Article pubs.acs.org/JAFC
Effects of Metabolites Produced from (−)-Epigallocatechin Gallate by Rat Intestinal Bacteria on Angiotensin I‑Converting Enzyme Activity and Blood Pressure in Spontaneously Hypertensive Rats Akiko Takagaki and Fumio Nanjo*
Downloaded by KAROLINSKA INST on September 13, 2015 | http://pubs.acs.org Publication Date (Web): September 11, 2015 | doi: 10.1021/acs.jafc.5b03676
Food Research Laboratories, Mitsui Norin Company, Ltd., 223-1 Miyabara, Fujieda-shi, Shizuoka 426-0133, Japan ABSTRACT: Inhibitory activity of angiotensin I-converting enzyme (ACE) was examined with (−)-epigallocatechin gallate (EGCG) metabolites produced by intestinal bacteria, together with tea catechins. All of the metabolites showed ACE inhibitory activities and the order of IC50 was hydroxyphenyl valeric acids > 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (1) > trihydroxyphenyl 4-hydroxyvaleric acid ≫ dihydroxyphenyl 4-hydroxyvaleric acid ≫ 5-(3,5-dihydroxyphenyl)-γ-valerolactone (2). Among the catechins, galloylated catechins exhibited stronger ACE inhibitory activity than nongalloylated catechins. Furthermore, the effects of a single oral intake of metabolites 1 and 2 on systolic blood pressure (SBP) were examined with spontaneously hypertensive rats (SHR). Significant decreases in SBP were observed between 2 h after oral administration of 1 (150 mg/kg in SHR) and the control group (p = 0.002) and between 4 h after administration of 2 (200 mg/kg in SHR) and the control group (p = 0.044). These results suggest that the two metabolites have hypotensive effects in vivo. KEYWORDS: epigallocatechin gallate, intestinal bacteria, metabolite, valerolactone, angiotensin I-converting enzyme, blood pressure, spontaneously hypertensive rat
■
INTRODUCTION Green tea catechins are well-known to show a variety of health benefit functions including antioxidative,1−4 blood cholesterol lowering,5 hypoglycemic,6 cancer preventive,7 and blood pressure lowering activities.8,9 Studies conducted in our laboratories have shown that long-term oral intake of a tea catechin mixture suppressed an increase in blood pressure in spontaneously hypertensive rats (SHR), delayed the outbreak of stroke, and extended the lifespan of stroke-prone SHR (SHRSP).8 In addition, we hypothesized that the mechanism of hypotensive action by tea catechins was due to the inhibitory activity against angiotensin I-converting enzyme (ACE).8 The hypotensive action of green tea polyphenols, including catechins, has been observed in human studies.10−12 Recently, it has been reported that chronic intake (3 weeks) of (−)-epigallocatechin gallate (EGCG), a major green tea polyphenol, reduced systolic blood pressure (SBP) in SHR and that the hypotensive effects may be due to endotheliumdependent vasodilation, which is caused by the augmented production of nitric acid (NO) through the activation of endothelial NO synthase (eNOS) by EGCG.13,14 However, it has been reported that the absorption rate of EGCG was 0.1−1.6% of the oral dose in rats.15 In a previous paper we also estimated the bioavailability of intact EGCG, including its conjugates, to be 0.26% after oral administration of [4-3H]EGCG in rats.16 Del Rio et al.17 have reported the bioavailability of tea catechins including EGCG, (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), and (−)-epicatechin (EC) would be 7) > trihydroxyphenyl-γ-valerolactone (1) > trihydroxyphenyl 4hydroxyvaleric acid (3) ≫ dihydroxyphenyl 4-hydroxyvaleric acid (4) ≫ dihydroxyphenyl-γ-valerolactone (2). With regard to ACE inhibitory activities of the catechins, galloylated catechins (EGCG, GCG, CG, and ECG) showed stronger inhibitory activities than nongalloylated catechins (EGC, EC, and C). Hypotensive Activities of Metabolites 1 and 2 after a Single Oral Dosage to SHR. All of the EGCG metabolites tested showed ACE inhibitory activity, although their activity was of varying degrees. To examine the hypotensive activity of these metabolites after a single oral administration to SHR, we selected 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (1) and 5(3,5-dihydroxyphenyl)-γ-valerolactone (2) because they are reported to be not only the dominant metabolites in the gut tract18 but also the major urinary metabolites in humans and rats,16,17,19,27 whereas the other metabolites were scarcely found in urine, if at all. Metabolite 1 was orally administered to SHR at a dosage of 100 or 150 mg/kg. Basal SBP values (mm of Hg) were 189.0 ± 4.7 for the control group versus 192.1 ± 3.7 for the 100 mg/kg dosage group and 193.9 ± 4.7 for the control group versus 193.4 ± 2.4 for the 150 mg/kg dosage group. The administration of 100 mg/kg of 1 brought about no significant decrease in SBP as compared with the control group. However, a significant decrease in SBP was observed between basal SBP (SBP before administration) and SBP 4 h after administration (−14.9 ± 5.6 mm of Hg, p = 0.029, Dunnett’s t test) in the 100 mg/kg dose group, but no significant change was observed in the control group as shown in Table 2. On the other hand, as shown in Figure 2A SBP significantly reduced 2 h after oral dosage of 150 mg/kg of 1 as compared with the control group C
DOI: 10.1021/acs.jafc.5b03676 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Downloaded by KAROLINSKA INST on September 13, 2015 | http://pubs.acs.org Publication Date (Web): September 11, 2015 | doi: 10.1021/acs.jafc.5b03676
Figure 2. Effects of a single oral administration of metabolites 1 and 2 on systolic blood pressure in SHR. SBP values are expressed as means ± SEM. Statistical analysis was performed using Student’s t test with the Bonferroni correction. Significant difference (p value) from the control group is shown.
metabolites 1 and 2 are at work remains unanswered. Further study is needed to clarify the mechanism of their hypotensive activity in vivo. It is well recognized that EGCG has a variety of physiological activities including suppression of cholesterol and triglyceride in the blood,5 suppression of glucose level in the blood,6 antihypertensive activity,8−10 antioxidant activity,1−4 and chemopreventive activity.7 Among the physiological activities, it is explainable to some extent that suppression of cholesterol in blood may be due to the prevention of cholesterol absorption through the small intestine by intact EGCG28 and that suppression of glucose levels in the blood may be due to the inhibition of α-amylase and sucrase by intact EGCG in the small intestine.29,30 However, it is doubtful in some cases whether intact EGCG actually acts as a major bioactive substance, because it has been reported to be poorly absorbed in the body.15−17 On the other hand, intestinal metabolites such as 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (1) and 5(3,5-dihydroxyphenyl)-γ-valerolactone (2) have been reported to be absorbed in large amounts in the body,16,17,19 leading us to suppose that the metabolites may act as in vivo bioactive substances. In this context, we previously examined the antioxidative activity of EGCG metabolites and demonstrated they possessed antioxidative activity at least equivalent to that of Trolox, although their activities were weaker than those of their original catechins.20 Among them, metabolites 1 and 2 may be the main antioxidant contributors in the body due to their good absorptivity. In this study, we observed that the EGCG metabolites had ACE inhibitory activity in vitro and that two major metabolites 1 and 2 had hypotensive effects in in vivo experiments. However, the relationship between ACE inhibitory activity and hypotensive effects remains to be investigated. Therefore, it seems reasonable to surmise that blood pressure lowering effects by catechin intake may be due, in part, to the hypotensive effects of metabolites 1 and 2. Thus, study on the biological activity of intestinal metabolites such as 1 and 2 likely makes it possible to explain some of the health beneficial effects of tea catechins in the body. Accordingly, further in vivo studies on the physiological activity of the metabolites will be required.
higher than that of EGCG based on their urinary excretion amounts.15−17,19 From the above observations, it was expected that these metabolites could show hypotensive effects in vivo. Accordingly, we examined hypotensive effects of metabolites 1 and 2 in in vivo experiments with SHR. The experiments clearly demonstrated hypotensive effects were observed after a single oral administration of the metabolites to SHR. In addition, our preliminary study on the metabolism of compound 1 showed that after a single oral administration of 5 mg/rat to SHR the compound peaked at 1 h, then decreased gradually, and at >4 h after dosage had almost disappeared from the rat plasma. During this period of time only a slight amount of degraded products derived from 1 was detected in plasma (data not shown). In the case of metabolite 2, we did not examine its metabolism, but it is presumed that results similar to those for metabolite 1 would be obtained, judging from the similarity of their chemical structures and results of our previous study.16 Therefore, it is likely that the two metabolites do actually possess hypotensive effects in vivo. With respect to the mechanism for hypotensive activity of metabolites 1 and 2 in vivo, we found that these metabolites possessed ACE inhibitory activity. Although a direct relationship between the ACE inhibitory activity and hypotensive effects of the metabolites has not yet been proven and the mechanism remains unclear, the possibility cannot be denied that ACE inhibitory activities may be associated with their hypotensive effects. Apart from the mechanism proposed above, nitric oxide (NO) released from endothelium is considered to function as an important factor for vasodilation and consequently to reduce blood pressure.13,14 Hence, the reduction of NO bioavailability by active oxygen species would cause endothelial dysfunction and, in the worst case, cardiovascular complications. Accordingly, antioxidants are thought to play an important role in the suppression of hypertension by preventing the reduction of NO bioavailability in arteries.11,14 Our previous study revealed that intestinal catechin metabolites including metabolites 1 and 2 possessed antioxidative activities and their activities were stronger than or nearly equal to those of Trolox, a vitamin E derivative.20 It is therefore possible that the metabolites may contribute as antioxidants, at least in part, to suppress the increase in blood pressure through the prevention of NO reduction by active oxygen species. As discussed above, the question of how mechanisms responsible for the hypotensive effects of D
DOI: 10.1021/acs.jafc.5b03676 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
■
myocardial I/R injury in SHR. Am. J. Physiol. Endocrinol. Metab. 2007, 292, E1378−E1387. (14) Grassi, D.; Desideri, G.; Di Giosia, P.; De Feo, M.; Fellini, E.; Cheli, P.; Ferri, L.; Ferri, C. Tea, flavonoids, and cardiovascular health: endothelial protection. Am. J. Clin. Nutr. 2013, 98 (Suppl.), 1660S− 1666S. (15) Chen, L.; Lee, M.-J.; Li, H.; Yang, C. S. Absorption, distribution, and elimination of tea polyphenols in rats. Drug Metab. Dispos. 1997, 25, 1045−1050. (16) Kohri, T.; Matsumoto, N.; Yamakawa, M.; Suzuki, M.; Nanjo, F.; Hara, Y.; Oku, N. Metabolic fate of (−)-[4-3H]epigallocatechin gallate in rats after oral administration. J. Agric. Food Chem. 2001, 49, 4102−4112. (17) Del Rio, D.; Calani, L.; Cordero, C.; Salvantore, S.; Pellegrini, N.; Brighenti, F. Bioavailability and catabolism of green tea flavan-3-ols in humans. Nutrition 2010, 26, 1110−1116. (18) Takagaki, A.; Nanjo, F. Metabolism of (−)-epigallocatechin gallate by rat intestinal flora. J. Agric. Food Chem. 2010, 58, 1313− 1321. (19) Li, C.; Lee, M.-J.; Sheng, S.; Meng, X.; Prabhu, S.; Winnik, B.; Huang, B.; Chung, J. Y.; Yan, S.; Ho, C.-T.; Yang, C. S. Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion. Chem. Res. Toxicol. 2000, 13, 177−184. (20) Takagaki, A.; Otani, S.; Nanjo, F. Antioxidative activity of microbial metabolites of (−)-epigallocatechin gallate produced in rat intestines. Biosci., Biotechnol., Biochem. 2011, 75, 582−585. (21) Lambert, J. D.; Rice, J. E.; Hong, J.; Hou, Z.; Yang, C. S. Synthesis and biological activity of the tea catechin metabolites, M4 and M6 and their methoxy-derivatives. Bioorg. Med. Chem. Lett. 2005, 15, 873−876. (22) Unno, T.; Tamemoto, K.; Yayabe, F.; Kakuda, T. Urinary excretion of 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, a ring-fission metabolite of (−)-epicatechin, in rats and its in vitro antioxidant activity. J. Agric. Food Chem. 2003, 51, 6893−6898. (23) Uhlenhut, K.; Högger, P. Facilitated cellular uptake and suppression of inducible nitric oxide synthase by a metabolite of maritime pine bark extract (pycnogenol). Free Radical Biol. Med. 2012, 53, 305−313. (24) Takagaki, A.; Nanjo, F. Catabolism of (+)-catechin and (−)-epicatechin by rat intestinal microbiota. J. Agric. Food Chem. 2013, 61, 4927−4935. (25) Takagaki, A.; Kato, Y.; Nanjo, F. Isolation and characterization of rat intestinal bacteria involved in biotransformation of (−)-epigallocatechin. Arch. Microbiol. 2014, 196, 681−695. (26) Seto, R.; Nakamura, H.; Nanjo, F.; Hara, Y. Preparation of epimers of tea catechins by heat treatment. Biosci., Biotechnol., Biochem. 1997, 61, 1434−1439. (27) Sang, S.; Lee, M.-J.; Yang, I.; Buckley, B.; Yang, C. S. Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition. Rapid Commun. Mass Spectrom. 2008, 22, 1567−1578. (28) 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. 1992, 1127, 141−146. (29) Hara, Y.; Honda, M. The inhibition of α-amylase by tea polyphenols. Agric. Biol. Chem. 1990, 54, 1939−1945. (30) Honda, M.; Hara, Y. Inhibition of rat small intestinal sucrase and α-glucosidase activities by tea polyphenols. Biosci., Biotechnol., Biochem. 1993, 57, 123−124.
AUTHOR INFORMATION
Corresponding Author
*(F.N.) E-mail:
[email protected]. Phone: +81-54-6390080. Fax: +81-54-648-2001. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We acknowledge the assistance of Andrea K. Suzuki (Mitsui Norin Co., Ltd.) in the review of the manuscript.
Downloaded by KAROLINSKA INST on September 13, 2015 | http://pubs.acs.org Publication Date (Web): September 11, 2015 | doi: 10.1021/acs.jafc.5b03676
■
ABBREVIATIONS USED ACE, angiotensin I-converting enzyme; SHR, spontaneously hypertensive rats; SBP, systolic blood pressure; EGCG, (−)-epigallocatechin gallate; EC, (−)-epicatechin; EGC, (−)-epigallocatechin; ECG, (−)-epicatechin gallate; C, (−)-catechin; GCG, (−)-gallocatechin gallate; CG, (−)-catechin gallate
■
REFERENCES
(1) Nanjo, F.; Honda, M.; Okushio, K.; Matsumoto, N.; Ishigaki, F.; Ishigami, T.; Hara, Y. Effects of dietary tea catechins on α-tocopherol 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) Nanjo, F.; Goto, K.; Seto, R.; Suzuki, M.; Sakai, M.; Hara, Y. Scavenging effects of tea catechins and their derivatives on 1,1diphenyl-2- picrylhydrazyl radical. Free Radical Biol. Med. 1996, 21, 895−902. (3) Nanjo, F.; Mori, M.; Goto, K.; Hara, Y. Radical scavenging activity of tea catechins and their related compounds. Biosci., Biotechnol., Biochem. 1999, 63, 1621−1623. (4) Higdon, J. V.; Frei, B. Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions. Crit. Rev. Food Sci. Nutr. 2003, 43, 89−143. (5) Muramatsu, K.; Fukuyo, M.; Hara, Y. Effect of green tea catechins on plasma cholesterol level in cholesterol-fed rats. J. Nutr. Sci. Vitaminol. 1986, 32, 613−622. (6) Matsumoto, N.; Ishigaki, F.; Ishigaki, A.; Iwashina, H.; Hara, Y. Reduction of blood glucose levels by tea catechin. Biosci., Biotechnol., Biochem. 1993, 57, 525−527. (7) Yang, C. S.; Lambert, J. D.; Hou, Z.; Ju, J.; Lu, G.; Hao, X. Molecular targets for the cancer preventive activity of tea polyphenols. Mol. Carcinog. 2006, 45, 431−435. (8) Hara, Y. Hypotensive action of tea polyphenols. In Green Tea: Health Benefits and Applications; CRC Press, Taylor & Francis Group: Boca Raton, FL, USA, 2001; pp 139−148. (9) Negishi, H.; Xu, J.-W.; Ikeda, K.; Njelekela, M.; Nara, Y.; Yamori, Y. Black and green tea polyphenols attenuate blood pressure increases in stroke-prone spontaneously hypertensive rats. J. Nutr. 2004, 134, 38−42. (10) Hodgson, J. M.; Devine, A.; Puddey, I. B.; Chan, S. Y.; Beilin, L. J.; Prince, R. L. Tea intake is inversely related to blood pressure in older women. J. Nutr. 2003, 133, 2883−2886. (11) Galleano, M.; Pechanova, O.; Fraga, C. G. Hypertension, nitric oxide, oxidants, and dietary plant polyphenols. Curr. Pharm. Biotechnol. 2010, 11, 837−848. (12) Medina-Remón, A.; Estruch, R.; Tresserra-Rimbau, A.; Vallverdú-Queralt, A.; Lamuela-Raventos, R. M. The effect of polyphenol consumption on blood pressure. Mini-Rev. Med. Chem. 2013, 13, 1137−1149. (13) Potenza, M.; Marasciulo, F. L.; Tarquinio, M.; Tiravanti, E.; Colantuono, G.; Federici, A.; Kim, J.; Quon, M. J.; Montagnani, M. EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against E
DOI: 10.1021/acs.jafc.5b03676 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
