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ORIGINAL PAPER
Potential anthelmintics: polyphenols from the tea plant Camellia
sinensis L. are lethally toxic to Caenorhabditis elegans
Daisuke Mukai Æ Noriko Matsuda Æ Yu Yoshioka Æ
Masashi Sato Æ Toru Yamasaki
Received: 16 April 2007 / Accepted: 31 August 2007 / Published online: 18 January 2008
Ó The Japanese Society of Pharmacognosy and Springer 2008
Abstract A novel gallate of tannin, (-)-epigallocatechin-
(2b?O?7
0
,4b?8
0
)-epicatechin-3
0
-O-gallate (8), together
with (-)-epicatechin-3-O-gallate (4), (-)-epigallocatechin
(5), (-)-epigallocatechin-3-O-gallate (6), and (+)-gallo-
catechin-(4a?8
0
)-epigallocatechin (7), were isolated from
the tea plant Camellia sinensis (L.) O. Kuntze var. sinensis
(cv., Yabukita). The structure of 8, including stereochem-
istry, was elucidated by spectroscopic methods and
hydrolysis. The compounds, along with commercially
available pyrogallol (1), (+)-catechin (2), and (-)-epicat-
echin (3), were examined for toxicity towards egg-bearing
adults of Caenorhabditis elegans. The anthelmintic
mebendazole (9) was used as a positive control. Neither 2
nor 3 were toxic but the other compounds were toxic in the
descending order 8, 7 & 6, 9, 4, 5, 1. The LC
50
(96 h)
values of 8 and 9 were evaluated as 49 and 334 lmol L
-1
,
respectively. These data show that many green tea poly-
phenols may be potential anthelmintics.
Keywords Camellia sinensis L. Novel tannin gallate
Caenorhabditis elegans LC
50
Anthelmintic
Introduction
Parasitic nematodes have a substantial impact on human
welfare, particularly through diseases they cause in ani-
mals and humans, and novel anthelmintics with little or
no side effects are still needed [1]. Nonfood gallotannins,
ellagitannins, and condensed tannins have been examined
for potency against the dog-roundworm Toxocara canis
[2] and the free-living nematode Caenorhabditis elegans
[3], and several have been identified as potential an-
thelmintics. This study focused on green tea polyphenols.
C. elegans has long been used to study antinematode
drugs, anthelmintics, and nematicides [4–7], as it has
many advantages, such as rapid growth [8]. The purpose
of this paper is to report the isolation and identification
of a new tannin gallate, with known polyphenols, from
the tea plant Camellia sinensis L. and their toxicities to
C. elegans, with special reference to novel anthelmintic
discoveries.
Materials and methods
General
Positive FAB-MS and HRFAB-MS spectra were acquired
with a Jeol JMS-SX 102 mass spectrometer using dithio-
threitol–dithioerythritol (5:1) as matrix.
1
H (400 MHz)
NMR,
13
C (100 MHz) NMR, DEPT,
13
C-
1
H and
1
H-
1
H
COSYs, HMBC, and NOESY spectra were acquired with a
Jeol JNM A-400 spectrometer. UV spectra were recorded
with a Hitachi U-2000, CD was measured with a Jasco J-
20C, and optical rotation was measured with a Jasco P-
1010. Open column chromatography was performed on
Sephadex LH 20 (25–100 lm, Pharmacia Biotech) and
MCI GEL CHP 20P (75–150 lm, Mitsubishi Chemical).
Compounds 1, 2, 3, and 9, and tannase from Aspergillus
oryza, were obtained from Wako Pure Chemical Industries;
2 was purified using Sephadex LH 20. The purity of 1, 2, 3,
and 9 was more than 99%.
D. Mukai N. Matsuda Y. Yoshioka M. Sato
T. Yamasaki (&)
Department of Applied Biological Science, Kagawa University,
2393 Miki-Ikenobe, Kagawa 761-0795, Japan
e-mail: yamasaki@ag.kagawa-u.ac.jp
123
J Nat Med (2008) 62:155–159
DOI 10.1007/s11418-007-0201-4
Plant material, extraction, and isolation
The leaves of C. sinensis (L.) O. Kuntze var. sinensis
(cv., Yabukita) were freshly collected in Kagawa Uni-
versity Farm in Kagawa Pref. on June 19, 2006. The plant
was identified by Dr M. Morokuma of Kagawa University
and a voucher specimen was deposited at the Department
of Applied Biological Sciences. The leaves (5.0 kg,
69.7% moisture) of the plant were immersed in acetone
(10.23 L) containing water (0.90 L). After 24 h the leaves
were further extracted with 70% aqueous acetone. The
extracts were evaporated under reduced pressure at less
than 30°C. After removal of the resulting viscous material
by centrifugation, the supernatant was concentrated in the
same manner. A portion (100 mL) of the concentrate,
equivalent to 33.36 g dry material, was divided into
fractions 1 (1.49 g), 2 (6.99 g), 3 (17.61 g), 4 (3.40 g),
and 5 (2.20 g) by chromatography on a Sephadex LH 20
column (5 9 95 cm) using EtOH, aqueous EtOH (90%
and 80%), EtOH–acetone–water (2:2:1), and 70% aqueous
acetone, respectively, as mobile phases. Fraction 3
(100 mg) was chromatographed on MCI GEL CHP 20P
(2 9 28 cm) with water and aqueous EtOH (20 and 40%)
as mobile phases. Five-milliliter eluent fractions were
collected, and 5 (11.5 mg), 6 (31.9 mg), and 4 (4.4 mg)
were isolated. Similarly, fraction 4 (100 mg) was chro-
matographed on CHP 20P (2 9 28 cm column) with
water and aqueous EtOH (20% and 40%) as mobile
phases. Five-milliliter eluent fractions were collected and
fractions 174–184 and 415–417 afforded 7 (4.7 mg) and
crude 8 (6.5 mg), respectively; 8 was purified using
Sephadex LH 20 with EtOH–acetone–water (3:1:1) as a
mobile phase.
Compound 8
Amorphous pale beige solid. HRFAB-MS m/z: 745.1466
[M + H]
+
(Calculated for C
37
H
29
O
17
; 745.1405). UV kmax
(MeOH) nm (log e): 205 (5.04), 277 (4.14). NMR data
are listed in Table 1. CD (MeOH): [h]
222
-87,300,
[h]
237
+83,300, [h]
274
-55,500. [a]
D
20
(c 0.2, MeOH) -38°.
Enzymatic hydrolysis of 8
Compound 8 (27 mg) in water (2 mL) was incubated with
tannase at 37°C for 35 min and freeze–dried. EtOH-soluble
products of the reaction mixture were chromatographed on
Sephadex LH 20 with aqueous EtOH and EtOH–acetone–
water (2:2:1) as mobile phases, giving 8a (17.8 mg) and
gallic acid (5.2 mg). FAB-MS of 8a: m/z 593 [M + H]
+
.
CD (MeOH) of 8a:[h]
213
-98,000, [h]
227
+70,500, and
[h]
270
-14,700; literature values [9]: [h]
211
-79,573 and
[h]
227
+119,573.
Egg-bearing adults of C. elegans and LC
50
In order to prepare egg-bearing adults of C. elegans var.
Bristol (strain N2), a stock culture was incubated under
standard conditions, and the dauer stage was induced by
starvation, followed by incubation of the dauer populations
on 3.5-cm NGM agar plates seeded with Escherichia coli
strain OP50 at 20°C[10].
Three-animal sets were incubated on E. coli in 200-lL
complete S medium containing one of the samples in 250-
lL hemiellipsoidal wells at 20°C[3, 11]. DMSO at con-
centrations up to 1% has no effect on the development of
C. elegans [12]. In tests on 9, water-insoluble, the final
concentration of DMSO was maintained at 0.8%. Twenty-
seven to thirty-six adults were used at each of the sample
concentrations (5–8 levels). LC
50
(96 h) values were cal-
culated [13].
Results and discussion
Novel gallate of green tea tannin and other polyphenols
Polyphenols 4–8 were isolated from a 70% aqueous ace-
tone extract of fresh leaves of C. sinensis by use of
Sephadex LH 20 and MCI GEL CHP 20P open column
chromatography (Fig. 1). Compound 4 was identical with
(-)-epicatechin-3-O-gallate [14], 5 with (-)-epigallocate-
chin [14], 6 with (-)-epigallocatechin-3-O-gallate [14],
and 7 with (+)-gallocatechin-(4a? 8
0
)-epigallocatechin
[15].
In the HRFAB-MS spectrum of 8, a molecular ion peak
[M + H]
+
was observed at m/z 745.1466, showing the
molecular formula to be C
37
H
29
O
17
(745.1405). The NMR
data (Table 1) suggested that 8 consists of two flavan-3-ol
units and one galloyl group. The chemical shift (d 6.73) of
H-10 was the same as that of H-14. Furthermore, long-
range correlations between H-10 and C-2 (d 100.1) and
between H-14 and C-2 were observed in the HMBC
spectrum of 8 (Fig. 2). The results showed that the upper
unit bears a pyrogallol-type B-ring. Other HMBC correla-
tions between H-10
0
(d 7.07) and C-2
0
(d 80.3) and between
H-14
0
(d 6.94) and C-2
0
, and a
1
H–
1
H correlation between
H-14
0
and H-13
0
(d 6.75), indicated that the lower unit has a
catechol-type B-ring. A diagnostic HMBC correlation
between H-4 (d 4.44) and C-8
0
(d 107.0) was evidence of
the presence of a C-4?C-8
0
inter-flavan linkage. A carbon
resonance, found relatively downfield (d 100.1), and a
singlet (d 6.10) were assigned to a ketal C-2 [16] and H-6
0
,
156 J Nat Med (2008) 62:155–159
123
respectively. The results suggested the presence of another
inter-flavan linkage, C-2?O?C-7
0
.
A trans relationship between H-3 and H-4 was shown by
the appearance of two doublets (J
3–4
3.4 Hz at d 4.07 due
to H-3 and J
4–3
3.4 Hz at d 4.44 due to H-4). In terms of
steric energy [16], it was rationalized that both inter-flavan
linkages occupy cis positions. Two broad singlets (H-2
0
, d
5.16; and H-3
0
, d 5.64) indicated that H-2
0
and H-3
0
are in
cis positions [17] and, furthermore, NOESY correlations
were observed between H-2
0
and H-3 and between H-2
0
and
Table 1
13
C NMR,
1
H NMR, DEPT, and
13
C–
1
H COSY data for 8 and 8a in MeOH-d
4
Position 88a
d
C
Multiplicity
a
d
H
d
C
Multiplicity
a
d
H
2 100.1 C 100.2 C
3 68.1 CH 4.07 d (3.4) 68.1 CH 4.04 d (3.4)
4 29.1 CH 4.44 d (3.4) 29.2 CH 4.40d (3.4)
5 157.2 C 156.6 C
6 98.1 CH 6.12 d (2.4) 98.3 CH 6.00d (2.4)
7 158.2 C 158.1 C
8 96.5 CH 6.09 d (2.4) 96.6 CH 6.07d (2.4)
4a 103.9 C 104.3 C
8a 154.1 C 154.2 C
9 131.7 C 131.7 C
10 107.4 CH 6.73 s 107.5 CH 6.72 s
11 146.4 C 146.4 C
12 134.6 C 134.6 C
13 146.4 C 146.4 C
14 107.4 CH 6.73 s 107.5 CH 6.72 s
2
0
80.3 CH 5.16 br. s 81.7 CH 4.98 br. s
3
0
69.3 CH 5.64 br. s 69.3 CH 4.24 br. s
4
0
27.4 CH
2
2.87 d (17.6), H
a
29.9 CH
2
2.76 d (17.0), H
a
3.09 dd (17.8, 4.9), H
b
2.95 dd (17.0, 4.9), H
b
5
0
156.4 C 157.0 C
6
0
96.5 CH 6.10 s 96.5 CH 6.09 s
7
0
152.6 C 152.2 C
8
0
107.0 C 107.1 C
4
0
a 101.8 C 102.4 C
8
0
a 151.9 C 152.1 C
9
0
130.4 C 131.1 C
10
0
115.5 CH 7.07 d (1.7) 115.9 CH 7.15 d (2.0)
11
0
146.4 C 146.0 C
12
0
146.4 C 146.4 C
13
0
116.2 CH 6.75 d (8.3) 116.0 CH 6.82 d (8.3)
14
0
120.0 CH 6.94 dd (8.3, 1.7) 120.4 CH 6.98 dd (8.3, 2.0)
1
00
121.4 C
2
00
110.5 CH 6.99 s
3
00
146.4 C
4
00
140.0 C
5
00
146.4 C
6
00
110.5 CH 6.99 s
7
00
167.6 C
Chemical shifts (d
C
and d
H
values) are expressed in ppm, and coupling constants (J values) are shown in parentheses.
1
H–
1
H COSY data were
useful for assignment of protons giving rise to broad singlets (br. s), doublets (d), or double doublets (dd)
a
Of carbons indicated by DEPT data
J Nat Med (2008) 62:155–159 157
123
H-4 (Fig. 2), suggesting that (-)-epicatechin constitutes
the lower unit. A high-amplitude positive Cotton effect of
[h]
237
= +83,300, observed in the CD spectrum of 8,
indicated that C-4 has the R configuration (b orientation)
[18]. Considering the cis/trans information including the
NOESY data, therefore, it is evident that other chiral
centers (C-2, C-3, C-2
0
and C-3
0
) also had the R configu-
ration. The specific rotation of 8 was -38°.
In addition to these results, a diagnostic HMBC correla-
tion between H-3
0
(d 5.64) and carbonylic C-7
00
(d 167.6) was
indicative of the presence of a C-3
0
-O-C-7
00
linkage in 8.
Enzymatic hydrolysis of 8 yielded 8a (Fig. 1) and gallic acid.
The hydrolysate 8a was identical with (+)-epigallocatechin-
(2b?O?7
0
,4b?8
0
)-epicatechin [9, 19]. In our
1
HNMR
spectrum of 8a (Table 1), a broad singlet at d 4.24 was
assigned to H-3
0
, whereas, in the case of 8, the relevant singlet
appeared at d 5.64. In our CD spectrum of 8a, a significant
positive Cotton effect of [h]
227
+70,500 was observed, con-
sistent with the value in the literature [9]. Thus, 8 was
identified as a novel gallate of green tea tannin, (-)-epi-
gallocatechin-(2b?O?7
0
,4b?8
0
)-epicatechin-3
0
-O-gallate.
Besides 8a,(-)-epigallocatechin-(2b? O?7
0
,4b?8
0
)-
epigallocatechin-3
0
-O-gallate [20] and (+)-epicatechin-
(2b?O?7
0
,4b?8
0
)-epicatechin [21] have been isolated
as analogues of 8 from oolong tea and peanut skins,
respectively.
Toxicities of green tea polyphenols to C. elegans
Compounds 4–8, together with commercially available
pyrogallol (1), (+)-catechin (2), and (-)-epicatechin (3)
(Fig. 1) were examined for toxicity to egg-bearing adults of
C. elegans (Table 2). The anthelmintic mebendazole,
methyl [(5-benzoyl-3H-benzoimidazol-2-yl)amino]formate
(9) (Fig. 1) was used as a positive reference. In all control
animals, no death was observed during an experimental
period of 96 h. Neither 2 nor 3 were toxic whereas 1 and 4–
9 were toxic. It is noticeable that 6–8 surpassed 9 in tox-
icity. The LC
50
(96 h) value for 8 was far lower than that
for 9; i.e., 49 versus 334 lmol L
-1
. On comparison of the
LC
50
values for 3 and 5 (or 4 and 6), the pyrogallol type B-
ring of 5 and 6 was found to enhance toxicity. Similarly (3
vs. 4,or5 vs. 6), the galloyl group of 4 and 6 has great
significance in causing toxicity. Further investigations
regarding green tea polyphenol toxicity in C. elegans are
needed. The present results strongly show that several
green tea polyphenols are potential anthelmintics.
Acknowledgments We are grateful to Dr Masahiro Morokuma of
Kagawa University for his identification of the plant material.
Fig. 1 Structures of the isolates (4–8), related compounds (1–3 and
8a) and the anthelmintic (9)
Fig. 2 Characteristic correlations observed in the HMBC and
NOESY spectra of 8
Table 2 Adult C. elegans LC
50
(96 h) values for 1–9
LC
50
values are expressed in
lmol L
-1
, and 95% confidence
intervals are shown in
parentheses
Compound LC
50
1 3,870 (3,361–
4,381)
2 [27,590
3 [17,240
4 399 (354–461)
5 1,110 (892–1,369)
6 237 (200–294)
7 201 (128–330)
8 49 (47–58)
9 334 (247–480)
158 J Nat Med (2008) 62:155–159
123
References
1. Horton J (2003) Global anthelmintic chemotherapy programs:
learning from history. Trends Parasitol 19:405–409
2. Kiuchi F, Tsuda Y, Kondo K, Yoshimura H, Nishioka I, Nonaka
G (1988) Studies on crude drugs effective on visceral larva
migrans III. The bursting activity of tannins on dog roundworm
larva. Chem Pharm Bull 36:1796–1802
3. Yamasaki T, Sato M, Mori T, Mohamed ASA, Fujii K, Tsukioka
J (2002) Toxicity of tannins towards the free-living nematode
Caenorhabditis elegans and the brine shrimp Artemia salina.
J Nat Toxins 11:165–171
4. Geary TG, Sangster NC, Thompson DP (1999) Frontiers in
anthelmintic pharmacology. Vet Parasitol 84:275–295
5. Rand JB, Johnson CD (1995) Genetic pharmacology: interactions
between drugs and gene products in Caenorhabditis elegans. In:
Epstein HF, Shakes DC (eds) Methods in cell biology 48. Cae-
norhabditis elegans: modern biological analysis of an organism.
Academic Press, San Diego, pp 187–204
6. Bennett JL, Pax RA (1986) Micromotility meter: an instrument
designed to evaluate the action of drugs on motility of larval and
adult nematodes. Parasitol 93:341–346
7. Simpkin KG, Coles GC (1981) The use of Caenorhabditis
elegans for anthelmintic screening. J Chem Tech Biotechnol
31:66–69
8. Wood WB (1988) Introduction to C. elegans biology. In: Wood
WB, the Community of C. elegans Researchers (eds) The nem-
atode Caenorhabditis elegans. Cold Spring Harbor Laboratory
Press, New York, pp 1–16
9. Barreiros ALBS, David JP, de Queiroz LP, David JM (2000)
A-type proanthocyanidin antioxidant from Dioclea lasiophylla.
Phytochemistry 55:805–808
10. Lewis JA, Fleming JT (1995) Basic culture methods. In: Epstein
HF, Shakes DC (eds) Methods in cell biology 48. Caenorhabditis
elegans: modern biological analysis of an organism. Academic
Press, San Diego, pp 3–29
11. Mori T, Mohamed ASA, Sato M, Yamasaki T (2000) Ellagitannin
toxicity in the free-living soil-inhabiting nematode, Caenorhab-
ditis elegans. J Pestic Sci 25:405–409
12. Spence AM, Malone KMB, Novak MMA, Woods RA (1982)
The effects of mebendazole on the growth and development of
Caenorhabditis elegans. Can J Zool 60:2616–2623
13. Litchfield JT, Wilcoxon F (1949) A simplified method of eval-
uating dose effect experiments. J Pharmacol Exp Ther 96:99–113
14. Coxon DT, Holmes A, Ollis WD, Vora VC, Grant MS, Tee JL
(1972) Flavanol digallates in green tea leaf. Tetrahedron
28:2819–2826
15. Foo LY, Porter LJ (1978) Prodelphinidin polymers: definition of
structural units. J Chem Soc Perkin Trans 1:1186–1190
16. Jacques D, Haslam E, Bedford GR, Greatbanks D (1974) Plant
proanthocyanidins Part II. Proanthocyanidin-A2 and its deriva-
tives. J Chem Soc Perkin Trans 1:2663–2671
17. Fletcher AC, Porter LJ, Haslam E, Gupta RK (1977) Plant pro-
anthocyanidins Part 3. Conformational and configurational
studies of natural procyanins. J Chem Soc Perkin Trans 1:1628–
1637
18. Barrett MW, Klyne W, Scopes PM, Fletcher AC, Porter LJ
(1979) Plant proanthocyanodins. Part 6. Chiroptical studies Part
95. Circular dichroism of procyanidins. J Chem Soc Perkin Trans
1:2375–2377
19. Ma C-M, Nakamura N, Hattori M, Kakuda H, Qiao J-C, Yu H-I
(2000) Inhibitory effects on HIV-1 protease of constituents from
the wood of Xanthoceras sorbifolia. J Nat Prod 63:238–242
20. Hashimoto F, Nonaka G, Nishioka I (1989) Tannins and related
compounds. XC. 8-C-Ascorbyl (-)-epigallocatechin 3-O-gallate
and novel dimeric flavan-3-ols, oolonghomobisflavans A and B,
from oolong tea (3). Chem Pharm Bull 37:3255–3263
21. Lou H, Yamazaki Y, Sasaki T, Uchida M, Tanaka H, Oka S
(1999) A-type proanthocyanidins from peanut skins. Phyto-
chemistry 51:297–308
J Nat Med (2008) 62:155–159 159
123
