Article pubs.acs.org/JAFC
Interaction between Tea Polyphenols and Bile Acid Inhibits Micellar Cholesterol Solubility Kazuki Ogawa, Sayumi Hirose, Satoshi Nagaoka, and Emiko Yanase* Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan S Supporting Information *
ABSTRACT: The molecular mechanism by which tea polyphenols decrease the micellar solubility of cholesterol is not completely clear. To clarify this mechanism, this study investigated the interaction between tea polyphenols (catechins and oolongtheanins) and cholesterol micelles. A nuclear magnetic resonance (NMR) study was performed on a micellar solution containing taurocholic acid and epigallocatechin gallate (EGCg), and high-performance liquid chromatography (HPLC) analysis was carried out on the precipitate and the supernatant that formed when EGCg was added to a cholesterol−micelle solution. The data indicated a regiospecific interaction of EGCg with taurocholic acid. Therefore, the ability of EGCg to lower the solubility of phosphatidylcholine (PC) and cholesterol in micellar solutions can be attributed to their elimination from the micelles due to interaction between taurocholic acids and EGCg. KEYWORDS: catechin, cholesterol, oolongtheanin, micelle
■
INTRODUCTION Tea (Camellia sinensis) is a globally popular beverage. Due to differences in the methods used for processing the tea leaves, tea is mainly classified into three types: green tea, oolong tea, and black tea. Catechins are the major polyphenols, present at approximately 30%, in tea leaves. There are four main types of catechins (Figure 1), namely, (−)-epicatechin gallate (ECg) and (−)-epigallocatechin gallate (EGCg; 1), having a gallate group at the 3-position, and (−)-epicatechin (EC) and (−)-epigallocatechin (EGC; 2), which have no gallate group. Catechins have attracted attention because of their various health benefits, including their antioxidant activity1 and antitumor activity.2 Furthermore, catechins are known to have blood cholesterollowering effects, and it has been reported that compound 1, which has a gallate group, is particularly potent in this respect.3−10 In our previous study, we synthesized oolongtheanins,11,12 which are the catechin dimers contained in oolong tea, and examined their ability to decrease the micellar solubility of cholesterol, using an in vitro micelle model. We found that oolongtheanin-3′-O-gallate (3) showed strong inhibitory activity on cholesterol solubility, comparable to that of 1.13 It is thought that the mechanism underlying the inhibition of cholesterol absorption by catechins involves inhibition of the solubility of the cholesterol in bile acid (Figure 2) micelles in the small intestine. The molecular mechanism by which 1 decreases the micellar solubility of cholesterol has been reported by Kobayashi et al.10 Those authors have concluded that 1 does not bind cholesterol directly, but forms a complex with phosphatidylcholine (PC), which then decreases the micellar solubility of cholesterol.10 However, the inhibitory mechanism is not completely elucidated, as the existence of such a complex has not yet been proven. In this study, to clarify the molecular mechanism by which the micellar solubility of cholesterol is decreased by tea © 2015 American Chemical Society
polyphenols, we investigated the interaction of catechins and oolongtheanins with cholesterol micelles.
■
MATERIALS AND METHODS
Materials. EGCg was a kind gift from Nagara Science Co. (Gifu, Japan). EGC was prepared from EGCg by enzymatic hydrolysis. Oolongtheanin-3′-O-gallate and desgalloyl oolongtheanin were synthesized according to our previous paper.13 PC from egg yolk, cholesterol, and sodium taurocholate were purchased from SigmaAldrich Co. LLC (St. Louis, MO, USA). Sodium glycocholate was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Sodium dodecyl sulfate (SDS) was purchased from Nacalai Tesque Co. (Kyoto, Japan). Deuterium oxide (D2O, 99.8% D) was purchased from Kanto Chemical Co. (Tokyo, Japan). Sample Preparation and 1H NMR Analysis. The micellar solubility of cholesterol in the presence of polyphenols was measured according to previously described methods.10,14 A bile salt micellar solution (in D2O) containing sodium taurocholate (6.6 mmol/L), PC (0.6 mmol/L), cholesterol (0.5 mmol/L), NaCl (132 mmol/L), and phosphate buffer (15 mmol/L, pH 7.0) was prepared by sonication. After storage at room temperature overnight, various concentrations of tea polyphenols were added to the micellar solution, and the mixtures were incubated for 1 h at room temperature. 1H NMR spectra were recorded on a JEOL ECA 600 (Tokyo, Japan) at 600 MHz. HPLC Analysis. Micellar solutions of sodium taurocholate were prepared as described above. The supernatant and precipitate were separated by centrifugation. High-performance liquid chromatography (HPLC) analyses of polyphenols were carried out with a JASCO PU2089 intelligent pump equipped with a JASCO MD-2010 detector and a JASCO CO-2065 column oven (Tokyo, Japan). The supernatant fluid and precipitate were analyzed on a C18 analytical column (NBODS-9 4.6 mm i.d. × 250 mm, Nagara Science Co., Ltd.). The mobile phase consisted of 15% CH3CN and 1% AcOH in H2O. The analyses were carried out at 35 °C, and the flow rate was set at 1.0 mL/min. Received: Revised: Accepted: Published: 204
October 20, 2015 December 10, 2015 December 10, 2015 December 10, 2015 DOI: 10.1021/acs.jafc.5b05088 J. Agric. Food Chem. 2016, 64, 204−209
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of catechins and oolongtheanins.
Figure 2. Structure of bile acid. HPLC analyses of PC and cholesterol were carried out with a DIONEX UltiMate 3000 intelligent pump equipped with a DIONEX corona charged aerosol detector (coronaCAD) and a DIONEX UltiMate 3000 column oven (Tokyo, Japan). The supernatant fluid and precipitate were analyzed on a C18 analytical column (Cosmosil 5C18-MS-II 4.6 mm i.d. × 250 mm, Nacalai Tesque Co., Ltd.). The mobile phase consisted of MeOH. The analyses were carried out at 35 °C, and the flow rate was set at 1.0 mL/min. Rotating Frame Nuclear Overhauser Effect Spectroscopy (ROESY) of Sodium Taurocholate/EGCg. A D2O solution containing 6.6 mmol/L sodium taurocholate and 1.0 mmol/L EGCg was prepared by sonication. ROESY spectra were recorded on a JEOL ECA 600 (JEOL, Japan) at 600 MHz. The typical acquisition parameters used were as follows: 64 scans; 0.028 and 0.9 s acquisition times in the f1 and f2 axes, respectively; mixing time of 0.25 s; and relaxation delay of 2 s. Effect of Different Bile Salts on Micellar Solubility of Cholesterol. Micellar solutions of sodium taurocholate, sodium glycocholate, and sodium dodecyl sulfate were prepared as described above. 1H NMR spectra were measured according to the method described above.
of 1 and 5, and no PC or cholesterol was observed. This indicated that the addition of 1 decreases the micellar solubility of PC and cholesterol. Furthermore, a change was observed in the chemical shift of 1, and the chemical shift of the 3-position was markedly changed. A similar result in the presence of bile salt micelles was reported by Kobayashi et al.10 The seven characteristic signals of 5 (positions at 3, 7, 12, 18, 19, 25, 26)15 were also shifted, and the methyl groups at positions 18 and 19 and the methylene group at position 26 changed markedly. Furthermore, a similar degree of change in the chemical shifts was observed in the mixture of 1 and 5 without PC and cholesterol. These results indicated that 1 interacts with 5, rather than with PC or cholesterol. The effect of the concentration of 1 on the chemical shifts of 5 was investigated. For all seven signals mentioned above, the degree of change in the chemical shifts increases as the concentration of 1 increases. We used 2, 3, and 4 in the same type of experiment. Figure 3 shows a graph that depicts the degree of change in the methyl group at position 18. The degree of change was 3 > 1 ≫ 4 ≫ 2, which was correlated with the difference in the capacity to inhibit cholesterol solubility.13 Furthermore, the change in the chemical shifts of 5 reached a constant level when the concentration of 1 exceeded 13.5 mM or when the concentration of 3 exceeded 10.0 mM. These results suggested that 5 interacts with 1 or 3 and becomes saturated at the above concentrations. In fact, we
■
RESULTS H NMR Measurement. Micellar solutions (in a buffer with D2O) prepared from PC, cholesterol, and sodium taurocholate (5) were analyzed by NMR. When 1 was added to the solution, a precipitate immediately formed. The 1H NMR measurement of the supernatant of these solutions showed only the presence 1
205
DOI: 10.1021/acs.jafc.5b05088 J. Agric. Food Chem. 2016, 64, 204−209
Article
Journal of Agricultural and Food Chemistry
Figure 3. Effect of the polyphenol concentration on the change in the chemical shift at the C18 position (methyl group) in sodium taurocholate (horizontal axis, mM; vertical axis, Δppm).
observed that the change in the chemical shifts depended on the concentration of 1. The degree of change in the chemical shifts correlated inversely with the concentration of 1, suggesting that an equilibrium exists between bound and uninteracted 1 at a high concentration, whereas all of 1 interacts with 5 at a low concentration. We propose that 1 molecule of sodium taurocholate interacts with 2 molecules of 1 or 1.5 molecules of 3, considering the concentration of sodium taurocholate in the micellar solution and the concentration of polyphenols at saturation. HPLC Analysis. HPLC analysis was carried out to examine whether 1, PC, and cholesterol were contained in the precipitate or supernatant following the addition of EGCG to bile salt micelles. First, we analyzed PC and cholesterol (Figure 4). When 2.5 mM 1 was added to the solution, approximately 40% of PC and cholesterol was contained in the supernatant. When the concentration was increased to 5 mM, these compounds disappeared from the supernatant and were contained quantitatively in the precipitate. Then, an analysis was performed for 1 (Figure S4). Most of 1 was found in the supernatant, and only a trace amount was found in the precipitate. The addition of 3 yielded similar results (Figures S3 and S5). From these results, it was suggested that there was no direct interaction of 1 with PC and cholesterol. ROESY of Sodium Taurocholate/EGCg. ROESY is a twodimensional NMR measurement method for detecting a correlation of the nuclear protons that are spatially in close proximity. It is possible to detect signals if the proton distance is within about 4.5 Å. The results we had found so far suggested that interaction between 5 and 1 was regiospecific. We therefore performed ROESY analysis to investigate the positional relationship between these two molecules. Figure 5 shows the ROESY spectrum of the mixture of 5 and 1. A number of intramolecular correlations were observed, including intermolecular correlation between the gallate group protons of 1 and the methyl groups (at positions 18 and 19) of 5. Furthermore, the ROESY spectrum of a mixture of 5 and 3 showed similar results (Figure S6). These results were in good agreement with the results obtained by observing the change in chemical shifts of 5 upon addition of 1. In addition, this result suggests the possibility of hydrogen bond formation between the galloyl group of EGCg and the OH group at the R3 position of 5, because the OH group at R3 is close to the methyl groups at positions 18 and 19. However, because of complex correlations, it was challenging to obtain details of the
Figure 4. HPLC analysis of cholesterol micellar solution after the addition of EGCg: (a) supernatant fluid; (b) precipitate (detector, coronaCAD; eluent, MeOH; column temperature, 35 °C; flow rate, 1.0 mL/min).
ROESY correlations for signals around 1.0−2.3 ppm. These results clearly indicated that the gallate group protons of 1 are in close proximity to the steroid skeleton of 5. Effect of Different Bile Salts on the Micellar Solubility of Cholesterol. The ROESY results revealed a spatially close relationship between 5 and 1. However, the actual sites on the two molecules involved in this interaction were not clear. Therefore, we examined the ability of 1 to interact with an amphiphilic substance with a structure similar to that of 5 (Figure 6). Sodium glycocholate (6), one of the constituents of bile acids with a steroid skeleton similar to that of 5, and sodium dodecyl sulfate (7), which has a sulfate group, were used as amphiphiles. Micelle solutions were prepared with 6 or 7 and sonicated, and NMR was performed; then, the formation of micelles was observed. When 1 was added to these samples, a change in the chemical shifts was seen for the solution containing 6, which was similar to that observed with 5; however, no such change in chemical shifts was observed with 7. From these results, we could deduce that the steroid skeleton is important for the interaction between 1 and 5, and the sulfate group is not involved. When we added 1 after generating micelles in the presence of 6 or 7, we noted precipitation in the solution containing 6. Thus, there appears to be no interaction between 1 and PC or cholesterol, or such an interaction is not involved in the decrease of the micellar solubility of cholesterol. On the other hand, hydrogen and hydrophobic bonds are well-known intermolecular forces. It is possible that the intermolecular interactions between 1 and 5 are mediated by hydrogen bonding, because of the three hydroxyl groups in 206
DOI: 10.1021/acs.jafc.5b05088 J. Agric. Food Chem. 2016, 64, 204−209
Article
Journal of Agricultural and Food Chemistry
Figure 5. ROESY spectrum of sodium taurocholate with EGCg at 297 K.
deduced from the NMR chemical shift changes and HPLC analysis of the components of the precipitate and supernatant. Furthermore, our findings revealed that the steroid skeleton of 5 is important for this interaction and that the gallate group of 1 is arranged in spatial proximity to this skeleton. Therefore, we propose that the mechanism by which the addition of 1 lowers the solubility of PC and cholesterol in micelles involves the interaction between 5 and 1. Although we have previously reported that 3 shows a potent inhibitory effect, comparable to that of 1, on the micellar solubility of cholesterol, there was no difference except the degree of change in the chemical shifts between 1 and 3.13 However, we could not observe changes in the chemical shifts, such as those caused by the addition of 1 or 3, or the formation of a precipitate, by addition of methyl gallate, which corresponds to the gallate moiety (data not shown). This suggests that steric factors may be involved in the strength of the inhibitory activity.
compound 5. Therefore, to clarify the role of hydrogen bonds in the intermolecular interaction between 1 and 5, 1H NMR analysis was performed for sodium taurodeoxycholate (8) or sodium taurochenodeoxycholate (9), which have fewer hydroxyl groups than 5 (Figure S8). The extent of change in the chemical shifts of 8 and 9 was approximately 33% less than that seen for 5. These results indicated that hydrogen bonding is involved in the interaction between 1 and 5.
■
DISCUSSION The reason for the decreased solubility of cholesterol micelles in the presence of 1 or oolongtheanins is assumed to be the interaction of these compounds with one or more components of the micelles (PC, cholesterol, and/or bile acid). A previous NMR study showed that the interaction between 1 and PC inhibited the solubility of cholesterol micelles.10 However, in this study, our data strongly suggested that 1 interacts with 5 regiospecifically, rather than with PC and cholesterol, as 207
DOI: 10.1021/acs.jafc.5b05088 J. Agric. Food Chem. 2016, 64, 204−209
Article
Journal of Agricultural and Food Chemistry
PC and cholesterol, as follows. The elimination of PC and cholesterol from bile acid micelle is caused by the capture of compound 5 by a hydrophobic space formed by molecules of compound 1 or 3. We consider that the result of this study contributes to the elucidation of the relationship between chemical structure and biological activity of tea polyphenols. Some of the complex signals in the NMR experiment overlapped, so that these data could not be fully analyzed. Hence, further studies using another approach should be performed to completely elucidate this mechanism.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05088. Figures S1−S8 (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*(E.Y.) Phone/fax: +08-58-293-2914. E-mail: e-yanase@gifu-u. ac.jp. Funding
This work was supported by a JSPS Kakenhi Grant (15K07427) and by the Toyo Institute of Food Technology. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Editage (www.editage.jp) for English language editing. We thank Prof. Kohei Nakano for his help to perform the coronaCAD HPLC analysis.
■
Figure 6. Effect of EGCg on the change in chemical shift of cholesterol micelles in the presence of three different bile salts (vertical axis, Δppm): (a) sodium taurocholate; (b) sodium glycocholate; (c) sodium dodecyl sulfate.
ABBREVIATIONS USED coronaCAD, corona charged aerosol detector; EC, epicatechin; ECg, epicatechin gallate; EGC, epigallocatechin; EGCg, epigallocatechin gallate; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; ROESY, rotating frame nuclear Overhauser effect spectroscopy; SDS, sodium dodecyl sulfate
Ishizu et al. reported an X-ray crystallographic analysis that was performed on a crystal obtained from an aqueous solution of EGCg and caffeine molecule. They revealed that the caffeine molecule was captured by a hydrophobic space formed by the A, B, and gallate rings of two EGCg molecules.16 In addition, an upfield shift of the proton signals in diketopiperazines cyclo (Pro-Gly) was observed in NMR measurement when EGCg was added, which was considered to result mainly from the magnetic anisotropic shielding effect of the ring current from 1. Similarly, in our NMR study, an upfield shift of the proton signals in 5 was observed, which suggested that 5 is captured by a hydrophobic space formed by the EGCg molecule(s).17 Furthermore, the NMR experiment indicated that interaction between 1 and 8 or 9 was weaker than that involving 5. This result suggests that bile acids are held strongly in the hydrophobic site via hydrogen bonding. In addition, Raederstorff et al. measured the micelle particle size using dynamic light scattering and reported that the diameter gradually increased when EGCg was added to the micellar solution.18 Their result indicates that the bile acid micelle structure is altered by the interaction with EGCg, and this is in agreement with our result. From these results, we propose a novel molecular mechanism by which compound 1 or 3 decreases the micellar solubility of
■
REFERENCES
(1) Nanjo, F.; Honda, M.; Okushio, K.; Matsumoto, N.; Ishigaki, F.; Ishigami, T.; Hara, Y. Effects of dietary tea catechins on alphatocopherol levels, lipid peroxidation, and erythrocyte deformability in rats fed on high palm oil and perilla oil diets. Biol. Pharm. Bull. 1993, 16, 1156−1159. (2) Okuda, T.; Mori, K.; Hayatsu, H. Effect of tannins on directacting mutagens. Chem. Pharm. Bull. 1984, 32, 3755−3758. (3) Ikeda, I.; Imasato, Y.; Sasaki, E.; Nakayama, M.; Nagao, H.; Takeo, T.; Yayabe, F.; Sugano, M. Tea catechins decrease micellar solubility and intestinal absorption of cholesterol in rats. Biochim. Biophys. Acta, Lipids Lipid Metab. 1992, 1127, 141−146. (4) Kajiya, K.; Kumazawa, S.; Nakayama, T. Steric effects on interaction of tea catechins with lipid bilayers. Biosci., Biotechnol., Biochem. 2001, 65, 2638−2643. (5) Yang, H.-M.; Wang, H.-C.; Chen, H.-L. Green, oolong and black tea extracts modulate lipid metabolism in hyperlipidemia rats fed highsucrose diet. J. Nutr. Biochem. 2001, 12, 14−20. (6) Ikeda, I.; Kobayashi, M.; Hamada, T.; Tsuda, T.; Goto, H.; Imaizumi, K.; Nozawa, A.; Sugimoto, A.; Kakuda, T. Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in
208
DOI: 10.1021/acs.jafc.5b05088 J. Agric. Food Chem. 2016, 64, 204−209
Article
Journal of Agricultural and Food Chemistry epigallocatechin gallate and epicatechin gallate. J. Agric. Food Chem. 2003, 51, 7303−7307. (7) Kobayashi, M.; Unno, T.; Suzuki, Y.; Nozawa, A.; Sagesaka, Y.; Kakuda, T.; Ikeda, I. Heat-Epimerized Tea Catechins Have the Same Cholesterol-Lowering Activity as Green Tea Catechins in CholesterolFed Rats. Biosci., Biotechnol., Biochem. 2005, 69, 2455−2458. (8) Toyoda-Ono, Y.; Yoshimura, M.; Nakai, M.; Fukui, Y.; Asami, S.; Shibata, H.; Kiso, Y.; Ikeda, I. Suppression of postprandial hypertriglyceridemia in rats and mice by oolong tea polymerized polyphenols. Biosci., Biotechnol., Biochem. 2007, 71, 971−976. (9) Ikeda, I.; Yamahira, T.; Kato, M.; Ishikawa, A. Black-tea polyphenols decrease micellar solubility of cholesterol in vitro and intestinal absorption of cholesterol in rats. J. Agric. Food Chem. 2010, 58, 8591−8595. (10) Kobayashi, M.; Nishizawa, M.; Inoue, N.; Hosoya, T.; Yoshida, M.; Ukawa, Y.; Sagesaka, Y. M.; Doi, T.; Nakayama, T.; Kumazawa, S.; Ikeda, I. Epigallocatechin gallate decreases the micellar solubility of cholesterol via specific interaction with phosphatidylcholine. J. Agric. Food Chem. 2014, 62, 2881−2890. (11) Hashimoto, F.; Nonaka, G.; Nishioka, I. Evaluation of the antioxidative effect (in vitro) of tea polyphenols. Chem. Pharm. Bull. 1988, 36, 1676−1684. (12) Matsuo, Y.; Tadakuma, F.; Shii, T.; Saito, Y.; Tanaka, T. Selective oxidation of pyrogallol-type catechins with unripe fruit homogenate of Citrus unshiu and structural revision of oolongtheanins. Tetrahedron 2015, 71, 2540−2548. (13) Ogawa, K.; Hirose, S.; Yamamoto, H.; Shimada, M.; Nagaoka, S.; Yanase, E. Synthesis of oolongtheanins and their inhibitory activity on micellar cholesterol solubility in vitro. Bioorg. Med. Chem. Lett. 2015, 25, 749−752. (14) Nagaoka, S.; Nakamura, A.; Shibata, H.; Kanamaru, Y. Soystatin (VAWWMY), a novel bile acid-binding peptide, decreased micellar solubility and inhibited cholesterol absorption in rats. Biosci., Biotechnol., Biochem. 2010, 74, 1738−1741. (15) Kim, H. J.; Yim, S. H.; Lee, I. S. A cholesterol biosynthesis inhibitor from Rhizopus oryzae. Arch. Pharmacal Res. 2004, 27, 624− 627. (16) 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. (17) Ishizu, T.; Tsutsumi, H.; Yokoyama, A. Chiral recognition of diketopiperazines cyclo(Pro-Gly) using (−)-epigallocatechin-3-Ogallate. Tetrahedron Lett. 2015, 56, 1111−1114. (18) Raederstorff, D. G.; Schlachter, M. F.; Elste, V.; Weber, P. Effect of EGCG on lipid absorption and plasma lipid levels in rats. J. Nutr. Biochem. 2003, 14, 326−332.
209
DOI: 10.1021/acs.jafc.5b05088 J. Agric. Food Chem. 2016, 64, 204−209
