Bioactive compounds from Rumex plants

Article abstract of DOI:10.1016/j.phytol.2010.05.005

Two new naphthalene acylglucosides, rumexneposides A (1) and B (2), together with 12 known compounds (3-14), were isolated from the roots of Rumex nepalensis. Their structures were established by chemical and spectroscopic methods. The biological activities of compounds 1-14 as well as an additional 11 compounds previously isolated from R. nepalensis and Rumex hastatus (15-25) were evaluated against Mycobacterium tuberculosis, para-aminobenzoic acid (pAba) pathway, and a panel of human cancer cell lines. The results showed that compound 15 was the most active against M. tuberculosis with an MIC value of 2.85 μM similar to that of isoniazid. Compound 5 could inhibit pAba synthetic pathway with an MIC value of 12.6 μM, comparable to that of positive control abyssomicin C, representing a new example of the rare pAba pathway inhibitors.

Full text of DOI:10.1016/j.phytol.2010.05.005

Phytochemistry Letters 3 (2010) 181–184
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Bioactive compounds from Rumex plants
a,c,1
b,1
d
c
a
Heng-Xing Liang
, Huan-Qin Dai , Hai-An Fu , Xiao-Ping Dong , Abiodun Humphrey Adebayo ,
b
a,
Li-Xin Zhang , Yong-Xian Cheng *
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China
b
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China
c
College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, People’s Republic of China
d
Department of Pharmacology, Emory University School of Medicine and Emory Chemical Biology Discovery Center, Atlanta, GA 30322, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new naphthalene acylglucosides, rumexneposides A (1) and B (2), together with 12 known
compounds (3-14), were isolated from the roots of Rumex nepalensis. Their structures were established
by chemical and spectroscopic methods. The biological activities of compounds 1-14 as well as an
additional 11 compounds previously isolated from R. nepalensis and Rumex hastatus (15–25) were
evaluated against Mycobacterium tuberculosis, para-aminobenzoic acid (pAba) pathway, and a panel of
human cancer cell lines. The results showed that compound 15 was the most active against M.
Received 23 February 2010
Received in revised form 10 May 2010
Accepted 11 May 2010
Available online 28 May 2010
Keywords:
tuberculosis with an MIC value of 2.85
mM similar to that of isoniazid. Compound 5 could inhibit pAba
Rumex nepalensis
Rumex hastatus
synthetic pathway with an MIC value of 12.6
mM, comparable to that of positive control abyssomicin C,
representing a new example of the rare pAba pathway inhibitors.
Polygonaceae
ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Rumexneposides A and B
Anti-tuberculosis
pAba pathway inhibitor
Cytotoxicity
1
. Introduction
Rumex species are widely distributed in China. Among these,
including the inhibition of Mycobacterium tuberculosis and para-
aminobenzoic acid (pAba) pathway, and against a panel of human
cancer cell lines. In this paper, we describe the clarification of the
structures from R. nepalensis and biological activities of com-
pounds 1–25.
the roots of Rumex nepalensis Spreng. are traditionally used for the
treatment of pain, inflammation, bleeding, tinea, tumor, and
constipation in Chinese folk medicine (Li, 1998; Zhang et al., 2008),
and the roots of Rumex hastatus are of medicinal significance for
cough, headache, and fever (Editorial Commission of China, 1998).
Previous studies revealed the presence of sterols in R. nepalensis
and anthraquinones in R. hastatus (Khetwal et al., 1987; Tiwari and
Sinha, 1980). During our search for bioactive metabolites from
Rumex plants, we have reported several anthraquinones, naphtha-
lenes, flavonoids and other phenolic compounds from R. nepalensis
and R. hastatus (Mei et al., 2009; Zhang et al., 2009). Further study
on the roots of R. nepalensis has resulted in the isolation of two new
naphthalene acylglucosides, designated rumexneposides A (1) and
B (2), together with 12 known compounds (3–14) (Fig. 1).
Compounds 1–14 together with previously isolated compounds
from R. nepalensis and R. hastus (15–25) (Mei et al., 2009; Zhang
et al., 2009) were screened for a broad spectrum of activities,
2. Results and discussion
The EtOH extracts of R. nepalensis roots were successively
2
partitioned with petroleum ether, EtOAc, and H O. From the
EtOAC-soluble fraction, two new (1 and 2) and 12 known
compounds were isolated. The known compounds were identified
as physcion (3) (Zhang et al., 2006), chrysophanol-8-O-
b-D-
glucopyranoside (4) (Yuan et al., 2000), torachrysone (5) (Demir-
ezer et al., 2001), emodin-8-O-
b
-
D
-glucopyranoside (6) (Zhang
0
et al., 2005), emodin-8-O-
b
-
D-(6 -O-acetyl)glucopyranoside (7) (Qi
et al., 2005), chrysophanol (8) (Yan et al., 2001), emodin (9) (Zhang
et al., 2005), citreorosein (10) (Xiang et al., 2001), resveratrol (11)
(
(
(
Sethi et al., 1980), nepodin-8-O-
Demirezer et al., 2001), torachrysone-8-O-
13) (Lemli et al., 1981), and chrysophanol-8-O-
b-D-glucopyranoside (12)
b
-
D
-glucopyranoside
0
b
-D
-(6 -O-acet-
yl)glucopyranoside (14) (Shi et al., 2001) based on comparisons
with data in the literature.
*
Corresponding author. Tel.: +86 871 5223048; fax: +86 871 5223048.
Rumexneposide A (1) was obtained as a yellow powder and its
E-mail addresses: lzhang03@gmail.com (L.-X. Zhang), yxcheng@mail.kib.ac.cn
(
Y.-X. Cheng).
molecular formula was deduced to be C23
negative high-resolution fast atom bombardment mass spectros-
26 9
H O on the basis of its
1
These authors contributed equally to this work.
1
874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2010.05.005

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(
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i
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_
1
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182
H.-X. Liang et al. / Phytochemistry Letters 3 (2010) 181–184
spectrum showed absorption bands attributable to hydroxyls
ꢀ
1
ꢀ1
1
3
(
3422 cm ) and carbonyls (1734 and 1715 cm ). The H and C
NMR data (Table 1) were very similar to those of 5, except that one
13
additional acetyl signal (
d
C
171.5, 20.5) was observed in the
C
NMR spectrum of 2. This was confirmed by the observed HMBC
0
0
00
0
00
interaction of H-2 with C-1 . The further HMBC of H-6 with C-1
0
indicates that the acetyl group is attached to C-6 . In the same
manner as used for 1, acid hydrolysis of 2 with 2% HCl–dioxane
(
1:1, v/v) produced D-glucose as the sugar residue, determined by
Fig. 1. Chemical structures of compounds 1–25.
GC analysis. Therefore, the structure of 2 was assigned as
torachrysone-8-O-
0
b
-D
-(6 -O-acetyl)glucopyranoside.
ꢀ
copy (HRFABMS) at m/z 445.1506 ([MꢀH] , calcd for 445.1498).
Notably, compounds 1, 2, 7, and 14 all have an acyl group on the
sugar moiety, which may be characteristic of the compounds from
R. nepalensis. Compounds 7, 10, 11, 13, and 14 were also isolated
from this plant for the first time.
Considering the traditional medical uses of Rumex plants,
compounds 1–25 were examined for their inhibitory effects on
infection- and tumor-associated targets, such as M. tuberculosis,
pAba biosynthetic pathway, and a panel of human cancer cell lines.
In an anti-tuberculosis assay, the results showed that com-
pound 1, 5, and 12–16 exhibited potent inhibitory activity towards
M. tuberculosis, with minimum inhibitory concentration (MIC)
The infrared (IR) spectrum showed absorption bands representa-
ꢀ1
tive of hydroxyl (3414 cm ) and carbonyl groups (1721 and
ꢀ
1
1
1
1
698 cm ). The H nuclear magnetic resonance (NMR) data (Table
) exhibited three methyls ( 2.48, s; 2.25, s; 1.94, d, J = 6.6 Hz),
6.97, s; 7.21, brd, J = 8.1 Hz; 7.03, dd,
J = 8.1, 7.6 Hz; 6.70, brd, J = 7.6 Hz), and a sugar moiety ( 2.71–
.46; 4.19, d, J = 7.2 Hz, H-1 ). The C NMR data (Table 1) were very
similar to those of nepodin-8-O- -glucopyrancoside (Demirezer
et al., 2001). However, two additional olefinic carbons ( 122.4,
45.4), one carbonyl ( 166.2), and one methyl group ( 18.1)
d
H
four aromatic protons (d
H
d
H
0
13
4
b
-D
d
C
1
d
C
3
d
C
1
were observed in the C NMR spectrum of 1, which indicate the
presence of a crotonoyl group in the molecule. The heteronuclear
values of 20.7, 6.1, 26.6, 8.9, 4.1, 2.85, and 10.2 mM, respectively,
when isoniazid was used as the positive control (MIC: 2.04
whereas the other compounds showed no activity (MIC > 40
(data not shown).
m
m
M),
M)
0
0
00
00
multiple bond correlations (HMBCs) of H-2 and H-3 with C-1 ,
0
0
00
00
and H-4 with C-2 and C-3 confirmed the presence of such a
0
0
00
moiety. The trans configurations of H-2 and H-3 were concluded
from the J value (15.5 Hz) of H-2 . The HMBC of H-6 with C-1
The pAba pathway exists in plants, algae, bacteria, fungi, and
parasites, but not in mammals. Therefore, this pathway is
considered to be an attractive target for the development of
new chemotherapeutic agents (McConkey, 1999; Roberts et al.,
1998). However, until now, only abyssomicin C, a marine natural
product, has been reported to inhibit pAba biosynthesis (Riedlinger
et al., 2004). Because the roots of R. nepalensis are renowned for the
treatment of tinea, the metabolites isolated from this material
together with previously isolated compounds (Mei et al., 2009)
were screened for their effects on pAba biosynthesis. Our results
show that 5 has significant inhibitory activity on the pAba
0
0
0
00
0
indicated attachment of the trans-crotonoyl group to C-6 . Acid
hydrolysis of 1 with 2% HCl–dioxane (1:1, v/v) produced D-glucose
as the sugar residue, determined by gas chromatography (GC)
analysis. Therefore, the structure of 1 was assigned as nepodin-8-
0
O-
b
-D
-(6 -O-trans-crotonoyl)glucopyranoside.
Rumexneposide B (2) was obtained as a yellow powder and its
molecular formula was deduced to be C22
HRFABMS at m/z 449.1441 ([MꢀH] , calcd for 449.1447). The IR
H
26
O
10 by negative
ꢀ
pathway, with an MIC value of 12.6
that of abyssomicin C (the MIC value of abyssomicin C in MP
medium is 8.3 M, used as a positive control in this study). Other
compounds, including analogues of 5, showed no inhibitory effects
at concentrations of up to 30 g/mL (data not shown). Like
mM (3.1 mg/mL), similar to
Table 1
1
13
m
3
NMR data for compounds 1 (500 MHz for H and 125 MHz for C, in CDCl ) and 2
1
13
(400 MHz for H and 100 MHz for C, in CDCl
3
).
m
1
2
abyssomicin C, this molecule represents another pAba biosynthesis
pathway inhibitor.
1
13
1
13
H
C
H
C
1
2
3
4
5
6
7
8
9
1
150.4 (s)
125.3 (s)
132.9 (s)
119.8 (d)
122.8 (d)
127.8 (d)
111.4 (d)
154.8 (s)
113.5 (s)
136.1 (s)
153.5 (s)
121.9 (s)
134.4 (s)
119.3 (d)
100.8 (d)
158.8 (s)
103.7 (d)
155.8 (s)
109.2 (s)
137.6 (s)
A cytotoxic assay of all the compounds was performed against
five cancer cell lines (A549, H522, MCF-7, MCF-10A, and SKBR3)
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) method, with cisplatin as the positive control.
Compounds 1, 11, 14, and 17 showed different cytotoxicities
towards the five cancer cell lines (Table 2), but the other
compounds showed no cytotoxic effects at concentrations of up
6.97 (s)
6.92 (s)
7.21 (brd, 8.1)
7.03 (dd, 8.1, 7.6)
6.70 (brd, 7.6)
6.63 (d, 2.2)
6.82 (d, 2.2)
to 40
mM. Compound 1, in particular, showed activity against a
0
broad spectrum of human cancer cells, including lung and breast
cancer cells. The cytotoxicity of 14 was cell-type-dependent, and
of the five cell lines tested, only MCF-10A cancer cells responded
to 14.
OH-1
9.70 (s)
COMe-2
COMe-2
Me-3
208.9 (s)
32.5 (q)
19.4 (q)
206.6 (s)
32.2 (q)
20.5 (q)
55.2 (d)
102.6 (d)
73.0 (d)
76.4 (d)
69.8 (d)
74.3 (d)
63.4 (t)
2.48 (s)
2.25 (s)
2.55 (s)
2.30 (s)
OMe-6
3.80 (s)
In this study, we isolated and characterized 2 new naphthalene
acylglucosides and 12 known compounds from R. nepalensis roots.
We evaluated the biological activities of 25 compounds, including
11 previously reported compounds from R. nepalensis and R.
hastatus, against M. tuberculosis, pAba pathway, and 5 human
cancer cell lines. Several compounds showed good activity against
M. tuberculosis, of which compound 15 was the most active, with an
MIC similar to that of isoniazid. Compound 5 was a potent pAba
synthetic pathway inhibitor, representing another example of the
rare pAba pathway inhibitors. Although most compounds showed
0
1
4.19 (d, 7.2)
3.70 (m)
103.3 (d)
73.6 (d)
76.0 (d)
69.9 (d)
74.6 (d)
62.8 (t)
4.90 (d, 7.9)
3.62 (m)
0
2
0
3
3.45 (m)
3.45 (m)
0
0
4
3.53 (m)
3.37 (m)
5
2.71 (m)
3.65 (m)
0
6
4.46 (brd, 11.0)
4.43 (dd, 12.0, 2.0)
4.25 (dd, 12.0, 7.2)
4.27 (dd, 11.0, 6.5)
0
0
0
0
0
0
0
0
1
2
3
4
166.2 (s)
122.4 (d)
145.4 (d)
18.1 (q)
171.5 (s)
20.5 (q)
5.93 (brd, 15.5)
7.05 (overlap)
1.94 (d, 6.6)
2.09 (s)

H.-X. Liang et al. / Phytochemistry Letters 3 (2010) 181–184
183
Table 2
Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences.
a
Cytotoxic activities of the compounds (IC50
m
M) .
b
b
c
c
c
Cell line
A549
H522
MCF-7
MCF-10A
SKBR3
3
.3. Extraction and isolation
1
2
3
4
5
6
7
8
9
31.0
15.7
21.8
>40
>40
>40
>40
>40
>40
>40
>40
>40
29.4
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
85.1
22.8
>40
>40
>40
>40
>40
>40
>40
>40
>40
12.3
>40
>40
9.6
20.7
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
19.9
>40
>40
>40
>40
>40
>40
>40
>40
67
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
The dried roots of R. nepalensis (15 kg, powdered) were
extracted with 95% EtOH (3 ꢁ 30 L) under reflux and concentrated
in vacuo to produce a crude extract, which was suspended in water
and then successively partitioned with petroleum ether (3 ꢁ 3 L)
and EtOAc (3 ꢁ 3 L). The EtOAC extract (488 g) was subjected to CC
over silica gel (200–300 mesh, 5 kg) and eluted with a gradient of
CHCl
to produce fractions A–G. Fraction C (26 g) was subjected to silica
gel CC eluted with a gradient of CHCl –Me CO (4:1, 3:1, 2:1, 1:1) to
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
3
–MeOH (100:0, 98:2, 96:4, 94:6, 90:10, 85:15, 80:20, 70:30)
27.8
>40
>40
>40
>40
>40
3
2
produce three fractions (fr. C.1–3). Fraction C.2 was subjected to
Sephadex LH-20 (MeOH) chromatography and repeated vacuum
liquid chromatography (VLC) over silica gel to yield 1 (31 mg) and
>40
>40
7.6
29.0
38.7
1
3 (187 mg). Fraction D (22 g) was subjected to silica gel CC with a
gradient of CHCl –Me CO (150:1, 120:1, 100:1, 80:1, 40:1, 20:1),
followed by gel filtration on Sephadex LH-20 (MeOH), MCI Gel
CHP20P chromatography (MeOH–H O, 9:1), and repeated VLC
silica gel) to produce 2 (32 mg), 3 (17 mg), 4 (104 mg), 8 (34 mg),
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
77
3
2
1
2
(
1
and 9 (75 mg). Fraction E (29 g) was fractionated by MCI Gel
CHP20P chromatography, eluted with a gradient of MeOH–H
3:7, 5:5, 7:3, 1:0), followed by Sephadex LH-20 (MeOH) filtration,
to produce 7 (20 mg), 10 (35 mg), 11 (76 mg), 12 (41 mg), and 14
76 mg). The combined chromatography of fraction F by silica gel
CC (CHCl –MeOH, 100:0, 80:1, 60:1, 40:1, 20:1) and Sephadex LH-
0 (MeOH) chromatography yielded 5 (136 mg). Compound 6
3
(21 mg) was purified from fraction G (23 g) by silica gel CC (CHCl –
2
O
d
Cisplatin
17.5
20.4
(
a
Previously isolates: aloesin (15), (ꢀ)-epicatechin-3-O-gallate (16), orientalo-
(18), orcinol glucoside (19), lyoniresinol -O-
glucopyranoside (20), (3,5-dimethoxy-4-hydroxyphenol)-1-O- -(6-O-galloyl)-
side (17), hastatuside
A
3
a
b-D-
(
b-D
3
glucose (21), (ꢀ)-epicatechin (22), nepalensides A (23) and B (24), and hastatuside
B (25).
2
b
Lung cancer cells.
c
Breast cancer cells.
MeOH, 40:1, 30:1, 15:1, 10:1, 6:1, 4:1, 2:1) and gel filtration over
Sephadex LH-20 (MeOH).
d
Positive control.
3.4. Nepalenside A (1)
2
D
1:6
no cytotoxicity against several human cancer cells, compound 1
exhibited broad activity against lung and breast cancer cells. MCF-
Yellow powder, mp 67–68 8C; ½
a
ꢂ
ꢀ45.1 (c 0.96, MeOH); UV
ꢀ1
13
l
max (CHCl
3
) nm (log
e
): 334 (3.54), 299 (3.57); IR (KBr)
3414, 1721, 1698, 1631, 1578, 1078; H and C NMR data, see
n
max cm
:
1
1
0A was sensitive to compounds 1, 11, 14, and 17.
ꢀ
Table 1; FABMS (negative) m/z: 445 [MꢀH] ; HRFABMS (negative)
m/z: 445.1506 [MꢀH]ꢀ (C23
25 9
H O , calcd for 445.1498).
3
. Experimental
3.5. Nepalenside B (2)
3
.1. General experimental procedures
2
D
2:1
Yellow powder, mp 151–153 8C; ½
a
ꢂ
ꢀ75.6 (c 0.97, MeOH);
max
Melting points were determined with an XRC-1 micromelting
UV
l
max (CHCl ) nm (log ): 323 (3.62), 262 (4.18); IR (KBr) n
cm : 3422, 1734, 1715, 1627, 1585, 1077; H and C NMR data,
3
e
ꢀ1
1
13
apparatus. Optical rotations were determined with a JASCO-20C
digital polarimeter. UV spectra were recorded on a Shimadzu UV-
2
Bruker Tensor 27 FT-IR spectrophotometer with KBr pellets. NMR
spectra were recorded on a Bruker AV-400 or a DRX-500
spectrometer. Electron impact mass spectrometry (EIMS) (70 eV)
and FABMS were recorded on a VG Auto Spec-3000 spectrometer.
ꢀ
see Table 1; FABMS (negative) m/z: 449 [MꢀH] ; HRFABMS
401PC spectrophotometer. IR spectra were obtained with a
(negative) m/z: 449.1441 [MꢀH]ꢀ (C22
H
25
O
10
,
calcd for
449.1447).
3.6. Acid hydrolysis of 1 or 2
The silica gel (200–300 mesh and 1–40
chromatography (CC) and GF254 for thin-layer chromatography
TLC) were obtained from the Qingdao Marine Chemical Factory,
m
m) for column
Compound 1 or 2 (5 mg each) was refluxed with 2% HCl–
dioxane (1:1, v/v, 4 mL) in a water bath (80 8C) for 6 h. The
reaction mixture was then evaporated to dryness, and parti-
(
Qingdao, China. Sephadex LH-20 was obtained from Amersham
Pharmacia Biotech, Sweden. The fractions were monitored by TLC
and the spots were visualized after spraying with 10% NaOH in
EtOH or anisaldehyde reagents, followed by heating.
tioned between CHCl
neutralized with Amberlite IRA-401 resin (OH form). The
filtrate was evaporated to dryness. The sugar residue was
3
and
H
2
O. The aqueous layer was
ꢀ
diluted in 2 mL of pyridine and treated with 1.5 mL of L-cysteine
methyl ester hydrochloride at 60 8C for 1 h, and then treated
with 1.5 mL of trimethylsilylimidazole at 60 8C for 30 min. The
supernatant was analyzed by GC under the conditions: 30QC/
AC-5 (30 m ꢁ 0.32 mm i.d.); detector, FID (250 8C); column
3
.2. Plant material
R. nepalensis roots were collected in the suburb of Kunming,
Yunnan Province, China, in July 2006. The voucher specimen (no.
CHYX0183) authenticated by Prof. Yun-Heng Ji in our institution
was deposited at the State Key Laboratory of Phytochemistry and
temperature, 180–280 8C, rate, 3 8C/min; t
-glucose), 19.856 (standard
1), or 19.412 ( -glucose from 2).
R
(s), 19.450 (standard
D
L
-glucose), 19.402 ( -glucose from
D
D

1
84
H.-X. Liang et al. / Phytochemistry Letters 3 (2010) 181–184
3.7. Assay for anti-M. tuberculosis (H37Rv) in its logarithmic growth
and cultured for 12 h. The medium was replaced with one
phase
containing the test compounds, and the cells were further cultured
at 37 8C. To evaluate the IC50 of all the compounds, the cells were
exposed to the test compounds at various concentrations. After
M. tuberculosis (MTB) assays were carried using a constitutive
green fluorescent protein (GFP) expression vector (pUV3583c–
GFP) with the direct fluorescence readout as a measure of bacterial
growth (Chan et al., 2002). The production of GFP by replicating
cells as a marker of live cells has been shown to result in the same
MIC as that measured by the direct enumeration of colony-forming
units (cfu) for all front-line antituberculosis agents (Changsen
et al., 2003). Briefly, MTB was grown in 50 mL of 7H9 broth (Difco)
incubation for 48 h, 10
each well and the cells were incubated under the same conditions
for 4 h until a purple precipitate was visible. DMSO (200 L) was
added and the optical density was measured at 570 nm in a
microplate reader (Bio-Tek Synergy HT). Cisplatin and DMSO were
used as the positive and negative controls, respectively.
mL of MTT solution (Sigma) was added to
m
containing 0.05% Tween 80 and 25
mg/mL kanamycin. The cultures
Acknowledgements
were incubated at 37 8C with rotary agitation, grown to mid-
exponential phase (optical density at 595 nm [OD595] of approxi-
mately 0.6–0.8), and harvested by centrifugation. The cell pellets
were resuspended in a small amount of enriched 7H9 medium. The
starter culture was grown to an OD595 of 0.3–0.5 and then diluted
This research was financially supported by the following grants:
National Natural Science Foundation of China (no. 20972165), ‘‘Xi-
Bu-Zhi-Guang’’ Project of the Chinese Academy of Sciences, Key
Project for Drug Innovation (2008ZX09401-004) from the Ministry
of Science and Technology of China, and ‘‘Talent Scholarship of
Yunnan Youth’’ (no. 2007PY01-48) from Yunnan Province.
4
with 7H9 medium to an OD595 of approximately 0.05 (1.5 ꢁ 10
cells). The compounds were prepared at 100 ꢁ stocks in dimethyl
sulfoxide (DMSO). Six serial dilutions of the compounds were
prepared in the same solvent and added to the wells in a 2
volume. Isoniazid (purchased from Sigma) was used as the positive
control for the MTB assay. Wells containing only the carrier solvent
mL
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were the negative controls. Each well received 80
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2
(
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4 h for three days using an EnVision 2103 multilabel reader
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.8. Assay for the inhibition of the biosynthetic pathway from
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Bacillus subtilis ATCC 66333 was grown on an agar plate on minimal
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ꢃ7H
NH SO 0.1% in deionized water. The B. subtilis strain was also
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pAba). Aliquots (2 L) of the compounds dissolved in DMSO were
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2
O
0
2
4
K
2
4
4
2
O
(
4
)
2
4
m
m
5
MM or MP medium to a final concentration of 1 ꢁ 10 cfu/mL. The
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3
.9. Cytotoxicity assay
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cell lung cancer), SKBR3, MCF-10A, MCF-7, and H522 cells (non-
small-cell lung carcinoma) was measured. The cells were grown in
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(50
2
mg/mL) in a humidified incubator under 5% CO at 37 8C. The
4
cells were seeded in 96-well microplates (1–2 ꢁ 10 cells/well)