ArticlePDF Available

Potential anthelmintics: Polyphenols from the tea plant Camellia sinensis L. are lethally toxic to Caenorhabditis elegans

Authors:

Abstract and Figures

A novel gallate of tannin, (−)-epigallocatechin-(2β→O→7′,4β→8′)-epicatechin-3′-O-gallate (8), together with (−)-epicatechin-3-O-gallate (4), (−)-epigallocatechin (5), (−)-epigallocatechin-3-O-gallate (6), and (+)-gallocatechin-(4α→8′)-epigallocatechin (7), were isolated from the tea plant Camellia sinensis (L.) O. Kuntze var. sinensis (cv., Yabukita). The structure of 8, including stereochemistry, was elucidated by spectroscopic methods and hydrolysis. The compounds, along with commercially available pyrogallol (1), (+)-catechin (2), and (−)-epicatechin (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 LC50 (96 h) values of 8 and 9 were evaluated as 49 and 334 μmol L−1, respectively. These data show that many green tea polyphenols may be potential anthelmintics.
Content may be subject to copyright.
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 [47], 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 48 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 48, 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 68 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 (48), related compounds (13 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 19
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
... Each of the five tested compounds harbored either a pyrogalloyl type B-ring or a galloyl group, but only EGCG has both groups, which might explain its high toxicity, it required higher LC 50 values than ECG. Mukai et al. [40] also reported that EGCG toxicity surpasses mebendazole. It was also shown in the same study that the pyrogalloyl type B-ring of EGC and EGCG enhances the toxicity compared to ECG. ...
... It was also shown in the same study that the pyrogalloyl type B-ring of EGC and EGCG enhances the toxicity compared to ECG. Similarly, the galloyl group of ECG and EGCG also has great significance in causing toxicity [40]. In another study, the toxicity of gallotannins and condensed tannins towards C. elegans has been shown to be dependent on the degree of galloylation and polymerization, respectively [41]. ...
Article
Acacia nilotica fruits with high tannin content are used in the northern parts of Cameroon as anti-filarial remedies by traditional healers. In this study, the hydro-alcoholic fruit extract (crude extract (CE)) and, one of the main constituents in its most active fractions, (+)-catechin-3-O-gallate (CG), as well as four related proanthocyanidins, (-)-epicatechin-3-O-gallate (ECG), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-O-gallate (EGCG), were assessed for their potential in vitro anthelmintic properties against the free-living model organism Caenorhabditis elegans and against the cattle filarial parasite Onchocerca ochengi. Worms were incubated in the presence of different concentrations of fruit extract, fractions and pure compounds. The effects on mortality were monitored after 48 h. The plant extract and all of the pure tested compounds were active against O. ochengi (LC50 ranging from 1.2 to 11.5 μg/mL on males) and C. elegans (LC50 ranging from 33.8 to 350 μg/mL on wild type). While high LC50 were required for the effects of the compounds on C. elegans, very low LC50 were required against O. ochengi. Importantly, tests for acute oral toxicity (lowest dose: 10 mg/kg) in Wistar rats demonstrated that crude extract and pure compounds were non-toxic and safe to use. Additionally, the results of cytotoxicity tests with the Caco-2 cell line (CC50 ranging from 47.1 to 93.2 μg/mL) confirmed the absence of significant toxicity of the crude extract and pure compounds. These results are in good accordance with the use of A. nilotica against nematode infections by traditional healers, herdsmen and pastoralists in Cameroon.
... Caenorhabditis elegans is a well-known, nonmammalian, alternative animal model that is extensively used in biomedical and toxicological research [17,18], including assessments 2 Evidence-Based Complementary and Alternative Medicine of toxicity. Several studies have tested for beneficial and/or adverse effects of compounds isolated from certain plants and Chinese medicines in C. elegans [19][20][21][22][23]. This is primarily due to the several advantages of using C. elegans, including their short life cycle, ease of handling, small body size, and high sensitivity to toxins and toxicants [17,[24][25][26]. ...
... C. elegans are invertebrates well suited for use as an animal model due to their short lifecycle, fast reproduction, and wellcharacterized genome [17]. This model has been successfully used in the assessment of toxicity of heavy metals [40][41][42][43][44], environmental pollutants [45,46], and specific components of plant extracts [21,23]. As a whole, these previous studies have resulted in the acceptance of the use of C. elegans as a bioindicator in toxicity studies [23,45,47,48]. ...
Article
Full-text available
Impatiens balsamina L. (Balsaminaceae), an annual herb found throughout China, has been extensively used in traditional Chinese medicine (TCM). However, our knowledge regarding the adverse effects of I. balsamina in vivo is very limited. In this present study, the nematode Caenorhabditis elegans model was employed to fully assess the adverse effects of hydroalcoholic (EtOH 55%) extracts of I. balsamina stems (HAEIBS) in vivo. After exposure to 10 mg/mL HAEIBS, the major organism-level endpoints of C . elegans of percent survival, frequency of head thrash and body bends, and reproduction had decreased by 24%, 30%, and 25%, respectively. The lifespan of C. elegans was also greatly reduced after HAEIBS exposure compared to the controls . The active compounds in HAEIBS were separated using high speed countercurrent chromatograph (HSCCC) and characterized by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Two compounds, lawsone and 2-methoxy-1,4-naphthoquinone (MNQ), and their adverse effects were then more thoroughly detailed in this study. It was found that lawsone is the major toxin in HAEIBS with a higher toxicity than MNQ in terms of negative impact on C. elegans mortality, locomotion, reproduction, and lifespan. Our data also suggests that the C. elegans model may be useful for assessing the possible toxicity of other Chinese medicines, plant extracts, and/or compounds.
... Eight compounds were isolated from Camellia sinensis and their anthelmintic activity was tested. One new gallate of tannin, (−)-epigallocatechin-(2β → O → 7 ,4β → 8 )-epicatechin-3 -O-gallate, showed the best activity with an IC 50 of 49 µM [91]. ...
Article
Full-text available
Intestinal parasitic nematodes infect approximately two billion people worldwide. In the absence of vaccines for human intestinal nematodes, control of infections currently relies mainly on chemotherapy, but resistance is an increasing problem. Thus, there is an urgent need for the discovery and development of new anthelmintic drugs, especially ones with novel mechanisms of action. Medicinal plants hold great promise as a source of effective treatments, including anthelmintic therapy. They have been used traditionally for centuries and are mostly safe (if not, their toxicity is well-known). However, in most medicinal plants the compounds active against nematodes have not been identified thus far. The free-living nematode C. elegans was demonstrated to be an excellent model system for the discovery of new anthelmintics and for characterizing their mechanism of action or resistance. The compounds discussed in this review are of botanical origin and were published since 2002. Most of them need further studies of their toxicity, mechanisms and structure-activity relationship to assess more fully their potential as drugs.
... When discussing the results of the present study, one must remember that the effects of phenolic compounds are not only negative. For example, polyphenols from Camelia sinensis (green tea) are lethal to the rhabditid Caenorhabditis elegans (Mukai et al., 2008), but Abbas and Wink (2009) showed that the life-span of C. elegans is prolonged by exposure to epigallo-catechin gallate (EGCG) from green tea. Similarly, no mortality or abnormality was noted in MH exposed to 200-ppm doses of the lentisk chloroform and hexane fractions and their F 1 offspring developed normally, though the proportions of life-stages different from the controls. ...
Article
Full-text available
Insects show adaptive plasticity by ingesting plant secondary compounds, such as phenolic compounds, that are noxious to parasites. This work examined whether exposure to phenolic compounds affects the development of insect parasitic nematodes. As a model system for parasitic life cycle, we used Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade) grown with Photorhabdita luminescens supplemented with different concentrations of plant phenolic extracts (0, 600, 1200, 2400 ppm): a crude ethanol extract of lentisk (Pistacia lentiscus) or lentisk extract fractionated along a scale of hydrophobicity with hexane, chloroform and ethyl acetate; and flavonoids (myricetin, catechin), flavanol-glycoside (rutin) or phenolic acids (chlorogenic and gallic acids). Resilience of the nematode to phenolic compounds was stage-dependent, with younger growth stages exhibiting less resilience than older growth stages (i.e., eggs < young juveniles < young hermaphrodites < infective juveniles < mature hermaphrodites). At high concentrations, all of the phenolic compounds studied were lethal to eggs and young juveniles. The nematodes were able to survive in the presence of medium and low concentrations of all studied compounds, but very few of those treatments allowed for reproduction beyond the infective juvenile stage and, at low concentrations, the crude 70% ethanol extract, chloroform and hexane extracts, and myricetin were associated with some impaired reproduction. The ethyl-acetate fraction and gallic acid were extremely lethal to the young stages and allowed almost no development beyond the infective juvenile stage. We conclude that exposure of infective juveniles to phenolics before they infect insects and post-infection exposure of other nematode developmental stages may affect the initiation of the infection, suggesting that the chemistry of dietary phenolics may limit H. bacteriophora's infection of insects.
... The most restrictive PNEC values reported in the literature, at the best of our knowledge were selected. In some cases, PNECs were estimated in this work using assessment factors suggested by the European Commission 11 as well as toxicological data available elsewhere [20][21][22][23][24] . Table 1 shows a list of the most commonly investigated emergent micropollutants in Brazil and PNEC values obtained in the literature or estimated in the present work. ...
Article
Full-text available
The present work intended to present a preliminary snapshot on the contamination of Brazilian surface waters by pharmaceuticals and personal care products. Data were collected elsewhere for Measured Environmental Concentrations (MEC) in surface waters and Predicted No-Effect Concentration (PNEC). Prioritization was based on risk assessment, where MEC/PNEC ratios higher than 1.0 indicate a positive risk, values below 0.1 indicate absence of risk and intermediate values, potential risk. Results revealed that hormones, antibiotics drugs and triclosan should be prioritized to subsidize the generation of water quality standards to protect aquatic life.
... In reality, both PAs and HTs may reduce feed intake, feed digestibility or even cause toxicity if consumed in large quantities, but neither is toxic when consumed in moderation (Katiki et al. 2013). Several screening studies have been conducted with plants known to be rich in HTs (Chandrawathani et al. 2006;Mukai et al. 2008;Manolaraki et al 2010;Waterman et al. 2010) but no unambiguous evidence on the relationship between the HTs and the anti-parasitic properties of these plants have been established. However, the few studies with focus on HTs have indicated potential anthelmintic activity (König et al. 1994;Mori et al. 2000;Yamasaki et al. 2002;Katiki et al. 2011Katiki et al. , 2013. ...
Thesis
Full-text available
Tannins, typically segregated into two major groups, the hydrolyzable tannins (HTs) and the proanthocyanidins (PAs), are plant polyphenolic secondary metabolites found throughout the plant kingdom. On one hand, tannins may cause harmful nutritional effects on herbivores, for example insects, and hence they work as plants’ defense against plant-eating animals. On the other hand, they may affect positively some herbivores, such as mammals, for example by their antioxidant, antimicrobial, anti-inflammatory or anticarcinogenic activities. This thesis focuses on understanding the bioactivity of plant tannins, their anthelmintic properties and the tools used for the qualitative and quantitative analysis of this endless source of structural diversity. The first part of the experimental work focused on the development of ultra-high performance liquid chromatography−tandem mass spectrometry (UHPLC-MS/MS) based methods for the rapid fingerprint analysis of bioactive polyphenols, especially tannins. In the second part of the experimental work the in vitro activity of isolated and purified HTs and their hydrolysis product, gallic acid, was tested against egg hatching and larval motility of two larval developmental stages, L1 and L2, of a common ruminant gastrointestinal parasite, Haemonchus contortus. The results indicated clear relationships between the HT structure and the anthelmintic activity. The activity of the studied compounds depended on many structural features, including size, functional groups present in the structure, and the structural rigidness. To further understand tannin bioactivity on a molecular level, the interaction between bovine serum albumin (BSA), and seven HTs and epigallocatechin gallate was examined. The objective was to define the effect of pH on the formation on tannin–protein complexes and to evaluate the stability of the formed complexes by gel electrophoresis and MALDI-TOF-MS. The results indicated that more basic pH values had a stabilizing effect on the tannin–protein complexes and that the tannin oxidative activity was directly linked with their tendency to form covalently stabilized complexes with BSA at increased pH.
Article
Onchocerciasis is a filarial disease caused by Onchocerca volvulus and transmitted through the bite of a blackfly of the genus Simulium. The microfilaricidal drug ivermectin has long been relied on to treat onchocerciasis. Nowadays, the focus is on the complete elimination of the disease through the development of macro-filaricidal drugs. In this study, a hydro-alcoholic extract from Annona senegalensis leaves was screened for micro- and macrofilaricidal activities against the cattle parasitic nematode Onchocerca ochengi and the free-living nematode Caenorhabditis elegans, both model organisms. Worms were assessed by in vitro motility/mortality assay studies and incubated with the various natural products and their effects on mortality were monitored after 48 h. The crude extract showed high activity with LC50 = 1450.2 µg/mL on the young adult of C. elegans wild type and 19.63 µg/mL on adult males of O. ochengi after 48 h post-exposure. The compounds, Quercetin-3-O-glucoside, (+)-Catechin, (-)-Epicatechin, Rutin and AS14.5, displayed LC50 of 179.3, 840.5, 1270.3, 118.2 and 129.7 µg/mL on C. elegans wild type (young adult), respectively, and 7.3, 17.5, 12.6, 6.6 and 11.5 µg/mL on O. ochengi (adult males), respectively, after 48 h. These data support the use of Annona senegalensis for the treatment of infections caused by Onchocerca volvulus.
Article
As gastro-intestinal nematodes (GINs) become increasingly resistant to chemical anthelmintics, and because consumers scrutinize chemical residues in animal products, the use of herbal anthelmintics and in particular, phenolic compounds, has become attractive. Most life stages of GINs cannot be grown in the lab as they are obligatory parasites, which limits our understanding of the effects of phenolic compounds on their parasitic stages of life. We hypothesized that a species phylogenetically close to GINs and grown in vitro, the insect-parasitic nematode Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade), when fed with Photorhabdus luminescens exposed to plant phenolics, can serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds. We compared the development of H. bacteriophora infective juveniles (IJ) and the exsheathment rate of L3 larvae of the strongyle Teladorsagia circumcincta and Trichostrongylus colubriformis when exposed to catechin, rutin, chlorogenic and gallic acids, and myricetin. Gallic acid had the highest impact in terms of IJ mortality but the highest impairment of IJ development to adulthood was imposed by myricetin. The studied compounds were not lethal to GINs stricto sensu but we consider that the practical implications of total exsheathment inhibition and mortality on GIN populations are similar. Catechin and rutin had similar effects on rhabditid and strongyles: they imposed ca. 90% lethality of IJs at concentrations higher than 1200 ppm and the remaining live IJs did not develop further, and they also totally inhibited strongyle L3 exsheathment in a dose-response fashion. Gallic acid was 100% lethal to IJs exposed above 300 ppm and chlorogenic acid caused 87% mortality above 1200 ppm, with no development for the surviving IJs but for all lower concentrations, all the IJs developed to adult stages. Likewise, gallic and chlorogenic acids did not affect the exsheatment of GIN L3 larvae. Therefore, a discrepancy between the effects of gallic and chlorogenic acids on the development of rhabditid IJs and exsheathment of GIN L3 larvae was found only when they were exposed to high concentrations. A dose-response of IJ lethality to myricetin was found, with no IJ development between 150 and 2400 ppm; but contrary to the other compounds, myricetin also impaired IJ development of IJs above 10 ppm in a dose-response manner and showed dose-responses in the L3 exsheathment. Apart for the high rates of lethality imposed on IJs by gallic and chlorogenic acids at high concentration, these results suggest that H. bacteriophora fed P. luminescens exposed to phenolics shows potential to serve as model in studies of the anthelmintic effects of phenolics in GIN.
Article
Covering: 2001 up to the end of 2016 Polyphenols comprise a structurally diverse class of natural products. As the development of new anthelmintic drugs against soil-transmitted helminthiases is an urgent need and polyphenols are widely used in the treatment of nematode infections in traditional medicine and modern science, we summarize the state of knowledge in the period of mainly 2001 up to the end of 2016 on plant extracts with known polyphenolic composition and of defined polyphenols, mainly from the classes of condensed and hydrolysable tannins, flavonoids, and phenylpropanoids. The diverse biological activity against different helminths and the underlying mechanisms are reviewed.
Article
Full-text available
Diallel designs are used in many breeding programmes because of the important genetic information they offer to plant breeders. Eight biochemical traits of Tea (Camellia sinensis (L.) O. Kuntze) were studied to investigate the underlying gene action, and estimate the general combining abilities (GCA) and specific combining abilities (SCA) of parents and crosses using diallel mating system. There were significant (p<0.05) differences among the genotypes for all the traits under study. The general combining ability (GCA) effects were significant for six of the traits, namely GA, EGC, Caffeine, ECG, EGCG and total catechin implying that these traits are governed by additive gene effects. SCA on the other hand was significant for EGC, Caffeine, EC, EGCG, and total catechin. Maternal effects were significant for EGC, EGCG and total catechin signifying importance of the choice of female parents in breeding programmes targeting these traits. Non-maternal effects were present in EGCG and total catechin. The study revealed that parents which would produce above average progenies for total catechins are AHP S15/10 and EPK TN14-3. The best combiners for total catechins were EPK TN14-3 x TREK 6/8 and AHP SI5/10 self This information, which has hitherto been lacking will be very valuable for tea breeding programmes targeting high black/green tea qualities.
Article
Full-text available
ミズキ科サンシュユの新鮮葉から精製エラジタンニン標品を得た. 線虫 Caenorhabditis elegans を同調培養した. 未成熟成虫と抱卵成虫をこのタンニン (10ppm濃度) に5日間暴露しても運動能に若干の低下が認められたものの, 未成熟成虫は死亡せず, 抱卵成虫の死亡率も7%に過ぎなかった. しかし, 未成熟成虫の性的成熟および抱卵成虫の繁殖能は阻害された. L1幼虫の運動能も低下しなかったが, その成長は阻害された. 同濃度下でさらに暴露試験を続けると, 抱卵成虫と未成熟成虫の死亡率は急激に高まり, 10日目で未成熟成虫の死亡率は56%, 抱卵成虫で78%に達した. しかし, L1幼虫は死亡しなかった. 1ppm濃度下でL1幼虫の成長が若干阻害された. 1000ppm濃度下でなお生存していた抱卵成虫の内臓は溶解・消失していた. しかし, 体内での卵割は認められた.
Article
A chemical examination of the polypenolic constituents in commercial oolong tea has led to the isolation of a new-flavan-3-ol, two novel dimeric flavan-3-ols named oolonghomobisflavans A and B eight new proanthocyanidins, together with twenty-one known polyphenols including proanthocyanidins, hydrolyzable tannins and red pigments. On the basis of chemical and spectroscopic evidence, the flavan-3-ol has been characterized as 8-C-ascorbyl (-)-epigallocatechin 3-O-gallate (22), while oolonghomobisflavans A (26) and B (27) have been determined to be dimeric flavan-3-ols in which two units are linked through a methylene bridge at the 8, 8'- and 8, 6'-positions, respectively. The structures of the new proanthocyanidins were elucidated mainly by tannase hydrolysis and thiolytic degradtion as epicatechin-(4β→8)-epigallocatechin 3-O-gallate (29), epicatechin 3-O-gallate-(4β→8)-epigallocatechin 3-O-gallate (30), catechin-(4α→8)-epigallocatechin 3-O-gallate (31), prodelphinidin B-4 3'-O-gallate (32), epicatechin 3-O-gallate-(4β→6)-epigallocatechin 3-O-gallate (33), epigallocatechin 3-O-gallate-(4β→6)-epicatechin 3-O-gallate (34), epiafzelechin 3-O-gallate-(4β→6)-epigallocatechin 3-O-gallate (35) and prodelphinidin B-2 3'-O-gallate (36).
Article
The flavanol class which is phytochemically characteristic of green tea leaf has been extended to include the novel natural flavanol-3,5-digallates III and IX in addition to the flavan-3-ols I, VI, VII, and XII and the flavanol-3-monogallates II and VIII.
Article
The 2,7″-epoxy-4,8″-biflavan structure (1) has been deduced for the natural proanthocyanidin-A2 on the basis of spectroscopic (13C and 1H n.m.r.) and chemical evidence. Spectroscopic data are also outlined which lead, in association with biogenetic arguments, to a proposal (1a) for the absolute stereochemistry of the natural product. Hydrogenolysis of proanthocyanidin-A2 leads to fission of the C–O and C–C inter-flavan bonds and formation of the phenols (20) and (22). The same technique also leads to rupture of the inter-flavan linkage of dimers of the B group and hence to a novel micro-procedure for their identification. The isolation and characterisation of three trimeric proanthocyanidins based on A2 is also discussed.
Article
This chapter discusses the interactions between drugs and gene products in Caenorhabditis elegans in genetic pharmacology. Caenorhabditis elegans has been a popular organism for the study of drug action. This chapter discusses the methods that used in compound-based studies of C. elegans and evaluates the effects of compounds on C. elegans growth, development, metabolism, and behavior. The strategies for the isolation, and analysis of drug-resistant and hypersensitive mutants are discussed. Studies combining bioactive compounds and C. elegans can be separated based on experimental strategy. The first strategy employs compounds with known modes of action to characterize particular aspects of C. elegans biology in wild type and mutant animals. The second strategy uses active compounds as screening or selective agents to isolate new drug-resistant or hypersensitive mutants and, thus, to identify genes with altered drug responses. Study of such compound-specific mutants can identify specific drug targets, and/or provide insight into the mechanism of drug action and the sites of drug action. The third strategy involves the use of C. elegans, both wild type and selected mutants, to analyze the mechanism of action of uncharacterized or poorly characterized compounds. This has led to the use of C. elegans as a primary screen for compounds active against parasitic nematodes.
Article
This chapter discusses the basic culture methods of the Caenorhabditis elegans (C. elegans) system. The nematode C. elegans is a small, rapidly growing organism that can easily be raised in the laboratory on the bacterium Escherichia coli. Because C. elegans is a self-fertilizing hermaphrodite, it is possible to grow large quantities of the organism in swirling liquid cultures, and possible to propagate severely incapacitated mutants. The rapidity of growth and the ability to self-fertilize necessitate special measures to establish a synchronous culture. C. elegans grows rapidly and is most fecund at 20°C. Growth at 16 and 25°C provides some control over the rate of growth and the facility to work with temperature-sensitive mutants. Gram quantities of nematodes can be prepared by growth on petri plates, or by rotary shaking in a flask at controlled temperatures. Petri plate methods are economical and require no special equipment. Large quantities of nematodes are more easily prepared in fermentor-like devices. Fementors offer the advantage of scalability, minimizing the number of parallel cultures required to grow a large quantity of worms.
Article
From a methanolic extract of the wood of Xanthoceras sorbifolia, two new compounds, 29-hydroxy-3-oxotirucalla-7,24-dien-21-oic acid (3, xanthocerasic acid) and epigallocatechin-(4 beta-->8,2 beta-->O-7)-epicatech (6), were isolated, together with 11 known compounds. Of the isolated compounds, 3-oxotirucalla-7,24-dien-21-oic acid (2), oleanolic acid (4), and 6 were found to be inhibitory substances against human immunodeficiency virus (HIV-1) protease, with their 50% inhibitory concentrations (IC50) being 20, 10, and 70 mu g/mL, respectively. Condensed tannins of high molecular weights with epicatechin and epiafzelechin as the main extender units were found to be the most active principles of this plant (IC50 values ca. 6.0 mu g/mL).
Article
Circular dichroism spectra of 26 procyanidins and their derivatives have been measured. All exhibit a strong positive or negative couplet at 200–220 nm and this has been correlated with the absolute stereochemistry at C-4 on the interflavan linkage.
Article
Means to generate the C-4 carbocations (3) and (4) corresponding to (+)-catechin (1) and (–)-epicatechin (2), respectively, are outlined, and the use of these intermediates for the synthesis of model procyanidins and for the biogenetically patterned synthesis of natural procyanidins is discussed. 13C N.m.r. data for model flavan systems and natural procyanidins are reported and analysed and the information is used to assign the 4R-configuration to four natural procyanidin dimers. The phenomenon of conformational isomerism is demonstrated for the natural procyanidin dimers, and two different forms of restricted rotation about the interflavan bond are proposed. The information is used to clarify many earlier structural anomalies, to predict preferred conformations, and to specify a C(4)–C(8) link for the four principal dimers (B-1–4). The properties of some procyanidin polymers are noted, and structures of opposite helicity are proposed for two of the major types found in nature.
Article
The two basic structural units of condensed prodelphinidin polymers have been defined by generation of the C-4 carbocations, corresponding in stereochemistry to (+)-gallocatechin (1) and (–)-epigallocatechin (2), and trapping them as their phloroglucinol adducts (4) and (7). Two naturally occurring dimeric prodelphinidins with stereochemistry based on that of gallocatechin have also been isolated.