Immunomodulatory proanthocyanidins from Ecdysanthera utilis

Article abstract of DOI:10.1021/np010414l

Two new A-type proanthocyanidins have been isolated from Ecdysanthera utilis and identified as epicatechin-(4β→8,2β→O→7)- epicatechin-(4β→8)-epicatechin (5) and epicatechin-(4β→8)-epicatechin- (4β→8,2β→O→7)-epicatechin- (4β→8)-epicatechin (6), respectively. The structure-related components epicatechin (1), procyanidin B2 (2), proanthocyanidin A1 (3), proanthocyanidin A2 (4), and aesculitannin C (7) were also isolated. All of these compounds were identified and evaluated for immunopharmacological activity. Human peripheral blood mononuclear cells (PBMC) were used as target cells, and cell proliferation was determined by ³H-thymidine uptake. The results indicated that compound 3 suppressed PBMC proliferation activated with phytohemagglutinin (PHA). The inhibitory mechanisms may involve the blocking of interleukin-2 (IL-2) and interferon-γ (IFN-γ) production, since compound 3 attenuated IL-2 and IFN-γ production of PBMC in a dose-dependent manner. Therefore, it is suggested that immunomodulatory agents are present in E. utilis.

Full text of DOI:10.1021/np010414l

J . Nat. Prod. 2002, 65, 505-508
505
Im m u n om od u la tor y P r oa n th ocya n id in s fr om Ecd ysa n th er a u tilis
Lie-Chwen Lin,* Yuh-Chi Kuo, and Cheng-J en Chou
National Research Institute of Chinese Medicine, 155-1, Sec. 2, Li-Nung Street, Taipei (112), Taiwan, Republic of China
Received August 22, 2001
Two new A-type proanthocyanidins have been isolated from Ecdysanthera utilis and identified as
epicatechin-(4âf8,2âfOf7)-epicatechin-(4âf8)-epicatechin (5) and epicatechin-(4âf8)-epicatechin-
(4âf8,2âfOf7)-epicatechin-(4âf8)-epicatechin (6), respectively. The structure-related components
epicatechin (1), procyanidin B2 (2), proanthocyanidin A1 (3), proanthocyanidin A2 (4), and aesculitannin
C (7) were also isolated. All of these compounds were identified and evaluated for immunopharmacological
activity. Human peripheral blood mononuclear cells (PBMC) were used as target cells, and cell proliferation
was determined by 3H-thymidine uptake. The results indicated that compound 3 suppressed PBMC
proliferation activated with phytohemagglutinin (PHA). The inhibitory mechanisms may involve the
blocking of interleukin-2 (IL-2) and interferon-γ (IFN-γ) production, since compound 3 attenuated IL-2
and IFN-γ production of PBMC in a dose-dependent manner. Therefore, it is suggested that immuno-
modulatory agents are present in E. utilis.
Ecdysanthera utilis Hayata & Kawakami (Apocynaceae)
Sephadex LH-20 and Cosmosil 140 C18-OPN to give seven
is a large climbing shrub scattered in forests at low
altitudes in Taiwan. Its roots and stems have been used
components. Compounds 1-4 were readily identified as
1
11
12
13
epicatechin,
procyanidin B2, proanthocyanidin A1,
as analgesic, antiphlogistic, and spasmolytic agents in
and proanthocyanidin A2,13 respectively, by comparison of
their spectral data with those of authentic samples.
2
Taiwan folk medicine. A literature survey shows that the
crude extract of E. utilis possessed antiinflammatory
3
effects. Previous chemical study of this plant has resulted
in the isolation of steroids and triterpenoids.4
The inflammatory response provides early protection in
restricting tissue damage at the site of infection or tissue
5
injury. Both leukocytes, including lymphocytes and mono-
cytes, and cytokines have important roles in clearance of
6
the antigen and healing of the tissue. Interferon-γ (IFN-
γ) is an important inflammatory factor for the attraction
7
of macrophages. Interleukin-2 (IL-2) stimulates prolifera-
tion of T-lymphocytes and also acts on natural killer cells
and macrophages to induce other cytokines involved in
8
inflammatory responses. Paradoxically, the inflammatory
response to invasive organisms, if sufficiently intense or
inappropriately prolonged, could aggravate the injury or
even cause death. The use of antiinflammatory medications
9
must therefore be done carefully. Blockade of lymphocyte
activation and proliferation is one antiinflammatory mech-
anism10 that has potential therapeutic benefit. In the
present study, human peripheral blood mononuclear cells
(PBMC) were used as target cells, and E. utilis was selected
for evaluation of immunopharmacological activity. A sys-
temic study of the bioactive part of E. utilis stems has
resulted in the isolation of five A-type proanthocyanidins
and one procyanidin and epicatechin. This paper deals with
the structure elucidation of the proanthocyanidins by
spectroscopic and chemical means. Furthermore, the re-
sults of the immunopharmacological evaluations of indi-
vidual components are also presented.
Compounds 5-7 yield an orange coloration on reaction
with anisaldehyde-sulfuric acid reagent, which is thought
14
to be characteristic of proanthocyanidins. Compounds 5
+
and 7 exhibited ion peaks at m/z 865 [M + H] and 863 [M
+
+
H] , respectively, in the fast atom bombardment mass
Resu lts a n d Discu ssion
spectra (FABMS), corresponding to a trimeric constitution
for both compounds. The 13C NMR spectrum of 5 shows
two signals at δ 80.0 and 78.6 attributable to flavan C-2
carbons. In addition, the appearance of a ketal carbon
signal at δ 100.0 indicated that 5 possesses an A-type unit
in the molecule. On degradation with acid in the presence
of R-toluenethiol, 5 gave epicatechin (1) and 4′-benzylthio-
proanthocyanidin A-2 (8). Therefore, 5 was determined to
be a coupled product of procyanidin A-2 and epicatechin.
E. utilis stems were extracted with acetone, and the
resulting extract was granulated by Celite. The granule of
extract was then eluted successively with n-hexane, CHCl ,
3
EtOAc, and acetone. The bioactive EtOAc extract fraction
was subjected to sequential column chromatography on
*
To whom correspondence should be addressed. Tel: 886-2-8201999, ext.
8
341. Fax: 886-2-28264276. E-mail: lclin@cma23.nricm.edu.tw.
1
0.1021/np010414l CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/14/2002

5
06 J ournal of Natural Products, 2002, Vol. 65, No. 4
Lin et al.
F igu r e 1. IL-2 production in PBMC cultures treated with compound
5
3
. PBMC (2 × 10 /well) were treated by 0, 6, 12.5, 25, 50, and 100 µM
of compound 3 with or without PHA (5 µg/mL) for 3 days. The cell
supernatants were then collected and IL-2 concentration was deter-
mined by EIA. Each point is the mean of three independent experi-
ments.
1
In the H NMR spectrum, signals of H-3 and H-2′′ were
apparent at δ 3.31 and 4.40, respectively. These remarkable
upfield shifts are attributable to the magnetic anisotropic
effects of the B′′-ring and the A′-ring, which can be located
just in front of the protons only when 5 possessed a (4′â-8)
linkage.15 Therefore, the structure of 5 was determined as
epicatechin-(4âf8,2âfOf7)-epicatechin-(4âf8)-epicat-
echin.16 The C NMR data of 7 showed a C-2 methine
signal at δ 81.4 and two ketal carbon signals at δ 100.0
and 100.7, indicating that trimeric 7 possesses an ad-
ditional a C-O interflavanoid linkage relative to 5. By
13
comparison of the published data, the structure of 7 was
F igu r e 2. Impairment of IFN-γ production in PBMC cultures treated
5
determined as aesculitannin C.17
with compound 3. PBMC (2 × 10 /well) were treated by 0, 6, 12.5, 25,
5
0, and 100 µM of compound 3 with or without PHA (5 µg/mL) for 3
days. The cell supernatants were then collected and IFN-γ concentra-
tion was determined by EIA. Each point is the mean of three
independent experiments.
echin (9), epicatechin-(4âf8,2âfOf7)-epicatechin-(4âf8)-
epicatechin (5), 4′-benzylthioproanthocyanidin A-2 (8), and
epicatechin (1). 13C NMR signals at δ 75.6, 70.4, and 36.8,
attributable to C-2, C-3, and C-4, respectively, were similar
to those of procyanidin B2 (2). These results implied that
6
possesses an epicatechin as the upper unit with the 4â-8
interflavonoid linkage to 5. Therefore, the structure of
was determined as epicatechin-(4âf8)-epicatechin-
4âf8,2âfOf7)-epicatechin-(4âf8)-epicatechin. The iso-
6
(
meric compounds of 5 and 6 had been isolated from
Cinnamomum zeylanicum.18
All of the isolated compounds were evaluated for their
effects on PBMC proliferation. With the exception of 3, no
inhibitory activity on PBMC proliferation was detected. The
results indicated that the 3 H-thymidine uptake in the
resting or PHA-stimulated states was not affected by
DMSO (0.1%) treatment. Although similar to cyclosporin
A in having little effects on 3 H-thymidine uptake in resting
PBMC, both cyclosporin A and 3 significantly suppressed
the enhanced uptake observable in activated cells. The IC50
of 3 on activated PBMC proliferation was 30.6 µM.
+
In the FABMS, 6 gave a [M + H] ion peak at m/z 1053
corresponding to a tetrameric constitution. Comparison of
1
13
the H and C NMR spectra of 6 with those of 5 revealed
that 6 contains an additional epicatechin molecule relative
to 5. On thiolytic degradation, 6 gave 4-benzylthioepicat-

Proanthocyanidins from Ecdysanthera
J ournal of Natural Products, 2002, Vol. 65, No. 4 507
To determine whether the impairment of activated
PBMC proliferation was related to cytokines production,
the cell supernatants were collected and the production of
IL-2 and IFN-γ were assayed by EIA. As shown in Figure
100.1 (C-2), 102.5 (C-10′), 104.0 (C-10), 107.1 (C-8′); ESI MS
m/z 697 [M - H] .
E p ica t ech in -(4âf8)-ep ica t ech in -(4âf8,2âfOf7)-ep i-
-
2
6
ca tech in -(4âf8)-ep ica tech in (6): amorphous powder; [R]
D
1
4
8.6° (c 1.83, acetone); H NMR (acetone-d
H-4′′′), 3.26 (1H, d, J ) 3.5 Hz, H-3′), 3.61 (1H, s, H-3′′′), 4.02
2H, s, H-3, -3′′), 4.08 (1H, s, H-2′′′), 4.16 (1H, d, J ) 3.5 Hz,
H-4′), 4.30 (1H, s, H-4′′), 4.32 (1H, s, H-4), 4.67 (1H, s, H-2),
.60 (1H, s, H-2′′), 5.83/5.89/5.91/6.02 (5H, H-6/-8/-6′/-6′′/-6′′′),
6
) δ 2.66 (2H, m,
1
and Figure 2, the stimulated production of IL-2 and IFN-γ
in activated PBMC was significantly inhibited by 3 in a
concentration-dependent manner. The IC50 of 3 on IL-2 and
IFN-γ production in PBMC activated with PHA were 25
and 5.1 µM, respectively. It is unlikely that 3 suppressed
PBMC proliferation through cytotoxic effects, because the
results of trypan blue staining indicated no significant
difference (data not shown) in cell viabilities of PBMC
treated with or without 3 for 4 days. The inhibitory
mechanisms may involve the impairments of IL-2 and
IFN-γ production. These results indicated that 3 is an
immunomodulatory agent.
(
5
1
3
6.31-7.32 (12H, C, C′, C′′, C′′′ ring protons); C NMR (acetone-
) δ 27.8 (C-4′), 29.0 (C-4′′′), 36.8 (C-4), 37.8 (C-4′′), 65.9 (C-
3′), 66.1 (C-3′′′), 70.4 (C-3), 71.0 (C-3′′), 75.6 (C-2), 77.6 (C-2′′),
9.2 (C-2′′′), 95.4/96.0/97.7 (C-6/-8/6′/6′′/6′′′), 98.1 (C-2), 99.3
d
6
7
+
(C-2′); FAB MS m/z 1153 [M + H] .
Th iolytic Degr a d a tion of 6. Treatment of compound 6
300 mg) with R-toluenethiol (2 mL) and acetic acid (3 mL) in
ethanol (10 mL) in the same manner as described for 5,
(
followed by separation on a Sephadex LH-20 column with 30%
MeOH-H
catechin-(4âf8,2âfOf7)-epicatechin-(4âf8)-epicatechin (5),
′-benzylthioproanthocyanidin A-2 (8), and epicatechin (1).
2
O as eluant, gave 4-benzylthioepicatechin (9), epi-
Exp er im en ta l Section
4
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined with a Yanaco micro melting point apparatus and
are uncorrected. IR spectra were obtained as KBr pellets on a
Nicolet Avatar 320 IR spectrometer. UV spectra were obtained
1
Compound 9: H NMR (CD
H-4), 3.95 (2H, s, -CH -S-), 4.04 (1H, d, J ) 2.0 Hz, H-3),
.22 (1H, s, H-2′), 5.89/5.96 (each 1H, d, J ) 2.0 Hz, H-6/-8);
3
OD) δ 3.84 (1H, d, J ) 2.0 Hz,
2
5
1
3
1
13
C NMR (CD
3
OD) δ 36.8 (C-4), 42.8 (-CH -S-), 70.5 (C-3),
2
on a Hitachi U-3200 spectrophotometer in MeOH. H, C, and
D NMR spectra were measured with a Varian Inova-500
7
-
4.4 (C-2), 94.6/95.7 (C-6/-8), 99.0 (C-10); ESI MS m/z 411 [M
2
-
H] .
spectrometer. EIMS, HREIMS, and APCIMS were recorded
on a Finnigan MAT 95S and a Finnigan LCQ spectrometer,
respectively.
P la n t Ma ter ia ls. The stems of E. utilis were collected at
Shihting, Taipei, Taiwan, in October 1999. A voucher specimen
2
6
Aescu lita n n in C (7): amorphous powder; [R]
D
64.3° (c
1
0
.42, acetone); H NMR (CD
3
OD) δ 2.83 (1H, d, J ) 15.5 Hz,
H-4′), 2.97 (1H, dd, J ) 15.5, 5.0 Hz, H-4′), 3.99 (1H, d, J )
3
H-3′′), 4.54 (1H, d, J ) 3.5 Hz, H-4′), 4.69 (1H, d, J ) 3.5 Hz,
H-4), 4.94 (1H, s, H-2′′), 6.03 (1H, d, J ) 2.0 Hz, H-6), 6.07
(
.5 Hz, H-3), 4.11 (1H, d, J ) 3.5 Hz, H-3′), 4.27 (1H, br s,
(No. 199508) has been deposited in the herbarium of the
Department of Botany of the National Taiwan University.
Extr a ction a n d Isola tion . The milled stems (10.0 kg) were
extracted with acetone (70 L × 3). The concentrated acetone
extract (378 g) was granulated with Celite (2.8 kg). The
granule of extract was then eluted successively with n-hexane
1H, d, J ) 2.0 Hz, H-8), 6.11 (1H, s, H-6′′), 6.18 (1H, s, H-6′),
13
6.79-7.43 (9H, C, C′, C′′ ring protons); C NMR (CD OD) δ
3
29.7/29.8/30.0 (C-4/-4′/-4′′), 67.1 (C-3′′), 68.1 (C-3′), 68.6 (C-3),
81.4 (C-2′′), 96.3/96.4/97.8/98.4 (C-6/-8/-6′/-6′′), 100.0 (C-2),
100.7 (C-2′), 102.4 (C-10′′), 103.7 (C-10), 105.9 (C-10′), 107.7
+
(8 L), CHCl
3
(8 L), EtOAc (8 L), and acetone (8 L). The bioactive
(C-8′′), 108.9 (C-8′); FAB MS m/z 863 [M + H] .
EtOAc extract fraction (38 g) was subjected to column chro-
P r ep a r a tion of P BMC. Heparinized human peripheral
blood (20 mL) was obtained from healthy donors. PBMC was
isolated by the Ficoll-Hypaque gradient density method as
described previously.19 The 20 mL of peripheral blood was
centrifuged at 2000 rpm, 4 °C for 10 min, to remove the
plasma. Blood cells were diluted with PBS buffer, then
centrifuged in a Ficoll-Hypaque discontinuous gradient at 1500
rpm for 30 min. The PBMC layers were collected and washed
with cold distilled water and 10× Hanks' buffer saline solution
(HBSS) to remove red blood cells. The cells were resuspended
matography on Sephadex LH-20 (10 × 100 cm) using a solvent
2
system (MeOH-H O) of increasing MeOH percentage yielding
eight fractions. Fraction 3 (10.3 g) was further chromatograph
on Cosmosil 140C18-OPN (4 × 60 cm) and eluted with 10%
2
MeOH-H O followed by increasing MeOH portion to give
compounds 1 (330 mg), 2 (194 mg), and 6 (644 mg). Fractions
(7.8 g), 6 (3.4 g), and 8 (2.5 g) were purified in the same
4
manner as described above. Compound 5 (5.08 g) was obtained
from fraction 4, compounds 3 (1.06 g) and 4 (28 mg) were
obtained from fraction 6, and compound 7 (16 mg) was
obtained from fraction 8, respectively.
Ep ica tech in -(4âf8, 2âfOf7)-ep ica tech in -(4âf8)-ep i-
ca tech in (5): amorphous powder; [R]
6
to a concentration of 2 × 10 cells/mL in RPMI-1640 medium
supplemented with 2% fetal calf serum (FCS), 100 U/mL
penicillin, and 100 µg/mL streptomycin.
2
6
D
63.5° (c 1.15, acetone);
Lym p h op r olifer a tion Test. The lymphoproliferation test
1
20
H NMR (CD
1H, s, H-3′′), 4.14 (1H, s, H-4), 4.17 (1H, d, J ) 3.0 Hz, H-3′),
.40 (1H, s, H-2′′), 4.57 (1H, s, H-4′), 5.72 (1H, s, H-2′), 5.82
1H, s, H-6′′), 5.98 (1H, d, J ) 2.0 Hz, H-8), 6.04 (1H, d, J )
.0 Hz, H-6), 6.12 (1H, s, H-6′), 6.73-7.33 (9H, C, C′, C′′ ring
3
OD) δ 2.91 (2H, m, H-4′′), 3.31 (1H, s, H-3), 3.87
was modified from that previously described. The density of
6
(
4
(
2
PBMC was adjusted to 2 × 10 cells/mL before use. A 100 µL
sample of cell suspension was divided into a 96-well flat-
bottomed plate (Nunc 167008, Nunclon, Raskilde, Denmark)
with or without 5 µg/mL PHA (Sigma, St. Louis, MO). Various
concentrations of components isolated from E. utilis were
1
3
protons); C NMR (CD
4
9
3
OD) δ 28.7 (C-4), 29.6 (C-4′′), 38.1 (C-
′), 67.0 (C-3), 67.3 (C-3′′), 72.3 (C-3′), 78.6 (C-2′), 80.0 (C-2′′),
6.0/96.5/98.3 (C-6/-8/6′/-6′′), 100.0 (C-2), 100.1 (C-10′), 104.9
2
added to the cells. The plates were incubated in 5% CO -air
3
humidified atmosphere at 37 °C for 3 days. Subsequently, H-
thymidine (1 µCi/well; NEN) was added into each well. After
16 h incubation, the cells were harvested on glass fiber filters
by an automatic harvester (Dynatech, Multimash 2000, Bill-
ingshurst, U.K.). Radioactivity in the filters was measured by
a scintillation counting. The inhibitory activity of each com-
ponent on PBMC proliferation was calculated by the following
formula:
(
C-10), 106.2 (C-10′′), 106.6 (C-8′′), 108.7 (C-8′); FAB MS m/z
+
8
65 [M + H] .
Th iolytic Degr a d a tion of 5. A solution of 5 (500 mg) in
EtOH (10 mL) containing R-toluenethiol (2 mL) and acetic acid
3 mL) was refluxed 20 h under N atmosphere. The reaction
1
5
(
2
mixture was concentrated to give an oily residue, which was
chromatographed over Sephadex LH-20. Elution with 30%
MeOH-H
2
O furnished epicatechin (1) and 4′-benzylthiopro-
anthocyanidin A-2 (8) as an off-white amorphous powder: 1
1
5
inhibitory activity (%) )
H
NMR (CD OD) δ 3.91 (1H, s, H-3′), 3.97 (2H, s, -CH -S-),
3
2
control group (CPM) - experiment group (CPM)
×
100
4
.06 (2H, m, H-3, 4′), 4.37 (1H, d, J ) 3.5 Hz, H-4), 5.28 (1H,
control group (CPM)
1
3
s, H-2′), 6.01/6.06/6.11 (each 1H, s, H-6/-8/-6′); C NMR
CD OD) δ 29.3 (C-4), 38.1 (-CH -S-), 44.4 (C-4′), 68.1 (C-
), 71.3 (C-3′), 77.5 (C-2′), 96.6 (C-8), 97.0 (C-6′), 98.3 (C-6),
(
3
2
Deter m in a tion of IL-2 a n d IF N-γ P r od u ction . PBMC
(2 × 10 cells/well) were cultured with PHA alone or in
5
3

5
08 J ournal of Natural Products, 2002, Vol. 65, No. 4
Lin et al.
combination with varying concentrations of compound 3 for 3
days. The cell supernatants were then collected and assayed
for IL-2 and IFN-γ concentrations by the enzyme immuno-
assays (EIA; R&D systems, Minneapolis, MN). The EIA assays
(7) Dalton, D. K.; Pitts-MeeK, S. Science 1993, 259, 1739-1742.
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(
9) Beutler, B.; Ceromi, A. Annu. Rev. Immunol. 1989, 7, 625-655.
(
10) Adams, D. O.; Hamilton, T. A. Annu. Rev. Immunol. 1984, 2, 283-
318.
used here are not reported to exhibit detectable cross-reactivity
(11) Porter, L. J .; Newman, R. H.; Foo, L. Y.; Wong, H. J . Chem. Soc.,
Perkin Trans. 1 1982, 1217-1221.
12) Nonaka, G. I.; Nishioka, I.; Nagasawa, T.; Oura, H. Chem. Pharm.
with the other cytokines we tested.21
(
(
(
(
(
(
(
(
Bull. 1981, 29, 2862-2870.
Ack n ow led gm en t. We are grateful to the National Sci-
ence Council, the Republic of China, for support of this
research under Grant NSC-89-2113-M-077-006.
13) Lou, H.; Yamazaki, Y.; Sasaki, T.; Uchida, M.; Tanaka, H.; Oka, S.
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4
338-4345.
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Chen, C. M. Chem. Pharm. Bull. 1991, 39, 55-59.
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Trans. 1 1982, 1209-1216.
(
1) Li, H. L.; Huang, T. C. In Flora of Taiwan, 2nd ed.; Editorial
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2
04.
4
717-4929.
(
(
(
2) Chiu, N. Y.; Chang, K. H. The Illustrated Medicinal Plants of Taiwan;
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3) Chen, C. Y.; Wang, J . P.; Ho, L. K.; Chang, Y. S. J . Chin. Med. 2001,
18) Nonaka, G. I.; Morimoto, S.; Nishioka, I. J . Chem. Soc., Perkin Trans.
1
1983, 2139-2145.
19) Yang, N. S.; Chou, C. J .; Lin, L. C.; Tsai, W. J .; Kuo, Y. C. J . Chin.
Med. 1999, 10, 179-188.
1
2, 109-117.
4) Wu, C. H. Studies on the constituents from the stems of Ecdysanthera
utilis. Master Thesis, Institute of Applied Chemistry, Providence
University, Taichung, Taiwan, R.O.C., 1991.
(20) Kuo, Y. C., Ou, J . C.; Tsai, W. J .; Wu, C. L.; Sun, C. M. J . Chin. Med.
1995, 3, 211-221.
(
5) J aneway, C. A.; Travers, P.; Hunt, S.; Walport, M. In Immunobiol-
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(21) Kuo, Y. C.; Yang, N. S.; Chou, C. J .; Lin, L. C.; Tsai, W. J . Mol.
Pharmacol. 2000, 58, 1057-1066.
(
6) Arai, K.; Lee, F.; Miyajima, A. Annu. Rev. Biochem. 1990, 59, 783-
8
36.
NP010414L