Isoeleutherin and eleutherinol, naturally occurring selective modulators

of Th cell-mediated immune responses

Jeong-Ho Hong
a,1
, Eun Sun Yu
b,1
, Ah-Reum Han
b
, Joo-Won Nam
b
, Eun-Kyoung Seo
b
, Eun Sook Hwang
b,
*
a
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea
b
College of Pharmacy and Division of Life and Pharmaceutical Sciences, Center for Cell Signaling & Drug Discovery Research, Ewha Womans University, 11-1 Daehyun-Dong,
Sudaemun-Ku, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
Article history:
Received 11 April 2008
Available online 22 April 2008
Keywords:
Naphthopyran
Naphthoquinone
Eleutherin
Isoeleutherin
Eleutherinol
IL-2
IFNc
T helper cell
a b s t r a c t
Natural compounds possessing naphthopyran moiety have been attracted by their anti-bacterial, anti-
fungal, and anti-viral activities, as well as anti-tumor activities. Although chemical structures were crit-
ical for the potential biological activities, the detailed functional mechanisms remained unclear. Here, we
have studied the effects of naphthopyran derivatives (eleutherin, isoeleutherin, and eleutherinol) on T
helper cell-mediated immune responses to understand the mechanisms of their anti-microbial and
anti-tumor activities. The study revealed that isoeleutherin, which has 1,4-naphthoquinone ring with
a-methyl group, selectively and specically stimulated IFNc production through the activation of T-bet
gene transcription, thus enhancing Th1-mediated immune responses. However, a natural naphthopy-
ran-4-one, eleutherinol dramatically inhibited both IFNc and IL-2 productions during Th cell activation
by suppressing the gene transcriptions of cytokines. Therefore, we suggest that the chemical modication
and chirality of naphthopyran moiety in isoeleutherin and eleutherinol may be critical for the selective
modulation of T helper cell-mediated immune responses.
2008 Elsevier Inc. All rights reserved.
Pyranonaphthoquinones are a diverse compoundfamily of naph-
thopyran derivatives, which are naturally occurring naphtho[2,3-
c]pyran-5,10-diones widespread in bacteria, fungi, aphides, and
higher plants [16]. They have been known to have important bio-
logical activities suchas anti-fungal, anti-viral, andanti-tumor activ-
ity as well as antibiotics [3,5,79]. A variety of naphthopyran
derivatives havebeenisolatedandidentiedas natural phytochemi-
cals [1,35]. Eleutherinandisoeleutherinare the simplest andmajor
naphthopyranpossessing1,4-naphthoquinonemoietyisolatedfrom
Eleutherine bulbosa [6] and Eleutherine americana (Iridaceae) [10].
Numerous numbers of pyranonaphthoquinones including eleuther-
in, isoeleutherin, elecanicin, eleutherol, and eleutherinone possess-
ing 1,4-naphthoquinone moiety and eleutherinol, a natural
naphthopyrone are known as natural products in Eleutherine genus
in Iridaceae family [3,5,6,11]. Plants classied to Eleutherine have
beenused as a folk medicine for treating heart diseases suchas angi-
na pectoris and intestinal infections [12,13] and reported to show
inhibitory activity against HIV infection [14]. In addition, Krishnan
and Bastow [15] described anti-tumor activity of eleutherin by the
inhibition of topoisomerase II with stereospecic and selective
inhibitory activity. The studies on chemistry of constituents in Ele-
utherine plants are well established and support therapeutic poten-
tials as anti-infectious and anti-tumor drugs, however, no relation
could be established between the chemical constituent and poten-
tial mechanisms for biological activities.
Activation of CD4+ T helper (Th) cells and differentiation into
effector Th1 cells are critically required for anti-bacterial, anti-
viral, and anti-fungal activities as well as inammation [16,17].
TCR engagement of CD4+ Th cells polarizes into two major sub-
sets of effector Th cells, such as Th1 and Th2 cells [1820]. Two
subsets, Th1 and Th2 cells, are distinguished from the master
cytokines they produce. While Th2 cells produce signature cyto-
kines such as IL-4, IL-5, and IL-13 and are involved in antibody-
mediated humoral immune responses, Th1 cells activate IFNc
expression and thus are responsible for the subsequent activa-
tion of macrophages and cytotoxic CD8+ T cells to kill microbes
[21]. Cell lineage commitment into either Th1 or Th2 cells is reg-
ulated by environmental cytokine milieu and also specic tran-
scription factors. GATA-3 is a well-known Th2-specic
transcription factor, which exclusively expressed in Th2 cells
and strongly activates gene transcription of Th2 cytokines [22].
On the other hand, T-bet is a master regulator of Th1 cells.
The facts that the ectopic expression or knockout of T-bet gene
dramatically increased or impaired IFNc productions [23,24] con-
vinced T-bet is critically required for both IFNc production and
Th1 cell differentiation.
0006-291X/$ - see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.04.060
* Corresponding author. Fax: +82 2 3277 3760.
E-mail address: eshwang@ewha.ac.kr (E.S. Hwang).
1
These authors equally contributed to this work.
Biochemical and Biophysical Research Communications 371 (2008) 278282
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
j our nal homepage: www. el sevi er . com/ l ocat e/ ybbr c
Here, we studied the effects of two representative pyranonaph-
thoquinones, eleutherin and isoeleutherin, and a naphthopyrone,
eleutherinol, on the activation and differentiation of Th cells. We
observed that isoeleutherin, a major constituent of E. americana
signicantly stimulated IFNc production and induced Th1 cell
differentiation, which may contribute to anti-microbial and
anti-tumor activities. Moreover, we found that a natural naphtho-
pyran-4-one, eleutherinol inhibited Th cell activation, suggesting
that the chemical modication and chirality of naphthopyran
moiety may be critical for the selective modulation of T helper
cell-mediated immune responses.
Materials and methods
Isolation and purication of eleutherin, isoeleutherin, and eleutherinol. The bulbs of
E. americana Merr. Et Heyne (Iridaceae) were collected at Batu Herba Medica Cen-
tre, East Java, Indonesia, in May 2005. Eleutherin, isoeleutherin, and eleutherinol
were puried from the methanol extracts by column chromatography and subse-
quent semi-preparative HPLC separation with the 98% purity. The structures were
conrmed by NMR and presented in Fig. 1A.
Reagents. Recombinant human IL-2, anti-CD3, and anti-CD28 antibodies and PE-
conjugated annexin V and IFNc antibodies were purchased from BD Pharmingen
(San Diego, CA). All cytokines and antibodies for ELISA were from BD Pharmingen.
Monensine was obtained from Sigma-Aldrich Inc. (St. Louis, MO).
Mice. Wild type C57BL6 and T-bet knockout/BL6 mice were housed in specic
pathogen-free conditions at Ewha Womans University. All mice handling and
experiments were done in accordance with Institutional Animal Care and Use Com-
mittee guidelines.
Isolation and activation of CD4+ Th cells in vitro. Single cells were isolated form
lymph node and spleen of wild type mice and incubated with mouse CD4 micro
beads for 30 min according to the manufacturers instruction (Miltenyi Biotech., Au-
burn, CA). Single cell suspensions of CD4+ Th cells were isolated and stimulated
with plate-bound anti-CD3 (2 lg/ml) and anti-CD28 (2 lg/ml). Recombinant human
IL-2 (100 U/ml) was added up to enhance T cell activation and proliferation.
Intracellular cytokine staining and annexin V staining. Cells were xed with 4%
paraformaldehyde solution, rinsed with permeabilization buffer (0.1% saponin,
0.1% sodium azide, 1% FBS in PBS), and incubated with either PE-conjugated anti-
IFNc Ab or PE-conjugated annexin V. Cells were washed twice with FACS buffer
A
10 M 30 M 10 M 30 M
vehicle
eleutherin
isoeleutherin
eleutherinol
B
O
H
3
CO O
O
R
24 h 48 h
eleutherin R= -CH
3
isoeleutherin R=-CH
3
O
O
HO
OH
eleutherinol
10
0
10
1
10
2
10
3
10
4
FL2-H
10
0
10
1
10
2
10
3
10
4
FL2-H
10
0
10
1
10
2
10
3
10
4
FL2-H
eleutherin isoeleutherin eleutherinol
c
o
u
n
t
s
c
o
u
n
t
s
c
o
u
n
t
s
0 M
10 M
30 M
0 M
10 M
30 M
0 M
10 M
30 M
C
Fig. 1. Effects of naphthopyran derivatives on Th cell activation upon TCR stimulation. (A) Structures of naphthopyran derivatives, eleutherin, isoeleutherin, and eleutherinol
isolated from E. americana. (B) Cell morphologies of Th cells upon TCR stimulation in the presence of pyranonaphthoquinones. CD4+ Th cells isolated from lymph node and
spleen were stimulated with anti-CD3 (2 lg/ml) and anti-CD28 (2 lg/ml) for 24 or 48 h with different concentrations of the compounds as indicated. (C) Apoptosis assay by
annexin V staining. Different doses of compounds were treated in Th cells triggered by TCR for 48 h. Activated Th cells were incubated with PE-annexin V and analyzed by
ow cytometric analysis.
h 4 2
h 8 4
A
n i r e h t u e l e o s i n i r e h t u e l e
I
L
-
2
(
n
g
/
m
l
)
0
2
4
6
8
10
12
14
16
18
e l c i h e v
B
0
2
4
6
8
10
12
14
16
I
F
N

(
n
g
/
m
l
)
n i r e h t u e l e o s i n i r e h t u e l e e l c i h e v
h 4 2
h 8 4
0 1
0
0 1
1
0 1
2
0 1
3
0 1
4
% 6 . 3
0 1
0
0
F
L
2
-
H
F
L
2
-
H
F
L
2
-
H
1
1
0 1
2
0 1
3
0 1
4
0
1
0
0
1
1
0
1
2
0
1
3
0
1
4
0
1
0
0
1
1
0
1
2
0
1
3
0
1
4
0
1
0
0
1
1
0
1
2
0
1
3
0
1
4
% 2 . 3
0 1
0
0 1
1
0 1
2
0 1
3
0 1
4
% 8 . 0 1
n i r e h t u e l e o s i n i r e h t u e l e
I
F
N

e l c i h e v
C
Fig. 2. Stimulation of IFNc production from Th cells by isoeleutherin. Isolated CD4+
Th cells were incubated with 10 lM concentration of either eleutherin or iso-
eleutherin concomitantly with TCR stimulation. Supernatants were collected from
24 to 48 h stimulated Th cells for measuring cytokines, IL-2 (A) and IFNc (B). (C)
Monensine was added to the activated Th cells 2 h prior to harvest and collected for
intracellular cytokine staining. Activated Th cells were incubated with PE-anti-IFNc
Ab followed by ow cytometric analysis.
J.-H. Hong et al. / Biochemical and Biophysical Research Communications 371 (2008) 278282 279
(1% FBS in PBS) and analyzed by FACS Calibur (BD Biosciences). Cell populations
were measured by CellQuest software (Tree Star, Ashland, OR). For intracellular
cytokine staining, monensine (2 lM) was treated to the activated Th cells for 2 h
prior to harvest.
ELISA. Cytokines were measured by ELISAas instructedinBDPharmingen. Briey,
culture supernatants were collected fromTh cells activated for 24 and 48 h and incu-
bated oncapture Ab-coated ELISAplate. Biotinylated anti-cytokine Abs and phospha-
tase-conjugated streptavidin were sequentially incubated after plate washing and
developed with phosphatase substrate. Color changes were read by ELISA plate read-
er (Molecular Devices, Palo Alto, CA). Puried and known concentrations of IL-2 and
IFNc were incubated parallel with unknown samples for standard curves.
Isolation of total RNA and real-time PCR. Total RNA was isolated fromthe activated
Th cells treated by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and treated
with DNase to remove any remnant genomic DNA. cDNA was reverse transcribed
from2 lg of total RNA by Superscript rst strand synthesis system(Invitrogen, Carls-
bad, CA) and used for quantitative real-time PCR using SYBR Green PCR master with
an ABI PRISM7000 Sequence Detection System(Applied Biosystems, Foster City, CA).
The following primers were used: b-actin-FWD 5
0
-aagcaggagtatgacgagtccg-3
0
, b-ac-
tin-REV 5
0
-cggaactaagtcatagtccgcc-3
0
; IFNc-FWD 5
0
-agcaacagcaaggcgaaaa-3
0
, IFNc-
REV 5
0
-ctggacctgtgggttgttga-3
0
; IL-2-FWD 5
0
-ctcctgagcaggatggagaatt-3
0
, IL-2-REV
5
0
-cgcagaggtccaagtgtagct-3
0
; T-bet-FWD 5
0
-cccccaaggaattgacagttg-3
0
, T-bet-REV 5
0
-
gggaaactaaagctcacaaac-3
0
; IL-4-FWD 5
0
-ggcattttgaacgaggtcaca-3
0
, IL-4-REV 5
0
-
aggacgtttggcacatccat-3
0
.
Results and discussions
Pyranonaphthoquinones affect CD4+ Th cell activation and
proliferation in vitro
A variety numbers of naphthopyran derivatives have been iden-
tied and grouped based on their structural moiety and are known
to have anti-fungal and anti-tumor functions [1,5,79]. Structures
of eleutherin, isoeleutherin, and eleutherinol are shown in Fig.
1A. While eleutherin and isoeleutherin are structurally epimeric
isomers derived from 1,4-naphthoquinone moiety, eleutherinol
(8,10-dihydroxy-2,5-dimethyl-4H-naphtho[1,2-b]-pyran-4-one)
contains naphthopyran-4-one moiety, which is different from 1,4-
naphthoquinone (Fig. 1A). We have treated these compounds with
different doses in Th cells, and assessed the activation and prolifer-
ation of Th cells upon TCR stimulation. Both eleutherin and
isoeleutherin partially inhibited cell proliferation at 30 lM concen-
tration, although there was no effect at lower concentration (Fig.
1B). However, Th cell proliferation upon TCR activation was not
at all affected by eleutherinol. Decreased cell populations by treat-
ment of naphthopyran derivatives prompted us to inspect the ef-
fects on cell apoptosis. Annexin V staining and ow cytometric
analyses determined that both eleutherin and isoeleutherin in-
creased apoptosis at 30 lM, but eleutherinol rarely induced apop-
totic cell death (Fig. 1C). These results indicated that 1,4-
naphthoquinone moiety in eleutherin and isoeleutherin may have
selective functions on cell apoptosis, remaining further studies on
the functional mechanisms. Since the high concentration of pyr-
anonaphthoquinones induced cell apoptosis, we chose lowconcen-
tration of naphthopyran derivatives for further studies.
Eleutherin and isoeleutherin differentially modulate IFNc production
in CD4+ Th cells upon TCR stimulation
In order to examine the effects of eleutherin and isoeleutherin
on Th cell activation and differentiation, we assessed cytokines
A
N F I
B t e b - T
r
e
l
a
t
i
v
e

t
r
a
n
s
c
r
i
p
t
s
(
x
1
0
-
2
)
0
5 . 0
1
5 . 1
2
5 . 2
0 3 0 1 0 3 0 1 0
n i r e h t u e l e o s i n i r e h t u e l e
(M)
0
1
2
3
4
0 3 0 1 0 3 0 1 0
n i r e h t u e l e o s i n i r e h t u e l e
(M)
r
e
l
a
t
i
v
e

t
r
a
n
s
c
r
i
p
t
s
(
x
1
0
-
2
)
C
D
e l c i h e v n i r e h t u e l e o s i
0 1
0
0 1
1
0 1
2
0 1
3
0 1
4
% 1 . 4 1
0 1
0
0 1
1
0 1
2
0 1
3
0 1
4
FL4-H FL4-H
% 5 . 4
n i r e h t u e l e
0 1
0
0 1
1
0 1
2
0 1
3
1 0 1
4
0
1
0
0
1
1
0
1
2
0
1
3
1
0
1
4
FL4-H
F
L
2
-
H
0
1
0
0
1
1
0
1
2
0
1
3
1
0
1
4
F
L
2
-
H
0
1
0
0
1
1
0
1
2
0
1
3
1
0
1
4
F
L
2
-
H
% 0 . 4
0
3
6
9
2 1
5 1
8 1
I
F
N

(
n
g
/
m
l
)
0
1
2
+ - n i r e h t u e l e o s i
t e b - T
+ / +
+ -
t e b - T
- / -
t e b - T
- / -
s n
I
F
N

Fig. 3. T-bet-dependent IFNc modulation mediated by isoeleutherin. Th cells were stimulated with anti-CD3 and anti-CD28 for 48 h with eleutherin or isoeleutherin as
indicated. Total RNA was isolated from the activated Th cells and used for determination of IFNc (A) and T-bet (B). (C) CD4+ Th cells were isolated from wild type C57BL6 and
T-bet knockout/B6 mice and stimulated with anti-CD3 and anti-CD28 concomitant with (+) or without () isoeleutherin (10 lM) for 48 h. Supernatants were collected for
measuring IFNc by ELISA. IFNc produced in T-bet-decient CD4+ Th cells is shown in the box with enlarged scale. Statistical signicance was indicated as ns (not signicant).
(D) TCR-triggered Th cells were incubated with 10 lM of compounds for 4 days and subsequently restimulated with PMA and ionomycin for 4 h. Cells were harvested and
stained for analyzing IFNc-producing cell populations.
280 J.-H. Hong et al. / Biochemical and Biophysical Research Communications 371 (2008) 278282
productions in Th cells upon treatment. Since TCR triggering signif-
icantly stimulated cytokine productions in CD4+ Th cells, both IL-2
and IFNc were comparatively increased in cell supernatants. Treat-
ment of 10 lM of either eleutherin or isoeleutherin in Th cells
seemed to have no effect on IL-2 production (Fig. 2A). Under the
same conditions, IFNc was signicantly increased by isoeleutherin
at 24 and 48 h post-TCR stimulation, but not induced by eleutherin
(Fig. 2B). Moreover, the intracellular cytokine staining showed con-
sistently increased IFNc-producing cell populations by isoeleuther-
in treatment, but not affected by eleutherin (Fig. 2C), insisting that
isoeleutherin may be a specic stimulator of IFNc production. Iso-
eleutherin has only one different chiral center from the structure of
eleutherin. Therefore, the activity of isoeleutherin seems to be dee-
ply related with the chirality at its pyran ring with the a-methyl
functionality.
Isoeleutherin activated IFNc gene transcription by the induction of
T-bet, a master Th1-specic transcription factor
Cytokines are critically regulated at the level of gene transcrip-
tion by specic transcription factors. IFNc production in Th cells is
known to be critically regulated by T-bet, a Th1-specic transcrip-
tion factor, which directly binds to IFNc gene promoter and enhan-
cer and plays a key role in Th1 cell differentiation. Quantitative
real-time PCR of IFNc and T-bet conrmed that isoeleutherin in-
creased both IFNc and T-bet gene transcription in dose-dependent
manner (Fig. 3A and B), suggesting that isoeleutherin modulates
IFNc expression in Th cells in T-bet-dependent manner. In order
to conrm the T-bet-dependent IFNc modulation by isoeleutherin,
we used T-bet-decient CD4+ Th cells, which decreased IFNc pro-
duction due to T-bet-deciency. Isoeleutherin increased IFNc pro-
duction in wild type Th cells, but failed to stimulate IFNc
expression in the absence of T-bet (Fig. 3C), insisting on the mech-
anism that isoeleutherin modulate IFNc production in T-bet-
dependent manner. Moreover, Th cell differentiation was more
prominently committed into Th1 cell lineages in the presence of
isoeleutherin (Fig. 3D), supporting the efcient modulator of
Th1-mediated immune responses of isoeleutherin.
Eleutherinol inhibits IL-2 and IFNc productions by suppressing gene
transcriptions
Eleutherinol possesses a naphthopyran-4-one moiety, which is
distinguished from 1,4-naphthoquinone moiety found in eleuther-
in and isoeleutherin as shown in Fig. 1A. As high concentration of
eleutherinol did not induce cell apoptosis, we measured the cyto-
kine productions during Th cell activation. Interestingly, IL-2 and
IFNc productions were signicantly impaired as the concentrations
of eleutherinol increased (Fig. 4A and B). Decreased cytokine pro-
ductions resulted from the reduced gene transcription levels of
IL-2 and IFNc by eleutherinol (Fig. 4C and D), whereas IL-4 gene
transcription was not reduced by eleutherinol, suggesting the
selective inhibition of IL-2 and IFNc (Fig. 4F). Moreover, semi-
quantitative RT-PCR convinced the impaired mRNA levels of cyto-
kines, which was mediated by eleutherinol (Fig. 4E).
Current studies of the effects of naphthopyran derivatives on Th
cell activation claried the critical function of isoeleutherin as a
stimulant of Th1 immune response and also uncovered novel
inhibitory functions of eleutherinol on cytokine productions during
Th cell activation. These ndings partly explain the mechanism
how E. americana were used as a folk medicine for treating micro-
bial infections and tumor. We strongly suggest that modulation of
0 3 0 1 0
(M) l o n i r e h t u e l e
0
2
4
6
8
0 1
I
F
N

(
n
g
/
m
l
)
A
0
2
4
6
8
0 1
2 1
4 1
6 1
8 1
I
L
-
2
(
n
g
/
m
l
)
0 3 0 1 0
l (M) o n i r e h t u e l e
h 4 2
h 8 4
B
0 3 0 1 0
(M) l o n i r e h t u e l e
0
4 0 . 0
8 0 . 0
2 1 . 0
N F I
0 3 0 1 0
(M) l o n i r e h t u e l e
r
e
l
a
t
i
v
e

t
r
a
n
s
c
r
i
p
t
s
t
o

-
a
c
t
i
n

(
x
1
0
-
2
)
1
2
3
4
0
1
2
4
3
5
0
2 - L I
r
e
l
a
t
i
v
e

t
r
a
n
s
c
r
i
p
t
s
t
o

-
a
c
t
i
n
D C
0 3 0 1 0
l (M) o n i r e h t u e l e
r
e
l
a
t
i
v
e
t
r
a
n
s
c
r
i
p
t
s
t
o

-
a
c
t
i
n
(
x
1
0
-
3
)
4 - L I
F E
2 - L I
N F I
n i t c a -
0 3 0 1 0
l (M) o n i r e h t u e l e
Fig. 4. Effects of eleutherinol on cytokine productions during Th cell activation. Th cells were co-incubated with eleutherinol with the indicated amount and time period and
supernatants were used for measuring cytokines, IFNc (A) and IL-2 (B). Activated Th cells were harvested for the isolation of total RNA and used for determination of IFNc (C),
IL-2 (D), and IL-4 (F). (D) Semi-quantitative RT-PCR was performed to detect IFNc and IL-2 in Th cells treated with eleutherinol and PCR products were resolved by agarose gel
electrophoresis.
J.-H. Hong et al. / Biochemical and Biophysical Research Communications 371 (2008) 278282 281
immune responses by pyranonaphthoquinone derivatives may
contribute to anti-tumor and anti-microbial activities.
Acknowledgments
This work was supported by Ewha Womans University Re-
search Grant of 2005, National R&D program for Cancer Control
of National Cancer Center (0620380) and in part by NCRC program
of MOST and KOSEF (R15-2006-020). T-bet-decient mice were
kindly provided by Dr. Laurie H. Glimcher at Harvard University.
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