes of the same mode as the 2:2 complex of EGCg and caffeine, X ray crystal analysis of the precipitate made from EGCg and 2-chloropyrimidine (Figure 8a) was performed [17].
Aqueous solution of 2-chloropyrimidine was poured into an aqueous solution of an equimolar amount of EGCg, and it afforded a precipitate as a colorless block crystal.
Based on an ORTEP drawing, this crystal contained two crystallographically different EGCgs and two 2-chloropyrimidines, and these formed a 2:2 complex which the aromatic ring of the 2-chloropyrimidine and the B’ rings of each EGCg faced (Figure 8). π-π Interactions formed between a six-membered ring of 2-chloropyrimidine and the B’ ring of EGCg, B rings of EGCg, and A rings of EGCg respectively.

Figure 8: 2-Chloropyrimidine in Hydrophobic space formed by the aromatic A, B, and B' rings of EGCg.

(a) 2-Chloropyrimidine.

(b) 2:2 complex of EGCg and 2-Chloropyrimidine Hydrogen atoms and crystal solvent are omitted for clarity.

It was found that 2-chloropyrimidine formed a 2:2 complex with EGCg in the same mode as the 2:2 complex of EGCg and caffeine. As a result, 2-chloropyrimidine molecules were captured by the hydrophobic space formed by the three aromatic rings of A, B, B’ rings of the EGCg as shown in Figure 8b, and it precipitated as the 2:2 complex from the aqueous solution of an equimolar amount of 2-chloropyrimidine and EGCg due to its high hydrophobicity.

Therefore, the precipitates were thought to be 2:2 complexes of EGCg and the heterocyclic compounds (Table 5) of the same mode as the 2:2 complex of EGCg and caffeine.

Molecular Capture of Heterocyclic Compounds Using Egcg

We investigated how much the heterocyclic compound could be captured in precipitate of the 2:2 complex from aqueous solution of EGCg and the heterocyclic compounds. The amount of the heterocyclic compounds were measured by an integrated value of corresponding proton signals in the quantitative 1H NMR spectrum in DMSO-d₆. The molecular capture abilities of various heterocyclic compounds using EGCg were evaluated by the ratio of the amount of heterocyclic compounds included in the precipitates to that of the total heterocyclic compounds used (Table 3) [17].

In Table 3, a heterocyclic compound having A (%) of 0% means that an aqueous solution of the heterocyclic compound and EGCg did not give a precipitate.

However, no correlation was found between the chemical structures of the heterocyclic compounds and the molecular capture ability.

Table 3: The molecular capture abilities of heterocyclic compounds using EGCg.

When an aqueous caffeine solution was poured into an aqueous solution of catechin mixture, precipitate by creaming down occurred. The gallated catechins EGCg and ECg were formed the precipitate of creaming down more predominantly rather than the non-gallated catechins, EC, EGC, CA, and GC.

X-ray crystallographic analysis showed that EGCg formed a 2:2 complex with caffeine, and EC formed 1:1 complex with caffeine. Upon the formation of the 2:2 complex, EGCg moieties formed the hydrophobic space surrounded by the top and bottom walls of the B’ rings and right and left walls of the A and B rings, whereas, upon the formation of the 1:1 complex, EC formed the hydrophobic space only using the top and bottom walls of the A rings. Therefore, it was thought that the hydrophobic effect in the 2:2 complex of EGCg and caffeine was stronger than that of the 1:1 complex of EC and caffeine, with the result that the 2:2 complex of the gallated catechin EGCg and caffeine precipitated as a precipitate formed by creaming down more predominantly than the 1:1 complex of the non-gallated catechin EC and caffeine.

Then we investigated molecular capture using EGCg, which formed a strong hydrophobic space in water. An aqueous solution of an equimolar amount of EGCg and a variety of heterocyclic compounds (Table 3) afforded sticky precipitates containing EGCg and the heterocyclic compounds of the 2:2 complex.

The molecular capture abilities of heterocyclic compounds using EGCg evaluated by the ratio of the amount of heterocyclic compounds included in the precipitates to that of total heterocyclic compounds used. However, no correlation was found between the chemical structures of the heterocyclic compounds and their molecular capture abilites.

Then the molecular capture abilities of diketopiperazine cyclo(Pro-Xxx) (Xxx: amino acid residue) were evaluated, and the correlation between the chemical structures of cyclo(Pro-Xxx) and the molecular capture abilites was investigated. A greater number of carbons in the side chain of the amino acid residue, such as Ala, Val, Pro, Leu, Ile, and Phe led to a higher molecular capture ability of cyclo (Pro-Yyy).

When cyclo(Pro-Xxx) was taken into the hydrophobic space formed by EGCg to form the 2:2 complex of EGCg, conformation of cyclo(Pro-Xxx) other than cyclo(D-Pro-L-Ala) still maintained. And conformation of cyclo(D-Pro-L-Ala) changed, that is, its 3-position methyl group changed from the axial position to the equatorial position due to steric hindrance by EGCg moieties.

Acknowledgements

All of the research in this review was conducted at the Laboratory of Organic and Bio-organic Chemistry in the Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University. The author is grateful to the members and graduates of the Laboratory of Organic and Bio-organic Chemistry. In particular, the author would like to thank Dr. Hiroyuki Tsutsumi, Faculty of Pharmaceutical Sciences, Fukuoka University, for making a great contribution to the research results related to this manuscript.

Conflict of Interest

The authors declare no conflict of interest.

1. Kuroda Y, Hara Y. Health Effects of Tea and Its Catechins. Kluwer Academic/Plenum Publishers, New York. 2004.
2. Hayashi N, Ujihara T. A Water-Soluble acyclic phane receptor recognizing 2,3-trans-gallate-type catechins. J Org Chem. 2008; 73: 4848-4854.
3. Okabe S, Suganuma M, Hayashi M, Sueoka E, Komori A, Fujiki H. Jpn J Cancer Res. 1997; 88: 639-643.
4. Tezuka M, Suzuki H, Suzuki Y, Hara Y, Okada S, Jpn J Toxicol Environ Health. 1997; 43: 311-315.
5. Mimura S, Watanabe J, Tomita T, Sano M, Tomita I, Biol Pharm Bull. 1994; 17: 1567-1572.
6. Hara Y, Watanabe M. Antibacterial action of tea polyphenols against Clostridium botulinum. Nippon Shokuhin Kogyo Gakkaishi. 1989; 36: 951-955.
7. Seshadri R, Dhanraj N. Proc. International Flavor Conference, Porto Karras Greece. 1987; 169-180.
8. Maruyama N, Suzuki Y, Sakata K, Yagi A, Ina K, Proc. International Symposium Tea Science 1991; 145-149.
9. Sato T, Kinoshita Y, Tsutsumi H, Yamamoto H, Ishizu T. Characterization of creaming precipitate of tea catechins and caffeine in aqueous solution. Chem Pharm Bull. 2012; 60: 1182-1187.
10. Davis AL, Cai Y, Davies AP, Lewis JR. Magn Reson Chem. 1996; 34: 887-890.
11. Muramatsu K. Chanokagaku Asakura Publishing. Tokyo; 1991: 124.
12. Karplus J. Contact Electron?spin coupling of nuclear magnetic moments. J Chem Phys. 1959; 30: 11.
13. Ishizu T, Kajitani S, Tsutsumi H, Sato T, Yamamoto H, Hirata C. Configurational studies of complexes of tea catechins with caffeine and various cyclodextrins. Planta Medica. 2011; 77: 1099-1109.
14. Tsutsumi H, Sato T, Ishizu T. Stereochemical structure of the complex of (−)-epigallocatechin 3-gallate and caffeine. Chem Lett. 2012; 41: 1669-1671.
15. Ishizu T, Tsutsumi H, Kinoshita Y, Mukaida H, Sato T, Kajitani S. Properties of precipitate of creaming down by (−)-Epigallocatechin-3-O-gallate and caffeine. Chem Pharm Bull. 2014; 62: 552-558.
16. Ishizu T, Sato T, Tsutsumi H, Yamamoto H. Stereochemical structure determination of caffeine complexes with galloylated and non-galloylated catechins. Chem Lett. 2010; 39: 607-609.
17. Tsutsumi H, Sato A, Fujino S, Fujioka Y, Ishizu T. Molecular capture using the precipitate of Creaming-Down by (−)-Epigallocatechin-3- O –gallate. Chem Pharm Bull. 2019; 67: 501-504.
18. Ishizu T, Tsutsumi H, Yokoyama A. Chiral recognition of diketopiperazines cyclo(Pro-Gly) using (−)-epigallocatechin-3-O-gallate. Tetrahedron Lett. 2015; 56: 1111-1114.
19. Ishizu T, Tsutsumi H, Yokoyama E, Tanabe H, Yokoyama A. Chiral recognition of diketopiperazine cyclo(Pro-Gly) and Propranolol Using (−)-Epigallocatechin-3-O-gallate. Chem Pharm Bull. 2016; 64: 142-149.
20. Ishizu T, Tokunaga M, Fukuda M, Matsumoto M, Goromaru T, Takemoto S. Molecular capture and conformational change of diketopiperazines containing proline residues by Epigallocatechin-3-O-gallate in Water. Chem Pharm Bull. 2021; 69: 585-589.