Inhibitory activity of catechin metabolites produced by intestinal microbiota on proliferation of heLa cells

Article abstract of DOI:10.1248/bpb.b17-00127

Eleven kinds of catechin metabolites produced from (?)-epigallocatechin (EGC) and (?)-epigallocatechin gallate (EGCg) by intestinal microbiota were evaluated for inhibitory activity on the proliferation of HeLa cells, which are human cervical cancer cells

Full text of DOI:10.1248/bpb.b17-00127

Vol. 40, No. 8
Biol. Pharm. Bull. 40, 1331–1335 (2017)
1331
Note
Inhibitory Activity of Catechin Metabolites Produced by Intestinal
Microbiota on Proliferation of HeLa Cells
Aya Hara-Terawaki*, Akiko Takagaki, Hirotsugu Kobayashi, and Fumio Nanjo*
Food Research Laboratories, Mitsui Norin Co., Ltd.; 223–1 Miyabara, Fujieda, Shizuoka 426–0133, Japan.
Received February 9, 2017; accepted April 18, 2017
Eleven kinds of catechin metabolites produced from (ꢀ)-epigallocatechin (EGC) and (ꢀ)-epigallocatechin
gallate (EGCg) by intestinal microbiota were evaluated for inhibitory activity on the proliferation of HeLa
cells, which are human cervical cancer cells. Among the catechin metabolites, 1-(3,4,5-trihydroxyphenyl)-
3-(2,4,6-trihydroxyphenyl)propan-2-ol (EGC-M2), 4-hydroxy-5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-
M7), and 5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M9) were found to show inhibitory activity on HeLa
cell proliferation as compared with control. The results suggested that three adjacent hydroxyl groups in the
phenyl moiety may play an important role in the inhibitory activity. In addition, the inhibitory activity was
also examined with four (ꢀ)-epicatechin (EC) metabolites possessing two adjacent hydroxyl groups in the
phenyl moiety. Only 5-(3,4-dihydroxyphenyl)valeric acid (EC-M9) showed inhibitory activity and therefore
valeric acid moiety likely contributes to the inhibitory activity. EGC-M9 showed the strongest inhibitory ac-
tivity with IC50 of 5.58µM. Thus, in this study it was found for the first time that several catechin metabolites
derived from EGC, EGCg, and EC inhibit the proliferation of cervical cancer cells.
Key words epigallocatechin gallate; metabolite; cervical cancer; catechin; 5-(3,4,5-trihydroxyphenyl)valeric
acid; HeLa cell
Tea catechins are natural constituents which have vari- treatments are urgently required since the rate of progress is
ous physiological functions such as antioxidative,1,2) blood rapid. In general, the main cervical cancer treatments are sur-
glucose-lowering,3) hypotensive,4,5) and anticancer activities.6) gery, radiation therapy and chemotherapy. The cervical cancer
However, it has been reported that the absorption rate of a treating agents used are bleomycin, paclitaxel, cisplatin, and
major tea catechin, (−)-epigallocatechin gallate (EGCg), was so on. These treatments can have anticancer effects on the
0.1 to 1.6% of the oral dose in rats.7) We also estimated the whole body, but they have the risk of side effects such as em-
bioavailability of EGCg including its conjugated forms to be esis, hair loss and leukopenia. Tea catechins including (+)-cat-
0.26% after oral dosage of [4-3H]EGCg in rats.8) In humans, echin (catechin hydrate) and EGCg have been reported to
the bioavailability of tea catechins has been reported to be inhibit proliferation of human cervical cells (SiHa and HeLa
less than 4%.9) Thus, intact tea catechins have been regarded cells) and their inhibitory mechanisms were discussed.24–27)
as being poorly absorbed in the body. On the other hand, tea However, anti-proliferative activity of catechin metabolites
catechins are recognized to be metabolized by intestinal mi- produced by intestinal microbiota against cervical cancer cells
crobiota after reaching the colon.10–13) The metabolites such as is not known.
5-hydroxyphenyl-γ-valerolactones, 4-hydroxy-5-hydroxyphenyl
In this study, we evaluated the effects of catechin metabo-
valeric acids, and 3-hydroxyphenyl propionic acids were lites on proliferation of HeLa cells as a model for human cer-
absorbed into the body and were detected in urine.14–18) Par- vical cancer.
ticularly, 5-hydroxyphenyl-γ-valerolactones were recognized
to be dominant urinary metabolites in rats and human urine
and their excretion amounts reached 6–39% of the ingested
catechins.8,9,19)
MATERIALS AND METHODS
Materials The following catechin metabolites were pre-
Catechin metabolites have been reported to show bioactivi- pared using the methods we reported previously.10–13)
ties such as antioxidative, anti-inflammatory, anti-thrombotic,
2-(3,5-Dihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-
and blood pressure lowering activities.18,20,21) Kim et al. re- 3,5,7-triol (EGC-M1), 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-
cently showed that 5-(3,5-dihydroxyphenyl)-γ-valerolactone, trihydroxyphenyl)propan-2-ol (EGC-M2), 1-(3,5-dihydroxy-
one of the major metabolites of EGCg, increased the activity phenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (EGC-M3), 4-
of CD4+ T cells and enhanced the cytotoxic activity of natural hydroxy-5-(3,5-dihydroxyphenyl)valeric acid (EGC-M4), 5-
killer cells.22) Thus, it is expected catechin metabolites may (3,5-dihydroxyphenyl)-γ-valerolactone (EGC-M5), 3-(3,5-
exhibit additional biological activities. With regard to anti- hydroxyphenyl)propionic acid (EGC-M6), 4-hydroxy-5-(3,4,5-
cancer effects, 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone has trihydroxyphenyl)valeric acid (EGC-M7), 5-(3,4,5-trihydroxy-
shown growth inhibition of human colon adenocarcinoma phenyl)-γ-valerolactone (EGC-M8), 5-(3,5-dihydroxyphenyl)-
cells (HT-29 and HCT-116) and esophageal squamous carci- valeric acid (EGC-M10), 1-(3,4-dihydroxyphenyl)-3-(2,4,6-
noma cells (KYSE150).23)
trihydroxyphenyl)propan-2-ol (EC-M1), 4-hydroxy-5-(3,4-
Cervical cancer is one of the most commonly occurring dihydroxyphenyl)valeric acid (EC-M3), 5-(3,4-dihydroxy-
malignant tumors in women. Recently, an increasing number phenyl)-γ-valerolactone (EC-M4). All other chemicals were
of younger women are contracting this disease and effective available products of analytical or HPLC grade.
*To whom correspondence should be addressed. e-mail: aya_hara@mitsui-norin.co.jp; nanjo@ric-shizuoka.or.jp
© 2017 The Pharmaceutical Society of Japan

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Biol. Pharm. Bull.
Vol. 40, No. 8 (2017)
Chemical Synthesis of 5-(3,4-Dihydroxyphenyl)levu-
increases to 100% B from 3 to 15min and held at 100% B for
3min, then eluted with 90% A and 10% B from 18 to 18.5min,
and finally equilibrated with 90% A and 10% B for 3.5min.
Finally, 0.02mmol (5.5mg) of 5-(3,4-dihydroxyphenyl)-
levulinic acid was obtained and its structure was confirmed
linic Acid (EC-M7) EC-M4 (0.49mmol, 102.7mg) and
N,N-diisopropylethylamine (1.96mmol, 510µL, Tokyo Chemi-
cal Ind. Co., Ltd. Tokyo, Japan) were dissolved in 2.0mL of
acetone. To the solution was added chloromethyl methyl ether
(1.00mmol, 158µL, Kanto Chemical Co., Inc., Tokyo, Japan)
1H, 400MHz: Bruker)
by NMR (Ultrashield 400 plus system:
and this was agitated for more than 48h at room temperature. analysis. 1H-NMR (methanol-d4) δ: 6.73 (1H, d, J=8.0Hz),
After the adjustment to pH 4.0 with 5% citric acid solution, 6.66 (1H, d, J=1.9Hz), 6.55 (1H, dd, J=8.0, 1.8Hz), 3.61 (2H,
the reaction solution was added to 3mL of distilled water and s), 2.76 (2H, t, J=6.3Hz), 2.50 (2H, t, J=6.5 Hz).
was extracted 3 times with 5mL of chloroform. The chloro-
Chemical Synthesis of 5-(3,4,5-Trihydroxyphenyl)valeric
form layer was washed with 5mL of distilled water and 5mL Acid (EGC-M9) Five milliliters of dichloromethane was
of the saturated NaCl solution, and was evaporated to dryness added to 3,4,5-tribenzyloxybenzylalcohol (2.23mmol, 950.5mg,
to obtain 5-(3,4-O-dimethoxymethylphenyl)-γ-valerolactone. Tokyo Chemical Ind. Co., Ltd.) and Dess–Martin Periodinane
The compound was dissolved in 1mL of methanol and 0.1mL (3.30mmol, 1.4g, Sigma-Aldrich Co., LCC) and the mixture
of 3M KOH solution was added. The reaction mixture was was stirred for 3h at room temperature. The mixture was
stirred for 1h at room temperature. To the mixture was added to 5mL of 5% sodium thiosulfate solution and was agi-
added 1M sodium formate buffer (pH 3.0) to adjust pH to tated for 30min at room temperature. Next, the mixture was
4.0. After the addition of 3mL of distilled water, the solution extracted with 20mL of ethyl acetate and the ethyl acetate
was extracted 3 times with 4mL of ethyl acetate. Triethyl- layer was washed with distilled water, the saturated NaHCO3
amine (0.1mL) was added to the ethyl acetate layer and the solution, and saturated NaCl solution. After that, the organic
solution was evaporated to dryness. 4-Hydroxy-5-(3,4-O- layer was dried with sodium sulfate and was filtered. The
dimethoxymethylphenyl)valeric acid trimethylamine salt was resulting filtrate was evaporated to dryness and the crude resi-
obtained. The valeric acid trimethylamine salt was dissolved due containing 3,4,5-tribenzyloxybenzaldehyde (1.24g) was
in 3mL of dichloromethane and then pyridine (1.83mmol, obtained. [3-(Ethoxycarbonyl)propyl]-triphenylphosphonium
145 µL) and Dess–Martin periodinane (0.92mmol, 400.0mg, Bromide (4.38mmol, 2.0g, Wako Pure Chemical Industries,
Sigma-Aldrich Co., LCC, Tokyo, Japan) was added. The reac- Ltd., Osaka, Japan) was added to 20mL of anhydrous tetra-
tion mixture was stirred for 4h at room temperature. To the hydrofuran and the mixture was stirred for 20min on ice.
reaction mixture was added 3mL of 5% sodium thiosulfate Tetrahydrofuran containing sodium bis(trimethylsilyl)amide
solution and this was agitated for 30min at room temperature. (5.83mmol, 3.07mL, Tokyo Chemical Ind. Co., Ltd.) was
After the adjustment to pH 3.0 by adding 5% citric acid solu- then added to the mixture and agitated for 1h on ice. To the
tion, the resulting solution was extracted 3 times with 10mL resulting solution was added 8mL of tetrahydrofuran contain-
of dichloromethane. The dichloromethane layer was washed ing 3,4,5-tribenzyloxybenzaldehyde (1.24g synthesized above)
with 10mL of 5% sodium thiosulfate solution and 10mL of and this was stirred for 48h at room temperature. After add-
saturated NaCl solution. The organic layer was dehydrated by ing 30mL of saturated ammonium chloride, the mixture was
sodium sulfate. After filtration, the solution was evaporated to extracted with 50mL of diethyl ether. The organic layer was
dryness. The residue was dissolved in 10mL of acetonitrile/ washed with distilled water, saturated NaHCO3 solution, and
distilled water/formic acid (5/95/0.1, v/v/v) and was subjected saturated NaCl solution and was dehydrated with sodium
to a preparative HPLC. The preparative HPLC was performed sulfate. After removal of sodium sulfate by filtration, the fil-
using CAPCELL PAK MG column (150mm×20mm i.d., trate was evaporated to dryness and the residue was added
5µm, Shiseido Co., Ltd., Tokyo, Japan) in a Preparative HPLC to the mixed solution (hexane/diethyl ether, 150/50, v/v) and
system PLC791 (GL Sciences Inc., Tokyo, Japan). The column kept overnight at 4°C. After removal of insoluble materi-
was eluted with mobile phase A (acetonitrile/distilled water/ als, the filtrate was evaporated. The residue was dissolved in
formic acid, 5/95/0.1, v/v/v) and mobile phase B (acetonitrile/ 1mL of the mobile phase (hexane/ethyl acetate (80/20, v/v))
distilled water/formic acid, 80/20/0.1, v/v/v) at a flow rate of and applied to a silica-gel chromatography for purification.
9.5mL/min at 40°C. Initially, the column was eluted with Silica-gel 60N (Kanto Chemical Co., Inc., Tokyo, Japan) in
60% A and 40% B for 3min, followed by linear increases to a column (20 i.d.×150mm) was washed with 100mL of the
100% B from 3 to 13min and held at 100% B for 3min, and mobile phase and then the sample was applied to the column.
then eluted with 60% A and 40% B from 16 to 16.5min, and The fractions were analyzed by the TLC plate developed with
finally equilibrated with 60% A and 40% B for 3.5min. The hexane/ethyl acetate (80/20, v/v) as mobile phase and detected
elution pattern was monitored by measuring the absorbance at with UV light at 302nm. The fractions containing ethyl-(Z)-
280nm. The 5-(3,4-O-dimethoxymethylphenyl)levulinic acid 5-(3,4,5-tribenzyloxyphenyl)-4-pentenoate were collected and
fraction was collected, concentrated to dryness and finally concentrated, and finally 1.91mmol (942.0mg) of the com-
the purified levulinic acid was obtained (0.06mmol, 19.1mg). pound was obtained. The pentenoate was dissolved in 4mL of
The compound was dissolved in a mixture of dichlorometh- methanol–tetrahydrofuran mixture. To the solution was added
M KOH and this was stirred for 5h at room tempera-
ane (1.5mL), distilled water (0.5mL) and trifluoroacetic acid 2mL of 3
ture. Then, the reaction mixture was adjusted to pH 4.0 with
2M HCl and extracted with 20mL of ethyl acetate. The ethyl
acetate layer was washed with distilled water and saturated
NaCl solution and was dehydrated with sodium sulfate. After
filtration, the filtrate was evaporated to dryness and 755.8mg
of the residue including (Z)-5-(3,4,5-tribenzyloxyphenyl)-
(1.0mL) and the solution was stirred for 1h at room tempera-
ture. The mixture was evaporated to dryness and the residue
was dissolved in 1mL of acetonitrile/distilled water/formic
acid (5/95/0.1, v/v/v) and then was purified by the same pre-
parative HPLC as above, except that the column was initially
eluted with 90% A and 10% B for 3min, followed by linear

Vol. 40, No. 8 (2017)
Biol. Pharm. Bull.
1333
4-pentenoic acid was obtained. The pentanoic acid was dis- (2.68mmol, 851.0mg, Wako Pure Chemical Industries,
solved in 8mL of ethyl acetate and to the solution was added Ltd.) for EC-M9 synthesis and m-Benzyloxybenzlaldehyde
600.7mg of Palladium (10wt% (dry) on carbon powder, wet, (5.18mmol, 1.1g, Wako Pure Chemical Industries, Ltd.) for
Sigma-Aldrich Co., LCC). The mixture was replaced with EGC-M11 synthesis as starting materials. Finally, 0.439mmol
argon gas and then was stirred for 6h at room temperature (92.1mg) of EC-M9 and 2.29mmol (445.2mg) of EGC-M11
by injecting hydrogen gas. After filtration, the filtrate was were obtained and their structures were confirmed by NMR
evaporated to dryness and the residue was dissolved in 10mL analysis.
1
of acetonitrile/distilled water/formic acid (5/95/0.1, v/v/v) and
EC-M9; H-NMR (methanol-d4) δ. 6.67 (1H, d, J=8.0Hz),
applied to the preparative HPLC system as described previ- 6.63 (1H, d, J=2.0Hz), 6.50 (1H, dd, J=8.0, 2.0Hz), 2.49 (2H,
ously. The preparative HPLC system, column, mobile phase, t, J=7.0Hz), 2.31 (2H, t, J=6.7Hz), 1.62 (4H, m). EGC-M11;
flow rate and absorbance were the same as the synthesis of 1H-NMR (methanol-d4) δ: 7.08 (1H, t, J=7.1Hz), 6.62 (3H, m),
5-(3,4-dihydroxyphenyl)levulinic acid. The column was ini- 2.57 (2H, t, J=7.1Hz), 2.32 (2H, t, J=7.0Hz), 1.64 (4H, m).
tially eluted with 90% A and 10% B for 3min, followed by
Assay for Inhibitory Activity against HeLa Cell Prolif-
linear increases to 100% B from 3 to 15min and held at 100% eration Catechin metabolites were assayed for cytotoxicity
B for 5min, then eluted with 90% A and 10% B from 20 to against HeLa cells according to the previous report.28) Simply
21min, and finally equilibrated with 90% A and 10% B for stated, HeLa cells (2×103 cells/well) were seeded in 96-well
5min. The 5-(3,4,5-trihydroxyphenyl)valeric acid fraction was microplate and allowed to grow for 24h. Then, each catechin
collected, concentrated and finally 1.47mmol (331.7mg) of the metabolite in dimethyl sulfoxide (DMSO) was added at a
compound was obtained and its structure was confirmed by final concentration range of 0.4 to 50µg/mL and incubated
1
NMR analysis. H-NMR (methanol-d4) δ: 6.23 (2H, s), 2.42 for 72h at 37°C under an atmosphere of 5% CO2. Adriamycin
(2H, t, J=6.8Hz), 2.31 (2H, t, J=6.7Hz), 1.59 (4H, m).
in DMSO as a positive control was used at a final concentra-
Chemical Synthesis of 5-(3,4-Dihydroxyphenyl)vale- tion range of 6.4×10−4 to 10µg/mL and DMSO was used as
ric Acid (EC-M9) and 5-(3-Hydroxyphenyl)valeric Acid a negative control. After the incubation, each well was added
(EGC-M11) EC-M9 and EGC-M11 were synthesized in to 50µL of 1mg/mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-
a manner similar to 5-(3,4,5-trihydroxyphenyl)valeric acid diphenyl-2H-tetrazolium bromide (MTT) in saline and further
(EGC-M9) except for using 3,4-dibenzyloxybenzaldehyde incubated for 3h. Then, the plate was centrifuged to remove
Fig. 1. Chemical Structures of Catechin Metabolites and Their Inhibitory Activity on HeLa Cell Proliferation
Inhibitory activity is noted in brackets and is expressed relative to negative control (DMSO) set at 100.

1334
Biol. Pharm. Bull.
Vol. 40, No. 8 (2017)
the supernatant, the cells were dissolved in 150µL of DMSO
In addition, since there are no metabolites possess-
and the absorbance was measured by a microplate reader at a ing two adjacent hydroxyl groups of the phenyl moiety
wavelength of 490nm.
among EGC and EGCg metabolites tested in this study, we
To determine IC50, HeLa cells were treated with EGC-M9, examined the inhibitory activity of EC metabolites hav-
the most effective inhibitor among catechin metabolites ing two adjacent hydroxyl groups in the phenyl moiety
against the proliferation of HeLa cells, and adriamycin was such as 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)-
used as a positive control. Final concentration was eight dif- propan-2-ol (EC-M1), 4-hydroxy-5-(3,4-dihydroxyphenyl)-
ferent ranges from 1.28×10−3 to 100µg/mL for the former valeric acid (EC-M3), 5-(3,4-dihydroxyphenyl)levulinic acid
and 1.28×10−4 to 10µg/mL for the later. The IC50 values were (EC-M7) and 5-(3,4-dihydroxyphenyl)valeric acid (EC-M9).
calculated from their inhibition curves.
As shown in Fig. 1, EC-M1, EC-M3, and EC-M7 did not show
inhibitory activity against HeLa cell proliferation whereas
EC-M9 inhibited proliferation. Thus, even though EC-M9 has
only two adjacent hydroxyl groups in phenyl moiety, it exhib-
ited the inhibitory activity. These results implied that aliphatic
side chain, valeric acid in this case, may play a certain role in
combination with two adjacent hydroxyl groups in the phenyl
moiety. Consequently, it is reasonable that EGC-M9 possess-
ing both valeric acid and three adjacent hydroxy groups in its
phenyl moiety showed strongest inhibitory activity on HeLa
cell proliferation.
RESULTS AND DISCUSSION
We first screened the inhibitory activities of eleven kinds of
metabolites produced from EGC and EGCg by intestinal mi-
crobiota on proliferation of human cervical cancer cells (HeLa
cells). Figure 1 illustrates the chemical structures of metabo-
lites. Among the metabolites, 1-(3,4,5-trihydroxyphenyl)-3-
(2,4,6-trihydroxyphenyl)propan-2-ol (EGC-M2), 4-hydroxy-5-
(3,4,5-trihydroxyphenyl)valeric acid (EGC-M7), and 5-(3,4,5-
trihydroxyphenyl)valeric acid (EGC-M9) were found to inhibit
value of EGC-M9 which showed the
We examined the IC50
proliferation of HeLa Cells (Fig. 1) at a final concentration strongest inhibitory activity, together with that of adriamycin
of 50µg/mL. On the other hand, their analogous metabolites as a positive control. IC50 value of EGC-M9 was calculated to
such as 1-(3,5-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)- be 5.58µM (1.26 µg/mL) and was extremely weak as compared
propan-2-ol (EGC-M3), 4-hydroxy-5-(3,5-dihydroxyphenyl)- with that 0.039µM (2.1×10−2 µg/mL) of adriamycin as shown
valeric acid (EGC-M4), 5-(3,5-dihydroxyphenyl)valeric acid in Fig. 2. However, the value of EGC-M9 was roughly 8.6
(EGC-M10), and 5-(3-hydroxyphenyl)valeric acid (EGC-M11) times higher than that of EGCg (47.9µM) which have reported
did not show any inhibitory activity against HeLa cell prolif- to exhibit the inhibitory activity against HeLa cell prolifera-
eration (Fig. 1). These observations suggested that three adja- tion.27) Thus, this compound can contribute to the cancer pre-
cent hydroxyl groups of the phenyl moiety in the metabolite ventive effect of green tea catechins.
structures play an important role in the inhibition of cell pro-
In this study, we newly discovered that EGC-M2, EGC-M7,
liferation. However, 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone EGC-M9, and EC-M9 formed from EGC, EGCg, and EC by
(EGC-M8) having the three hydroxyl groups in its phenyl intestinal microbiota inhibit the proliferation of cervical can-
moiety did not exhibit the inhibitory activity, making it an cer HeLa cells. The metabolites may be responsible for the
exceptional instance. We have no reasonable explanation for cancer preventive activity of green tea as well as EGCg.25–27)
this anomaly but speculate that the valerolactone moiety may Indeed, oral intake of tea catechin has been reported to show
be responsible for decreasing affinity for HeLa cells.
a chemopreventive effect against cervical intraepithelial
Fig. 2. Measurement of IC50 Values of EGC-M9 (○) and Adriamycin (□) against HeLa Cell Proliferation
Intersections of a solid line and a dotted line indicate the IC50 values of EGC-M9 and adriamycin, respectively.

Vol. 40, No. 8 (2017)
Biol. Pharm. Bull.
1335
neoplasia in human.29) On the other hand, Lambert et al.23)
reported that EGC-M8 inhibited the cell growth in esopha-
geal squamous carcinoma cells (KYSE150) and human colon
adenocarcinoma cells (HT-29), but indicated inhibition of pre-
malignant cells by this metabolite could be selective. In our
study EGC-M8 showed no inhibitory activity on HeLa cell
proliferation, suggesting that selectivity may also be a factor
with HeLa cells. Further research is needed not only to evalu-
ate the anticancer effect of catechin metabolites with respect
to different cancer cells but also to confirm the effect in in
vivo experiments.
catechin, (−)-catechin, and (+)-catechin by (−)-epigallocatechin-
metabolizing bacteria. Biol. Pharm. Bull., 38, 789–794 (2015).
14) Das NP, Sothy SP. Studies on flavonoid metabolism. Biliary and
urinary excretion of metabolites of (+)-[U-14C]catechin. Biochem.
J., 125, 417–423 (1971).
15) Das NP. Studies on flavonoid metabolism. Absorption and metabo-
lism of (+)-catechin in man. Biochem. Pharmacol., 20, 3435–3445
(1971).
16) Llorach R, Garrido I, Monagas M, Urpi-Sarda M, Tulipani S,
Bartolome B, Andres-Lacueva C. metabolomics study of human
urinary metabolome modifications after intake of almond (Prunus
dulcis (Mill.) D.A. webb) skin polyphenols. J. Proteome Res., 9,
5859–5867 (2010).
17) van der Hooft JJJ, de Vos RCH, Mihaleva V, Bino RJ, Ridder L,
de Roo N, Jacobs DM, van Duynhoven JPM, Vervoort J. Structural
elucidation and quantification of phenolic conjugates present in
human urine after tea intake. Anal. Chem., 84, 7263–7271 (2012).
18) Monagas M, Urpi-Sarda M, Sánchez-patán F, Llorach R, Garrido
I, Gómez-Cordovés C, Andres-Lacueva C, Bartolomé B. Insights
into the metabolism and microbial biotransformation of dietary
flavan-3-ols and the bioactivity of their metabolites. Food Funct., 1,
233–253 (2010).
Acknowledgments We acknowledge Prof. S. Matsunaga
(Department of Aquatic Bioscience, the University of Tokyo)
for MTT assay of HeLa cell proliferation and the assistance of
Ms. Andrea K. Suzuki (Mitsui Norin Co., Ltd.) in the proof-
reading of the manuscript.
Conflict of Interest The authors declare no conflict of
interest.
19) Li C, Lee MJ, Sheng S, Meng X, Prabhu S, Winnik B, Huang B,
Chung JY, Yan S, Ho CT, Yang CS. Structural identification of
two metabolites of catechins and their kinetics in human urine and
blood after tea ingestion. Chem. Res. Toxicol., 13, 177–184 (2000).
20) Takagaki A, Otani S, Nanjo F. Antioxidative activity of microbial
metabolites of (−)-epigallocatechin gallate produced in rat intes-
tines. Biosci. Biotechnol. Biochem., 75, 582–585 (2011).
21) Takagaki A, Nanjo F. Effects of metabolites produced from (−)-epi-
gallocatechin gallate by rat intestinal bacteria on angiotensin I-
converting enzyme activity and blood pressure in spontaneously
hypertensive rats. J. Agric. Food Chem., 63, 8262–8266 (2015).
22) Kim YH, Won YS, Yang X, Kumazoe M, Yamashita S, Hara A,
Takagaki A, Goto K, Nanjo F, Tachibana H. Green tea catechin
metabolites exert immunoregulatory effects on CD4+ T cell and
natural killer cell activities. J. Agric. Food Chem., 64, 3591–3597
(2016).
23) Lambert JD, Rice JE, Hong J, Hou Z, Yang CS. Synthesis and
biological activity of the tea catechin metabolites, M4 and M6 and
their methoxy-derivatives. Bioorg. Med. Chem. Lett., 15, 873–876
(2005).
24) Al-Hazzani AA, Alshatwi AA. Catechin hydrate inhibits prolifera-
tion and mediates apoptosis of SiHa human cervical cancer cells.
Food Chem. Toxicol., 49, 3281–3286 (2011).
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., 16, 1156–1159
(1993).
2) Higdon JV, Frei B. Tea catechins and polyphenols: health effects,
metabolism, and antioxidant functions. Crit. Rev. Food Sci. Nutr.,
43, 89–143 (2003).
3) Matsumoto N, Ishigaki F, Ishigaki A, Iwashina H, Hara Y. Reduc-
tion of blood glucose levels by tea catechin. Biosci. Biotechnol.
Biochem., 57, 525–527 (1993).
4) Hara Y. Hypotensive action of tea polyphenols. Green Tea. Health
Benefits and Application, CRC Press, Taylor & Francis Group, Boca
Raton, FL, pp. 139–148 (2001).
5) Negishi H, Xu JW, 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., 134, 38–42
(2004).
6) Yang CS, Lambert JD, Hou Z, Ju J, Lu G, Hao X. Molecular targets
for the cancer preventive activity of tea polyphenols. Mol. Car-
cinog., 45, 431–435 (2006).
7) Chen L, Lee MJ, Li H, Yang CS. Absorption, distribution, and
elimination of tea polyphenols in rats. Drug Metab. Dispos., 25,
1045–1050 (1997).
25) Chakrabarty S, Ganguli A, Das A, Nag D, Chakrabarti G. Epigal-
locatechin-3-gallate shows anti-proliferative activity in HeLa cells
Targeting tubulin-microtubule equilibrium. Chem. Biol. Interact.,
242, 380–389 (2015).
26) Sharma C, Nusri QEIA, Begum S, Javed E, Rizvi TA, Hussain A.
(−)-Epigallocatechin-3-gallate induces apoptosis and inhibits inva-
sion and migration of human cervical cancer cells. Asian Pac. J.
Cancer Prev., 13, 4815–4822 (2012).
8) 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., 49, 4102–4112
(2001).
27) Qiao Y, Cao J, Xie L, Shi X. Cell growth inhibition and gene
expression regulation by (−)-epigallocatechin-3-gallate in human
cervical cancer cells. Arch. Pharm. Res., 32, 1309–1315 (2009).
28) Ueoka R, Fujita T, Iwashita T, van Soest RWM, Matsunaga S.
Inconspicamide, new N-acylated serinol from the marine sponge
Stelletta inconspicua. Biosci. Biotechnol. Biochem., 72, 3055–3058
(2008).
29) Bettuzzi S, Brausi M, Rizzi F, Castagnetti G, Peracchia G, Corti A.
Chemoprevention of human prostate cancer by oral administration
of green tea catechins in volunteers with high-grade prostate in-
traepithelial neoplasia: A preliminary report from a one-year proof-
of-principle study. Cancer Res., 66, 1234–1240 (2006).
9) Del Rio D, Calani L, Cordero C, Salvatore S, Pellegrini N, Brigh-
enti F. Bioavailability and catabolism of green tea flavan-3-ols in
humans. Nutrition, 26, 1110–1116 (2010).
10) Takagaki A, Nanjo F. Metabolism of (−)-epigallocatechin gallate by
rat intestinal flora. J. Agric. Food Chem., 58, 1313–1321 (2010).
11) Takagaki A, Nanjo F. Catabolism of (+)-catechin and (−)-epi-
catechin by rat intestinal Microbiota. J. Agric. Food Chem., 61,
4927–4935 (2013).
12) Takagaki A, Kato Y, Nanjo F. Isolation and characterization of rat
intestinal bacteria involved in biotransformation of (−)-epigallocat-
echin. Arch. Microbiol., 196, 681–695 (2014).
13) Takagaki A, Nanjo F. Bioconversion of (−)-epicatechin, (+)-epi-