Epicatechin conjugated with fatty acid is a potent inhibitor of DNA polymerase and angiogenesis

Article abstract of DOI:10.1016/j.lfs.2007.01.049

Anti-cancer and anti-angiogenesis effects of green tea catechins have been demonstrated. It has been found that chemical modification of tea catechins improves their biological activities. We examined the chemical modification of epicatechin enhanced anti

Full text of DOI:10.1016/j.lfs.2007.01.049

Life Sciences 80 (2007) 1578–1585
www.elsevier.com/locate/lifescie
Epicatechin conjugated with fatty acid is a potent inhibitor of DNA
polymerase and angiogenesis
Kiminori Matsubara a,b, ,1, Akiko Saito c, Akira Tanaka d, Noriyuki Nakajima e,
⁎
Reiko Akagi a, Masaharu Mori f, Yoshiyuki Mizushina b,g
a
Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan
b
Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
c
Biotechnology Center, Toyama Prefecture, Kosugi, Toyama 939-0398, Japan
d
Department of Bioresources Science, College of Technology, Toyama Prefectural University, Kosugi, Toyama 939-0398, Japan
e
Biotechnology Research Center, Toyama Prefectural University, Kosugi, Toyama 939-0398, Japan
f
Department of Nursing, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan
g
Department of Nutritional Science, Kobe-Gakuin University, Kobe, Hyogo 651-2180, Japan
Received 28 August 2006; accepted 18 January 2007
Abstract
Anti-cancer and anti-angiogenesis effects of green tea catechins have been demonstrated. It has been found that chemical modification of tea
catechins improves their biological activities. We examined the chemical modification of epicatechin enhanced anti-cancer and anti-angiogenic
effects. Epicatechin conjugated with fatty acid (acyl-catechin) strongly inhibited DNA polymerase activity, HL-60 cancer cell growth and
angiogenesis. Epicatechin conjugated with palmitic acid ((2R,3R)-3′,4′,5,7-tetrahydroxyflavan-3-yl hexadecanoate, epicatechin-C16) was the
strongest inhibitor in DNA polymerase α, β, λ and angiogenesis assays. Epicatechin-C16 also suppressed human endothelial cell (HUVEC) tube
formation on reconstituted basement membrane, suggesting that it affected not only DNA polymerase activity but also the signal transduction
pathways needed for the tube formation in HUVECs. These results suggest that acylation of epicatechin is an effective chemical modification to
improve the anti-cancer activity of epicatechin.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Angiogenesis; Aortic ring; Epicatechin; Endothelial cells; Fatty acid
Introduction
numerous efforts to develop more effective angiogenesis
inhibitors have been conducted (Cao, 2004). We have found
some angiogenesis inhibitors in natural products including
vitamin B6, algal polysaccharides and phenolic compounds
(Matsubara et al., 2001, 2003, 2005a,b). Recently, we have
reported that a polyphenolic DNA polymerase inhibitor,
petasiphenol, has strong inhibitory effect on angiogenesis in
vitro (Matsubara et al., 2004). DNA polymerases, especially
DNA polymerase α, are regarded as the target of some anti-
cancer drugs because DNA polymerases play central roles in
DNA replication which is indispensable for the proliferation of
cancer cells. Thus, it should be noted that polyphenolic DNA
polymerase inhibitor has anti-angiogenic activity.
Natural polyphenols distributed in plants have diverse
biological activities: antioxidant, anti-cancer, and anti-inflam-
mation activities (Yang et al., 2001). In addition, their inhibitory
effect on angiogenesis has been revealed (Cao et al., 2002).
Angiogenesis is involved in tumor growth and metastasis,
atherosclerosis and diabetic retinopathy (Folkman, 1995). Thus
⁎
Corresponding author. Department of Nutritional Science, Faculty of Health
and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-
1197, Japan. Tel./fax: +81 866 94 2149.
E-mail address: kmatsuba@fhw.oka-pu.ac.jp (K. Matsubara).
New Address (after April 1st, 2007): Department of Human Life Sciences
We have synthesized various derivatives of polyphenols,
intending to improve biological activities of natural polyphe-
nolic compounds. Tea catechins have been studied for their
1
Education, Graduate School of Education, Hiroshima University, Hiroshima
739-8524, Japan.
0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2007.01.049

K. Matsubara et al. / Life Sciences 80 (2007) 1578–1585
1579
biological activities. The main compound of tea catechins,
epigallocatechin-3-gallate (EGCG) has been the most exten-
sively studied, and anti-angiogenic activity of EGCG has been
revealed (Cao and Cao, 1999). On the other hand, other
catechins including epicatechin are weaker in anti-angiogenic
activity than EGCG (Lamy et al., 2002). Thus, our efforts have
been conducted on synthesized potent epicatechin derivatives.
We have succeeded in synthesizing 3-O-acylepicatechin
derivatives and examined their effect on DNA polymerase
activity and angiogenesis. Interestingly, 3-O-acylepicatechin
derivatives exerted stronger inhibitory effects both on DNA
polymerase activity and angiogenesis than epicatechin, sug-
gesting the possibility of developing new anti-cancer agents.
The characteristics of the acylated epicatechin as DNA
polymerase and angiogenesis inhibitors are demonstrated in
this report.
(5 mg) and fatty acid chlorides (lauroyl chloride, miristoyl
chloride, and palmitoyl chloride, 1.5 eq.) at 0 °C. After stirring
for 6–12 h at rt, the reaction mixture was quenched with water
and extracted with CH2Cl2. The organic phase was washed with
water and brine and dried (Na2SO4). Filtration, concentration
and silica gel column chromatography (hexane/EtOAc, 6/1)
gave a pure ester.
General procedure for the hydrogenation of benzyl protecting
group of acyl-epicatechins
A solution of a benzylated ester (2–4) in THF/MeOH/H2O
(20/1/1) was hydrogenated over 20% Pd(OH)2/C (5 mg) for
12 h at rt. Filtration and concentration afforded a colorless oil,
which was purified by Cosmosil 75C-18 OPN column
chromatography (MeOH–H2O) to give a pure debenzyrated
acyl-epicatechins.
Materials and methods
(2R,3R)-3′,4′,5,7-Tetra-O-benzylflavan-3-yl dodecanoate (2)
Materials
White powder. [α]D25 =−19.5 (c 0.94, CHCl3); 1H NMR
(400 MHz, CDCl3) 7.47–7.28 (20H, m), 7.11 (1H, d,
J=1.7 Hz), 6.96 (1H, dd, J=1.7, 8.3 Hz), 6.92 (1H, d,
J=8.3 Hz), 6.28 (1H, d, J=2.2 Hz), 6.27 (1H, d, J=2.2 Hz),
5.45–5.41 (1H, m), 5.17 (1H, d, J=12.0 Hz), 5.15 (2H, s), 5.14
(1H, d, J=12.0 Hz), 5.02 (4H, s), 4.99 (1H, br s), 3.02 (1H, dd,
J=4.6, 18.1 Hz), 2.95 (1H, dd, J=2.0, 18.1 Hz), 2.19–2.07
(2H, m), 1.45–1.37 (2H, m), 1.32–1.10 (16H, m), 0.86 (3H, t,
J=5.6 Hz); 13C NMR (100 MHz, CDCl3) 173.1, 158.7, 157.9,
155.5, 148.9, 148.7, 137.2, 136.9, 131.1, 128.6–127.1 (C×14),
119.7, 114.7, 113.6, 100.8, 94.6, 93.8, 77.2, 71,4, 71.3, 70.1,
69.9, 67.5, 34.2, 31.9, 29.62, 29.60, 29.4, 29.3, 29.26, 28.98,
26.0, 24.8, 22.7, 14.1; IR (neat, cm−1) 3065 (m), 3034 (m),
2924 (s), 2855 (s), 2361 (w), 2338 (w), 1950 (w), 1871 (w),
1819 (w), 1782 (s), 1618 (s), 1593 (s), 1518 (s), 1454 (s), 1379
(s), 1269 (s), 1217 (s), 1184 (s), 1152 (s), 1115 (s), 1020 (s), 945
(w), 810 (m), 735 (s); FAB-MS (m/z) 856 (11), 855 ([M+Na]+,
17), 834 (3.6), 833 ([M+H]+, 7.5), 633 (21), 632 (28), 610 (54),
609 (100); FAB-HRMS calcd for C55H61O7 [M + H]+,
833.4417; found: 833.4448.
Human recombinant vascular endothelial growth factor
(VEGF) was obtained from R&D systems (MN, U.S.A).
Other reagents were special grade as commercially available.
Synthesis
Optical rotation was measured with a Horiba SEPA-300
spectrometer (Horiba, Kyoto). H NMR (400 MHz) and 13C
1
NMR (100 MHz) spectra were taken with a JEOL LA-400
1
spectrometer (Tokyo, Japan). CDCl3 (δ7.26 for H and δ77.0
for 13C NMR) and acetone-d6 (δ2.00 for H and δ30.3 and
1
206.0 for 13C NMR) were used as the internal standards,
respectively. 1H and 13C NMR signals were assigned by means
of 1D proton decoupling technique, 2D HH-COSY, and CH-
COSY. FT-IR spectra were taken with a Shimadzu FT-IR DR-
8000 (Shimadzu, Kyoto). FAB mass spectra were taken with a
JEOL JMS-AX500 instrument.
Synthesis of tetra-benzylated epicatechin
To a solution of (2R,3R)-3′,4′,5,7-tetrahydroxyflavan-3-ol
(10 g, 0.035 mol) and K2CO3 (26.2 g, 0.19 mol) in N,N-
dimethylformamide (500 ml) was added benzyl bromide
(29.5 g, 0.17 mol) at 0 °C. After stirring for 48 h at rt, the
reaction mixture was quenched with cold water and extracted
with EtOAc. The organic phase was washed with water and
brine and dried (Na2SO4). Filtration, concentration and silica
gel column chromatography (hexane/EtOAc, 6/1) gave a 10.2 g
of tetra-benzylated epicatechin (0.016 mol, 45%) as a white
powder.
(2R,3R)-3′,4′,5,7-Tetrahydroxyflavan-3-dodecanoate,
epicatechin-C12
Colorless amorphous solid. [α]D24 =−54.0 (c 0.58, EtOH); 1H
NMR (400 MHz, CDCl3) 6.91 (1H, br s), 6.73 (2H, br s), 5.94
(1H, d, J=2.4 Hz), 5.91 (1H, d, J=2.4 Hz), 5.33–5.32 (1H, m),
4.93 (1H, br s), 2.90 (1H, dd, J=4.7, 17.6 Hz), 2.78 (1H, d,
J=17.6 Hz), 2.19–2.14 (2H, m), 1.44–1.39 (2H, m), 1.30–1.09
(16H, m), 0.88 (3H, t, J=6.6 Hz); 13C NMR (100 MHz, CDCl3)
175.0, 157.83, 157.79, 157.1, 145.99, 145.96, 131.3, 119.0,
115.9, 114.9, 99.1, 96.5, 95.8, 78.2, 69.9, 35.2, 33.1, 30.72,
30.70, 30.51, 30.47, 30.3, 29.9, 26.6, 26.0, 23.7, 14.4; FAB-MS
(m/z) 496 (5.5), 495 ([M+Na]+, 17), 494 (5.8), 474 (6.3), 473
([M+H]+, 20), 275 (13), 274 (55), 273 (100), 272 (22); FAB-
HRMS calcd for C27H37O7 [M+H]+, 473.2539; found:
473.2542.
General procedure for the esterification of benzylated
flavan-3-ols
To a solution of (2R,3R)-3′,4′,5,7-tetra-O-benzylflavan-3-ol
(1) (500 mg) in CH2Cl2 (10 ml) was added Et3N (3 eq.), DMAP

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K. Matsubara et al. / Life Sciences 80 (2007) 1578–1585
(2R,3R)-3′,4′,5,7-Tetra-O-benzylflavan-3-yl tetradecanoate (3)
MS (m/z) 912 (12), 911 ([M+Na]+, 19), 891 (15), 890 ([M+
H]+, 21), 889 (7.2), 634 (19), 633 (59), 632 (78), 631 (19), 320
(22), 319 (100); FAB-HRMS calcd for C59H69O7 [M+H]+,
889.5043; found: 889.5089.
White powder. [α]D23 =−20.4 (c 0.72, CHCl3); 1H NMR
(400 MHz, CDCl3) 7.47–7.30 (20H, m), 7.11 (1H, d,
J=1.7 Hz), 6.96 (1H, dd, J=1.7, 8.3 Hz), 6.92 (1H, d,
J=8.3 Hz), 5.46–5.41 (1H, m), 5.17 (1H, d, J=11.9 Hz), 5.15
(2H, s), 5.13 (1H, d, J=11.9 Hz), 5.12 (4H, s), 4.99 (1H, br s),
3.02 (1H, dd, J=4.6, 16.1 Hz), 2.94 (1H, d, J=16.1 Hz), 2.19–
2.05 (2H, m), 1.42–1.38 (2H, m), 1.29–1.10 (20H, m), 0.88
(3H, t, J=6.8 Hz); 13C NMR (100 MHz, CDCl3) 173.1, 158.7,
157.9, 155.4, 148.8, 148.7, 137.2 (×2), 136.9, 136.8, 131.1,
128.6, 128.5, 128.4 (×2), 128.0, 127.9, 127.8, 127.7, 127.5,
127.4, 127.2, 127.1, 119.7, 114.7, 113.6, 100.8, 94.6, 93.8,
77.2, 71.4, 71.2, 70.1, 69.9, 67.5, 34.2, 31.9, 29.7, 29.63, 29.61
(×2), 29.4, 29.3, 29.2, 29.0, 25.9, 24.8, 22.7, 14.1; IR (neat,
cm−1) 2924 (s), 2853 (s), 1732 (s), 1653 (s), 1558 (s), 1456 (s),
1385 (s), 1339 (m), 1151 (s), 1115 (s), 1078 (m), 1028 (m), 902
(w), 810 (w), 733 (m); FAB-MS (m/z) 885 (9.4), 884 (24), 883
([M+Na]+, 36), 862 (14), 861 ([M+H]+, 29), 860 (7.9),
695 (12), 694 (44), 693 (100); FAB-HRMS calcd for C57H65O7
[M+H]+, 861.4730; found: 861.4730.
(2R,3R)-3′,4′,5,7-Tetrahydroxyflavan-3-yl hexadecanoate,
epicatechin-C16
White powder. [α]D25 =−42.0 (c 1.22, CH3COCH3); 1H
NMR (400 MHz, CD3COCD3-D2O, 10:1) 6.94 (1H, br s), 6.76
(2H, br s), 6.01 (1H, d, J=2.2 Hz), 5.91 (1H, d, J=2.2 Hz),
5.32–5.31 (1H, m), 4.95 (1H, br s), 2.91 (1H, dd, J=4.9,
17.5 Hz), 2.72 (1H, dd, J=2.0, 17.5 Hz), 2.16–2.10 (2H, m),
1.41–1.34 (2H, m), 1.26–1.10 (24H, m), 0.82 (3H, t,
J=6.8 Hz); 13C NMR (100 MHz, CD3COCD3-D2O, 10:1)
173.4, 157.4, 157.2, 156.5, 145.3 (×2), 130.7, 118.6, 115.3,
114.5, 98.4, 96.2, 95.3, 77.5, 68.9, 34.5, 32.3, 30.4–29.2
(C×10), 26.2, 25.3, 23.0, 14.2; FAB-MS (m/z) 552 (11), 551
([M+Na]+, 29), 550 (6.1), 530 (4.4), 529 ([M+H]+, 11), 275
(15), 274 (77), 273 (100), 272 (44); FAB-HRMS calcd for
C31H45O7 [M+H]+, 529.3165; found: 529.3167.
(2R,3R)-3′,4′,5,7-Tetrahydroxyflavan-3-yl tetradecanoate,
DNA polymerase assays
epicatechin-C14
DNA polymerase α was purified from calf thymus by
immuno-affinity column chromatography as described by
Tamai et al. (1988). Recombinant rat DNA polymerase β was
purified from E. coli JMpβ5 as described by Date et al. (1988).
The cDNA encoding the full-length human DNA polymerase λ
was constructed and purified as described previously (Mizushina
et al., 2002; Shimazaki et al., 2002). The reaction mixtures for
DNA polymerases were described previously (Mizushina et al.,
1996, 1997). The substrates of DNA polymerases were used poly
(dA)/oligo(dT)12–18 and deoxythymidine triphosphates (dTTP)
as template-primer DNA and nucleotide substrate, respectively.
The synthesized compounds were dissolved in dimethyl
sulfoxide (DMSO) at various concentrations and sonicated for
30 s. Four microliter of sonicated samples were mixed with 16 μl
of each enzyme (final 0.05 units) in 50 mM Tris–HCl (pH 7.5)
containing 1 mM dithiothreitol, 50% glycerol and 0.1 mM EDTA,
and kept at 0 °C for 10 min. These inhibitor-enzyme mixtures
(8 μl) were added to 16 μl of each of the enzyme standard reaction
mixtures, and incubation was carried out at 37 °C for 60 min. The
activity without the inhibitor was considered 100%, and the
remaining activities at each concentration of inhibitor were
determined as percentages of this value. One unit of each DNA
polymerase activity was defined as the amount of enzyme
that catalyzes the incorporation of 1 nmol of deoxyribo-
nucleotide triphosphates (i.e. dTTP) into synthetic template-
primers (i.e. poly(dA)/oligo(dT)12–18, A/T=2/1) in 60 min at
37 °C under the normal reaction conditions for each enzyme
(Mizushina et al., 1996, 1997).
Colorless oil. [α]D25 =−38.3 (c 0.38, CH3COCH3); 1H NMR
(400 MHz, CD3COCD3-D2O, 10:1) 6.94 (1H, br s), 6.76 (2H,
br s), 6.02 (1H, d, J=2.2 Hz), 5.91 (1H, d, J=2.2 Hz), 5.33 (1H,
br s), 4.96 (1H, br s), 2.92 (1H, dd, J=4.3, 17.3 Hz), 2.73 (1H,
d, J=17.3 Hz), 2.13–2.10 (2H, m), 1.40–1.36 (2H, m), 1.25–
1.08 (20H, m), 0.82 (3H, t, J=6.8 Hz); 13C NMR (100 MHz,
CD3COCD3-D2O, 10:1) 173.5, 157.4, 157.3, 156.5, 145.3 (×2),
130.7, 118.6, 115.3, 114.5, 98.4, 96.2, 95.3, 77.5, 68.9, 34.5,
32.3, 30.4–29.1 (C×8), 26.2, 25.3, 23.0, 14.2; FAB-MS (m/z)
524 (15), 523 ([M+Na]+, 30), 522 (6.8), 502 (6.0), 501 ([M+
H]+, 14), 275 (28), 274 (90), 273 (100), 272 (43); FAB-HRMS
calcd for C29H41O7 [M+H]+, 501.2852; found: 501.2861.
(2R,3R)-3′,4′,5,7-Tetra-O-benzylflavan-3-yl hexadecanoate (4)
White powder. [α]D24 =−16.1 (c 1.34, CHCl3); 1H NMR
(400 MHz, CDCl3) 7.46–7.27 (20H, m), 7.12 (1H, d,
J=1.7 Hz), 6.95 (1H, dd, J=1.7, 8.3 Hz), 6.91 (1H, d,
J=8.3 Hz), 6.29 (1H, d, J=2.2 Hz), 6.27 (1H, d, J=2.2 Hz),
5.48–5.41 (1H, m), 5.17 (1H, d, J=12.2 Hz), 5.14 (2H, s), 5.13
(1H, d, J=12.2 Hz), 5.01 (4H, s), 4.98 (1H, br s), 3.01 (1H, dd,
J=4.4, 17.8 Hz), 2.95 (1H, dd, J=2.2, 17.8 Hz), 2.19–2.06
(2H, m), 1.45–1.37 (2H, m), 1.35–1.05 (24H, m), 0.88 (3H, t,
J=6.8 Hz); 13C NMR (100 MHz, CDCl3) 171.1, 158.7, 157.9,
155.4, 148.8, 148.7, 137.2, 136.84, 136.82, 131.1, 128.54 (×2),
128.48, 128.39 (×2), 127.9, 127.8, 127.74, 127.70, 127.5,
127.3, 127.2, 127.1, 119.6, 114.6, 113.5, 100.8, 94.6, 93.8,
77.1, 71.4, 71.2, 70.0, 69.8, 67.5, 34.2, 31.9, 29.64 (×2), 29.60
(×2), 29.6, 29.4, 29.3, 29.2, 28.9, 25.9, 24.8, 22.7, 21.0, 14.1;
IR (neat, cm−1) 3063 (w), 3034 (w), 2923 (s), 2853 (s), 1948
(w), 1809 (w), 1782 (s), 1618 (s), 1593 (s), 1520 (s), 1379 (s),
1184 (s), 1151 (s), 1028 (s), 943 (w), 810 (m), 733 (m); FAB-
Cell culture and measurement of cell viability
A human promyelocytic leukemia cell line, HL-60, was
obtained from the Health Science Research Bank (Osaka, Japan).

K. Matsubara et al. / Life Sciences 80 (2007) 1578–1585
1581
Scheme 1. Synthesis of acyl-epicatechins. Reagents: (a) RCOCl, Et3N, DMAP, CH2Cl2, (b) H2, Pd(OH)2/C, THF-MeOH–H2O (20/1/1).
The cells were cultured in RPMI 1640 medium supplemented
with 10% fetal bovine serum, penicillin (100 units/ml) and
streptomycin (100 μg/ml) at 37 °C in a humid atmosphere of 5%
CO2/95% air. For the cell growth assay, HL-60 cells were plated at
3×105 cells into each well of 96-well microplates with various
concentrations of acyl-epicatechin compound. These compounds
were dissolved in dimethyl sulfoxide (DMSO) at a concentration
of 10 mM as a stock solution. The stock solutions were diluted to
the appropriate final concentrations with growth medium as 0.5%
DMSO just before use. The cell viability was determined by
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide) assay (Mosmann, 1983).
mixed gently at 4 °C. Each aortic segment was placed in the
center of a well on a 6-well culture plate and covered with 0.5 ml
of gel matrix solution reconstituted as described. The solution
was allowed to gel at 37 °C for 20 min. Culture medium
(RPMI 1640 medium (Gibco, New York, U.S.A.) containing 1%
of ITS+ (Becton Dickinson Labware, MA, U.S.A.)) with various
concentration of acyl-epicatechin or vehicle (DMSO) was
prepared as the same way described above. Then, the collagen
gel was overlaid with 2 ml of culture medium. Incubation was
carried out for 10 days in a fully humidified system of 5% CO2 in
the air at 37 °C. The medium was changed on day 7 of the
culture. An estimation of the length of the capillary was
performed under phase-contrast microscopy by measuring the
distance from the cut end of the aortic segment to the
approximate mean point of capillary. Microscopic fields were
photographed with a digital camera (Nikon, COOLPICKS 950).
The length of the capillary was measured using Adobe
Photoshop software. Each reported value represents the average
of three culture samples.
Ex vivo angiogenesis assay
Male Wistar rats (6 week old, Clea Japan, Inc., Tokyo, Japan)
were housed in metal cages in a room with controlled
temperature (24 1 °C) and a 12-h light:dark cycle (lights on,
08:00–20:00 h). They had free access to diets and deionized
water. The rats were maintained according to the “Guide for the
Care and Use of Laboratory Animals” established by Okayama
Prefectural University. The ex vivo angiogenesis assay was
performed according to slightly modified methods as described
before (Mori et al., 1988; Kawasaki et al., 1989). Briefly, a male
Wistar rat (body weight ∼200 g) was sacrificed by bleeding
from the right femoral artery under anesthesia with diethyl ether.
A thoracic aorta was removed and washed with RPMI 1640
medium to avoid contamination with blood. It was then turned
inside out, and cut into short segments about 1–1.5 mm.
Collagen gel (gel matrix solution) was made with 8 volumes of
porcine tendon collagen solution (3 mg/ml) (Cellmatrix Ia, Nitta
Gelatin Co., Osaka, Japan), 1 volume of 10× Eagle's MEM
(Gibco, New York, U.S.A.), 1 volume of reconstitution buffer
(0.08 M NaOH and 200 mM HEPES). These solutions were
Endothelial cells
Human umbilical vein endothelial cells (HUVECs) were
purchased from Kurabo Industries (Osaka, Japan). Cells were
grown in the medium, HuMedia EG-2 (Kurabo Industries,
Osaka, Japan), which was modified MCDB 131 medium
containing 2% fetal bovine serum (FBS), 10 ng/ml recombinant
human epidermal growth factor (EGF), 1 μg/ml hydrocortisone,
50 μg/ml gentamicin, 50 ng/ml amphotericin B, 5 ng/ml
recombinant human basic fibroblast growth factor (bFGF) and
10 μg/ml heparin, at 37 °C in a humidified 5% CO2. Subcultures
were obtained by treating the HUVEC cultures with 0.025%
trypsin–0.01% EDTA solution. HUVECs at passage three to
five were used in this experiment.
Fig. 1. Structures of acyl-epicatechins.

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K. Matsubara et al. / Life Sciences 80 (2007) 1578–1585
Table 2
HUVEC tube formation assay
Ki values of epicatechin-C16 for mammalian DNA polymerases
DNA
polymerase
poly(dA)/oligo(dT)12–18
dTTP
Tube formation assay was performed using BD Matrigel™
(Becton, Dickinson and Company, Tokyo, Japan). Briefly, solid
gels were prepared according to the manufacture manual on a 96-
well tissue culture plate. HUVECs (1×105 cells/ml) in HuMedia
EG-2 medium containing acyl-epicatechin (1–100 μM) or vehicle
(DMSO) were seeded 100 μl per well onto the surface of the solid
gel, BD Matrigel™. The cells were incubated for 12 h at 37 °C in
a CO2 incubator. Tube formation was observed under an inverted
light microscope at 40× magnification. Microscopic fields were
photographed with a digital camera (Nikon, COOLPICKS 950).
The total length of tube structures in each photograph was
measured using Adobe Photoshop software. Each reported value
represents the average of three samples.
Ki (μM)
Inhibitory mode
Ki (μM)
Inhibitory mode
α
β
λ
5.7
7.8
1.1
Competitive
Competitive
Competitive
8.4
9.8
1.4
Non-competitive
Non-competitive
Non-competitive
The inhibitory mode and Ki values of epicatechin-C16 for DNA polymerases
were determined with respect to each of poly(dA)/oligo(dT)12–18 (DNA
template-primer) and dTTP (nucleotide substrate) from Dixon plots.
than that on other polymerases. On the other hand, both
epicatechin and acetylated epicatechin-C12Ac were not effective
for these inhibitory activities. This result suggested that the acyl
chain group and hydroxyl group is essential for the inhibitory
effect.
Statistical analysis
Next, to elucidate the mechanism of inhibition of epicate-
chin-C16 on mammalian DNA polymerases, the extent of
inhibition as a function of DNA template-primer or dNTP
substrate concentrations was studied. In kinetic analysis, poly
(dA)/oligo(dT)12–18 and dTTP were used as the template-primer
DNA and dNTP substrate, respectively. Double reciprocal plots
of the results showed that the epicatechin-C16-induced
inhibition of DNA polymerase activities was competitive and
non-competitive with the DNA template and the dNTP
substrate, respectively (Table 2), suggesting that the compound
directly binds to the DNA template binding site of the enzyme.
The inhibition constant (Ki) values of epicatechin-C16,
obtained from Dixon plots, were found to be 1.1–7.8 μM and
1.4–9.8 μM for the DNA template and substrate dTTP,
respectively (Table 2). Because the Ki values for the DNA
template-primer were smaller than those for the dNTP substrate,
the affinity of epicatechin-C16 is greater for the enzyme–DNA
template-primer binary complex than for the enzyme–nucleo-
tide substrate complex.
Values are presented as means SD. Data were analyzed by
one-way analysis of variance (ANOVA). Differences with
pb0.05 were considered significant.
Results
Synthesis of 3-O-acylepicatechin derivatives
3-O-Acylepicatechin derivatives (epicatechin-C12–epicate-
chin-C16) were synthesized as shown in Scheme 1. Benzylated
(+)-epicatechin (1) was acylated with each fatty acid chlorides
(C12, C14, and C16) to afford benzylated-3-O-acyl-flavan-3-
ols 2–4 in good to excellent yields. Following hydrogenation
with Pd(OH)2/C gave epicatechin-C12–epicatechin-C16 in
satisfactory yields. The chemical structures are shown in Fig. 1.
Effects of acyl-epicatechin compounds on DNA polymerase
activities
Effects of acyl-epicatechin compounds on cancer cell growth
The effect of synthesized acyl-epicatechin compounds on
mammalian DNA polymerase α, β and λ was investigated. DNA
polymerase α and β, λ are replicative and repair-related DNA
polymerases in nuclei, respectively (Bebenek and Kunkel, 2004).
As shown in Table 1, epiatechin-C12 to epicatechin-C16 inhibited
the activity of calf DNA polymerase α (pol α), rat DNA
polymerase β (pol β) and human DNA polymerase λ (pol λ).
Epicatechin-C16 was the strongest inhibitor of these enzymes,
with 50% inhibition doses of 2.2–15.0 μM. The inhibitory effect
of the compound on DNA polymerase λ was 6.7-fold stronger
Epicatechin-C12 to epicatechin-C16 (10 μM each) also
inhibited human promyelocytic leukemia (HL-60) cell growth
for 2 days (48 h), and epicatechin-C16 was the strongest of the
acyl-epicatechin compounds tested (Fig. 2). The IC50 values of
epicatechin-C12, -C14 and -C16 were 10.8, 5.9, and 3.5 μM,
respectively. The measurement of IC50 values was 0.1–200 μM.
The human cell growth inhibitory effect of acyl-epicatechin
Table 1
IC50 values of acyl-epicatechins on DNA polymerase α, β and λ activities
Sample
IC50 values (μM)
pol α
pol β
N200
28.0
16.6
14.8
pol λ
N200
12.5
5.6
2.2
Epicatechin
N200
30.1
19.1
15.0
N200
Epicatechin-C12
Epicatechin-C14
Epicatechin-C16
Epicatechin-C12Ac
Fig. 2. Effect of acyl-epicatechins (10 μM each) on the proliferation of HL-60 cells.
After incubation for 2 days (48 h) with compound, cell proliferation was determined
by MTT assay. Data are shown as means of four independent experiments.
N200
N200

K. Matsubara et al. / Life Sciences 80 (2007) 1578–1585
1583
Fig. 3. Effect of acyl-epicatechins (100 μM each) on ex vivo angiogenesis using
a rat aortic ring. Representative result of the inhibitory effect of epicatechin-C16
(100 μM) (A). Bar equals 500 μm. Microvessel length was measured on day 7 of
culture. Values are means SD (n=3) (B).
Fig. 5. Effect of epicatechin-C16 on HUVEC tube formation on reconstituted
basement membrane. Cells were plated on reconstituted gel and observed 12 h
later (A). Capillary length was measured, and values are means SD (n=3).
compounds was the same tendency as the inhibitory activity of
mammalian DNA polymerases.
⁎
Means with an asterisk ( ; pb0.01) are significantly different from control (B).
Effects of acyl-epicatechin compounds on ex vivo angiogenesis
the ends of the aortic rings after 2 to 3 days, and they spread in
the collagen gel. Microvessels appeared from the ends of aortic
rings after 5 to 6 days and elongated (Fig. 3A). Since the
amounts of synthesized acyl-epicatechins were limited, we
could examine epicatechin-C14 and -C16, which yielded
enough amounts for angiogenesis assays. Inhibitory effects of
acyl-epicatechins were stronger than epicatechin (Fig. 3B).
Epicatechin-C16 was the most effective inhibitor to angiogen-
esis within the compounds so far examined. The inhibitory
effect of epicatechin-C16 was in a dose-dependent manner and
statistically significant at higher than 50 μM (Fig. 4).
It has been shown that tea catechins have anti-angiogenic
activity (Cao and Cao, 1999). Epigallocatechin-3-gallate
(EGCG) is the most effective anti-angiogenic catechin among
them. We also examined the effect of acyl-epicatechins on
angiogenesis in a rat aortic ring model. This angiogenesis
method is widely used to evaluate anti-angiogenic agents
(Nicosia and Ottinetti, 1990; Kruger et al., 2000; Zogakis et al.,
2002). In this model, fibroblastic fusiform cells migrated from
Effect of epicatechin-C16 on HUVEC tube formation
The effect of epicatechin-C16 was examined in in vitro
angiogenesis model using HUVECs. HUVECs inoculated on
reconstituted basement membrane (Matrigel™) migrated, then
attached each other, and finally formed tube structures (Fig. 5A).
Epicatechin-C16 suppressed HUVEC tube formation in a dose-
dependent manner (Fig. 5A, B).
Taken together, epicatechin-C16 exerted the anti-angiogenic
activity through inhibiting DNA polymerase and endothelial
cell tube formation.
Discussion
Fig. 4. Suppressive effect of epicatechin-C16 in ex vivo angiogenesis model.
Various biological activities of natural polyphenols have
been reported. It has been suggested that catechins in green tea
Microvessel length was measured on day 7 of culture. Values are means SD
⁎
(n=3). Mean with an asterisk ( ; pb0.01) is significantly different from control.

1584
K. Matsubara et al. / Life Sciences 80 (2007) 1578–1585
could lower the risk of cancer and cardiovascular diseases.
Epigallocatechin-3-gallate (EGCG) is the most abundant
compound present in green tea and has been the most
extensively studied. EGCG has strong anti-cancer and anti-
angiogenic effects (Cao et al., 2002). In addition, it has been
shown that esterification of epigallocatechin increases anti-virus
activity (Uesato et al., 2000) and that epicatechin and its
metabolite have a protective effect on human fibroblast cells
from oxidative-stress-induced cell death (Spencer et al., 2001).
These observations suggest that chemical modification of green
tea catechins could improve the biological activities. In this
study, we have examined the effects of acyl-epicatechins as anti-
cancer agents.
We have synthesized acyl-epicatechins (epicatechin-C12 to
C16) and found that acylation of epicatechins enhances their
inhibitory effects on DNA polymerase activity, cancer cell
growth and angiogenesis. These observations suggest that acyl-
epicatechins might be useful as potent anti-cancer agents.
Among acyl-epicatechins, epicatechin-C16, the longest acyl-
chain epicatechin, exerted the strongest inhibitory effects on all
assays showing that the length of acyl-chain is critical for their
biological activities. The exact mechanism of how acyl-
epicatechins inhibit DNA polymerase in an acyl-chain length
dependent manner is not clear at this moment. However, the
present study suggests that the stereochemistry of the fatty acid
moiety is also important in showing the inhibitory effect on
DNA polymerase, by affecting the template-primer binding or
the configuration of catalytic domain (Murakami-Murofushi et
al., 1995; Mizushina et al., 2005). Acyl-epicatechins exert a
suppressive effect on HL-60 cancer cell growth by inhibiting
DNA polymerases, which are critical enzymes for cell
proliferation. The inhibitory effects of acyl-catechins on DNA
polymerase and angiogenesis were correlated with the length of
acyl-chain, suggesting that their anti-angiogenic effects would
be exerted by suppressing endothelial cell proliferation. In
addition, epicatechin C-16 strongly inhibits HUVEC tube
formation suggesting that epicatechin C-16 could target other
molecules involved in neovascularization.
EGCG, a tea catechin having anti-angiogenic activity, also
inhibits HUVEC tube formation. As a mechanism, it has been
demonstrated that EGCG inhibits tyrosine phosphorylation of
vascular endothelial growth factor (VEGF) receptor. VEGF is a
highly pro-angiogenic protein. Epicatechin (50 μM) does not
inhibit phosphorylation of VEGF receptor induced by VEGF,
however, epicatechin-3-gallate inhibits the phosphorylation in
the same manner as EGCG (Lamy et al., 2002). These
observations suggest that acyl-epicatechin could attenuate the
phosphorylation of VEGF receptor. To clarify the exact
mechanism of the anti-angiogenic effect of acyl-epicatechin,
further study will be conducted.
From our results that acyl-epicatechins not only inhibit
cancer cell growth directly but also inhibit angiogenesis, they
are likely to be potent anti-cancer agents. Recently, anti-
angiogenic effects of natural polyphenols have considerable
attention, because they are small molecules and act as
angiogenesis inhibitors by oral administration (Cao and Cao,
1999; Bråkenhielm et al., 2001). Their characteristics would
have advantages for long period anti-angiogenic therapy to
arrest tumors during their dormant stage. Natural polyphenol-
derivatives with enhanced their anti-angiogenic activity, as
shown in this study, would be useful for anti-angiogenic
therapy. To reveal the advantage of acyl-epicatechins, we should
address the issues of the derivatives: specificity for endothelial
cells and stability and metabolic fate in vivo in the future.
Acknowledgements
This work was supported by grant from Japan Ministry of
Education, Culture, Sports, Science and Technology (MEXT)
No. 18700608 (K. M.) and in part by “Academic Frontier” Project
for Private Universities (Kobe-Gakuin University): matching
fund subsidy from MEXT, 2006–2010, (K. M. and Y. M.).
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