Bioactive dammarane triterpenes from the mangrove Plant Bruguiera gymnorrhiza

Article abstract of DOI:10.1021/np058112x

Three new dammarane triterpenes, bruguierins A-C (1-3), were isolated from a petroleum ether extract of the flowers of Bruguiera gymnorrhiza. Their structures were determined on the basis of physical and spectroscopic data interpretation. With stably transfected HepG2 cells, the three isolates activated antioxidant response element (ARE luciferase activation) with EC₅₀ values of 7.8, 9.4, and 15.7 μM, respectively. Bruguierin A (1) also inhibited phorbol ester-induced NFκB (nuclear factor-κB) luciferase activation with an IC₅₀ value of 1.4 μM and selectively inhibited cyclooxygenase-2 (COX-2) activity with an IC₅₀ value of 0.37 μM. Compounds 2 and 3 were not active in these bioassays.

Full text of DOI:10.1021/np058112x

J. Nat. Prod. 2006, 69, 421-424
421
Bioactive Dammarane Triterpenes from the Mangrove Plant Bruguiera gymnorrhiza⊥
†
,†
‡
§
#
Sudarat Homhual, Nuntavan Bunyapraphatsara,* Tamara Kondratyuk, Angkana Herunsalee, Wongsatit Chaukul,
‡
|
,|
John M. Pezzuto, Harry H. S. Fong, and Hong-Jie Zhang*
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol UniVersity, Bangkok 10400, Thailand, College of Pharmacy, Nursing, and
Health Sciences, Purdue UniVersity, 575 Stadium Mall DriVe, West Lafayette, Indiana 47907-2091, Department of Medical Sciences, The
Ministry of Public Health, Nontaburi 11000, Thailand, Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol UniVersity,
Bangkok 10400, Thailand, and Program for CollaboratiVe Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and
Pharmacognosy (M/C 781), College of Pharmacy, UniVersity of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois 60612
ReceiVed October 14, 2005
Three new dammarane triterpenes, bruguierins A-C (1-3), were isolated from a petroleum ether extract of the flowers
of Bruguiera gymnorrhiza. Their structures were determined on the basis of physical and spectroscopic data interpretation.
With stably transfected HepG2 cells, the three isolates activated antioxidant response element (ARE luciferase activation)
with EC50 values of 7.8, 9.4, and 15.7 µM, respectively. Bruguierin A (1) also inhibited phorbol ester-induced NFκB
(
2
nuclear factor-κB) luciferase activation with an IC50 value of 1.4 µM and selectively inhibited cyclooxygenase-2 (COX-
) activity with an IC50 value of 0.37 µM. Compounds 2 and 3 were not active in these bioassays.
Bruguiera gymnorrhiza (L.) Savigny, a mangrove plant of the
family Rhizophoraceae, is native to many countries of southern
1
and eastern Africa, Asia, and northern Australia. In Thailand, B.
2
gymnorrhiza flowers are used as a vegetable. The fruit and bark
of this plant have been used traditionally for diarrhea in mainland
3
4
China, and its roots and leaves are used for treating burns. Previous
studies have reported the presence of a wide range of phytochemi-
cals, including phenol, flavonoid, steroid, sulfur-containing, and
terpenoid compounds.5
-9
As part of a cancer chemoprevention study, we investigated
previously a CHCl -soluble extract of the flowers of B. gymnorrhiza,
3
collected in Thailand, and found stimulation of ARE-linked
luciferase activity by cyclic dithiols, bruguiesulfurol, brugierol, and
isobrugierol.10 In the present study, we have evaluated a petroleum
ether-soluble extract of the same plant material and found activation
of ARE-linked luciferase activity (EC50 0.7 µg/mL) and inhibition
of NFκB-linked luciferase activation (IC50 19.7 µg/mL). Further
investigation of this extract has led to the isolation of three new
dammarane triterpene fatty acid esters with biological activity,
bruguierins A-C (1-3), as reported herein.
A crude petroleum ether extract of the dried and milled flowers
of B. gymnorrhiza was fractionated by repeated flash column
chromatography on silica gel to afford triterpenoids 1-3.
Bruguierin A (1) was obtained as a white wax from ethyl acetate,
carbonyl carbon (δ 173.7). The side chain in this dammarane
triterpene (1) was confirmed by a 1D TOCSY NMR experiment.
On selective excitation of the olefinic proton signal at δ 5.12 (H-
24), the proton signals of the side chain spin system [δ 1.47 (H-
2
2), 2.06 (H-23), 1.69 (H-26), and 1.62 (H-27)] were clearly
and its molecular formula was established as C48
H
86
O
4
by HR-
24,25
observed. A double bond was assigned to ∆
in the side chain
+
FABMS ([M + Na] m/z 749.6438, calcd 749.6424). Its structure
was determined to be a dammarane triterpene by analysis of its
NMR data as well as by comparison with the spectroscopic data
due to the presence of HMBC correlations of C-24 and C-25
resonances with the 26,27-geminal methyl protons (Figure 1). The
1
H NMR spectrum of 1 displayed a downfield singlet at δ 1.14 for
11,12
of known dammarane triterpenes.
The complete assignments
of the H and C NMR spectra were achieved as shown in Table
. The 13C NMR (DEPT-90 and -135) and HSQC spectra were
a methyl group at the C-21 position. Moreover, the resonances of
these methyl protons with C-17 and C-20 were observed. The
appearance of the downfield quarternary 13C at δ 75.6 was attributed
to C-20. Furthermore, the C-20 configuration of 1 was established
to be S on the basis of a comparison of the 13C NMR chemical
shifts of C-20 through C-22 with those published for dammarenediol
II (20S) [δ 75.4 (C-20), 24.9 (C-21), and 40.5 (C-22)] and
1
13
1
used to characterize eight tertiary methyls and one methyl, four
methines, two oxygenated methines (δ 78.5 and 77.1), one olefinic
methine (δ 124.9), four quaternary carbons, an olefinic quaternary
carbon (δ 131.8), an oxygenated quaternary carbon (δ 75.6), and a
dammarenediol I (20R) [δ 75.8 (C-20), 23.5 (C-21), and 41.8 (C-
⊥
Dedicated to Dr. Norman R. Farnsworth of the University of Illinois
12
22)]. The two oxygenated methine carbons in 1 were assigned as
at Chicago for his pioneering work on bioactive natural products.
*
C-1 and C-3, respectively, on the basis of the observation that in
To whom correspondence should be addressed. Tel: 66-2-6444566
1
1
the H- H COSY spectrum of 1 both the proton signals of H-1
(
(
B.N.); 1-312-996-7868 (Z.H.). Fax: 66-2-6444566 (B.N.); 1-312-996-7107
Z.H.). E-mail: pynby@mahidol.ac.th; zhanghj@uic.edu.
3
and H-3 coupled with the signal of H-2 at δ 1.72. The J long-
†
Department of Pharmacognosy, Mahidol University.
Purdue University.
Ministry of Public Health.
Department of Pharmaceutical Botany, Mahidol University.
University of Illinois at Chicago.
range correlations of Me-19 to C-1 and Me-28, Me-29 to C-3 were
also observed in the HMBC spectrum. The correlations of H-1 to
H-2 and H-3 were confirmed by a 1D TOCSY experiment.
Irradiation at H-1 caused the splitting of signals of the two protons
‡
§
#
|
1
0.1021/np058112x CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/25/2006

4
22 Journal of Natural Products, 2006, Vol. 69, No. 3
Notes
Table 1. NMR Spectroscopic Data of Compounds 1-3
1a
2b
3b
position
δH
δC
δH
δC
δH
δC
1
2
3
4
5
6
7
8
9
3.53 dd (10.9, 4.4)
1.72 m
4.50 dd (13.0, 4.4)
78.5 d
34.5 t
77.1 d
38.1 s
53.8 d
18.1 t
40.5 t
3.54 dd (11.3, 4.5)
1.70 m
4.51 dd (13.5, 4.5)
78.7 d
34.7 t
77.3 d
38.3 s
54.0 d
18.3 t
35.3 t
41.4 s
51.8 d
43.8 s
25.1 t
28.0 t
42.3 d
50.7 s
32.3 t
25.5 t
50.7 d
16.1 q
12.5 q
75.5 s
26.0 q
44.0 t
127.6 d
137.7 d
82.4 s
24.5 q
24.9 q
28.2 q
16.5 q
16.8 q
173.9 s
35.1 t
1.63 m
4.51 dd (10.0, 6.3)
24.1 t
80.4 d
39.1 s
56.4 d
18.2 t
0.74 m
1.55 m
1.28 m
0.75 m
1.60 m
1.58 m
1.51 m
1.22 m
35.1 t
36.1 t
41.2 s
51.7 d
43.7 s
22.8 t
41.0 s
55.9 d
38.6 s
71.4 d
40.3 t
40.9 d
50.3 s
31.0 t
1.53 m
1.53 m
1.55 m
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
2
3
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
1.60 m
1.57 m
1.61 m
3.98 m
1.42 m
1.80 m
1.30 m
1.85 m
1.65 m
25.3 t
41.9 d
50.5 s
31.6 t
1.10 m
1.38 m
1.55 m
1.05 m
1.68 m
1.75 m
0.98 s
0.94 s
27.8 t
25.2 t
1.73 m
0.97 s
0.93 s
50.1 d
15.9 q
12.3 q
75.6 s
25.5 q
41.0 t
49.9 d
17.0 q
16.9 q
75.3 s
26.0 q
40.8 t
0.98 s
1.07 s
1.14 s
1.47 t (8.0)
2.06 m
1.16 s
1.44 m
2.05 m
5.12 t (6.3)
1.14 s
2.25 m
5.78 m
5.62 d (15.8)
22.9 t
22.8 t
5.12 t (6.5)
124.9 d
131.8 s
25.9 q
17.9 q
28.0 q
16.3 q
16.6 q
173.7 s
34.9 t
124.8 d
132.0 s
25.8 q
17.9 q
28.5 q
16.5 q
16.8 q
173.9 s
35.1 t
6
7
8
9
0
′
1.69 s
1.62 s
0.84 s
0.84 s
0.88 s
1.77 s
1.69 s
0.86 s
0.88 s
0.92 s
1.36 s
1.35 s
0.85 s
0.85 s
0.88 s
′
2.29 t (8.0)
1.25 brs
0.88 t (6.0)
2.29 t (7.1)
1.26 brs
0.88 t (6.5)
2.32 t (7.3)
1.26 brs
0.88 t (5.8)
′-17′
8′
29.8-29.9 t
14.3 q
29.8-29.9 t
14.3 q
30.0-30.1 t
14.5 q
a
Recorded at 400 MHz. b Recorded at 300 MHz; coupling constants (Hz) are shown in parentheses.
HRFABMS ([M + Na]+ m/z 749.6396, calcd 749.6424 for
C
48
H
86
4
O ). The similarity of the NMR spectroscopic data (Table
1) of these two compounds suggested that 2 is a positional isomer
of 1. The only difference between the two structures was that the
second hydroxy group in 2 was located at C-11 instead of C-1 as
in 1. The chemical shift of H-11 was shifted downfield from δ 1.6
in 1 to δ 3.98 in 2. The COSY experiment revealed correlations of
resonances of the methine proton (δ 3.98) at the C-11 position with
the methine proton of H-9 and the methylene protons of H-12. In
addition to the 13C NMR chemical shift of the oxymethine, C-11
was observed to be at much higher field for 2 (δ 71.4) than for 1
Figure 1. Selected HMBC correlations for compound 1.
1
3
(δ 78.5), which is in accordance with literature. On basic
of H-2 at δ 1.67 (m) and 1.87 (m), while the proton of H-3 was
observed as a doublet of doublets (J ) 13.0, 4.4 Hz) at δ 4.5. The
multiplicity of the H-2 protons was observed as a doublet of
doublets (J ) 11.1 Hz) at δ 1.67 and multiplet at δ 1.87 when H-3
was irradiated. The 1H NMR spectrum of 1 showed a broad,
intensive singlet signal at δ 1.25 due to methylene protons,
indicating the presence of a long fatty acid ester chain moiety. By
subtraction of the dammarane triterpene unit from the molecular
formula of 1, the fatty acid ester moiety was calculated as stearic
acid. On basic hydrolysis and GC-MS analysis, the fatty acid side
chain of 1 was confirmed to be octadecanoic acid. The stearic acid
ester moiety was attached to C-3 due to the presence of the HMBC
correlations between H-3 and the carbonyl carbon signal of the
fatty acid ester chain. Finally, both oxygen-containing groups at
C-1 and C-3 were determined to be â-oriented due to the large
coupling constants of H-1 (J ) 10.9 Hz, 4.4 Hz) and H-3 (J )
hydrolysis and GC-MS analysis, the fatty acid side chain of 2 was
again confirmed to be octadecanoic acid. The hydroxy group of
C-11 in 2 was determined to be R-oriented by comparison of the
NMR data with those of the known compound dammar-24-en-
3
â,11R,20(S)-triol, which possesses the same dammarane triterpene
13
nucleus. Thus, 2 was determined to be 11R,20(S)-dihydroxydam-
mar-24(25)-ene-3-O-stearate and has been given the trivial name
bruguierin B.
Bruguierin C (3) was obtained as a colorless gum. The HR-
+
FABMS data ([M + Na] m/z 781.6322, calcd 781.6322) was used
1
13
to establish its molecular formula as C48
H
86
O
6
. The H and
C
NMR spectra of 3 closely resembled those of 1, especially the
signals from the fatty acid moiety and rings A-D. Analysis of the
MS data revealed that the fatty acid moiety is the same stearic acid
moiety of 1 and also was assigned to C-3 according to the HMBC
spectroscopic data (Figure 2). Compound 3 differed from 1 only
by the C-17 side chain. In 3, the double bond was positioned at
1
3.0 Hz, 4.4 Hz).11 Thus, compound 1 was determined to be 1â,20-
S)-dihydroxydammar-24(25)-ene-3â-O-stearate and has been given
(
2
3,24
1
1
the trivial name bruguierin A.
Bruguierin B (2) was isolated as white needles from ethyl acetate,
and its molecular formula was shown to be the same as 1 by
∆
, as evidenced by an analysis of the H- H COSY and HMBC
1
data. The H NMR spectrum of 3 showed two olefinic proton signals
at δ 5.62 (d, J ) 15.8 Hz, H-24) and 5.78 (m, H-23), which was

Notes
Journal of Natural Products, 2006, Vol. 69, No. 3 423
The plant material was identified by one of us (W.C.). Voucher
specimens (nos. PBM 3757, 3758, and 3759) have been deposited in
the Herbarium of the Department of Pharmaceutical Botany, Mahidol
University, Bangkok, Thailand.
Extraction and Isolation. The air-dried and milled flowers of B.
gymnorrhiza (1.2 kg) were extracted exhaustively in a Soxhlet apparatus
with petroleum ether, cooled to room temperature, filtered, and
evaporated to dryness under vacuum to yield 40 g of a crude extract.
A portion of the extract (13 g) was fractionated by flash column
chromatography over a silica gel G60 (0.063-0.2 mm, 150 g) column
developed by solvent systems containing petroleum ether and increasing
concentrations of chloroform and/or ethyl acetate to afford 21 fractions
Figure 2. Selected HMBC correlations for compound 3.
[
petroleum ether-CHCl
3
/10:0 (eluates F1, F2, each 0.5 L), 9.5:0.5
due to a double bond at C-23, C-24 from the presence of HMBC
correlations between H-23/C-20, H -26/C-24, and H -27/C-24. The
3 3
(eluates F3, F4, each 1.2 L), 8.5:1.5 (eluate F5, 0.8 L), 8:2 (eluate F6,
0
0
(
(
.4 L), 6:4 (eluates F7-F9, each 0.8 L), 3:7 (eluates F10, F11, each
.8 L), 1.5:8.5 (eluate F12, 1.4 L), respectively; CHCl
eluates F13-F15, each 2 L), 8:2 (eluates F17, F18, each 0.8 L), 6:4
eluate F19, 0.4 L), 0:10 (eluates F20, F21, each 1 L), respectively].
double bond (H-23, H-24) was assigned an E configuration due to
the large coupling constant (J ) 15.8 Hz) between its two protons.
The effect of the hydroperoxyl group downfield of the quaternary
carbon and the allylic carbon by 10 and 5 ppm, respectively, was
reported.14 This phenomenon was observed in compound 3. The
low-field signals at δ 82.4 and 127.6 were assigned as quarternary
3
-EtOAc/10:0
Fraction F15 (1.61 g) was rechromatographed on a silica gel G flash
column (0.063-0.2 mm, 29 g), using a stepwise gradient system of
CHCl -EtOAc, to give 12 fractions (F15.1-F15.12). Compound 3 (15
3
13
C-25 and allylic 13C-23, respectively. The correlations of C-25
mg) was obtained from fraction F15.9 as a colorless gum. Fraction
F16 (2.84 g) was subjected to repeated flash column chromatography
to methyl protons at the 26 and 27 positions and olefinic proton at
the 23 position were elucidated by a HMBC experiment. Further-
more, the comparison of the molecular formula of compound 1 to
that of compound 3 showed two additional oxygen atoms, consistent
with a hydroperoxyl group at the low-field quarternary C-25. On
basic hydrolysis and GC-MS analysis, the fatty acid side chain of
on silica gel G (0.063-0.2 mm, 30 g), eluting with CHCl
9.5:0.5), to afford seven fractions (F16.1-F16.7). Fraction F16.3 was
further fractionated on a prepacked Combi flash silica gel column (35-
3
-EtOAc
(
6
0 µm, 12 g) using CHCl -EtOAc (8:2) to afford compound 1 as a
3
white wax (6.3 mg) by precipitation from EtOAc. Fraction F17 (0.47
g) was rechromatographed on a silica gel G (0.063-0.2 mm, 45 g)
3
column, eluting sequentially with CHCl and increasing volume of
3
was confirmed to be octadecanoic acid. Thus, the structure of 3
was assigned as 1â,20(S)-dihydroxy-25-hydroperoxydammar-23-
24)-ene-3-O-stearate and was given the trivial name bruguierin
C.
EtOAc to afford six fractions (F17.1-F17.6). Fraction F17.4 was further
(
separated by a prepacked Combi flash silica gel column (35-60 µm,
3 g) using CHCl -EtOAc (7:3) to afford compound 2. Compound 2
3
was obtained as white needles (15 mg) by crystallization from EtOAc.
The three dammarane triterpene stearic acid esters (1-3) were
20
Bruguierin A (1): white wax; [R]
D
3
+31.5 (c 0.37, CHCl ); IR
subjected to COX-1, COX-2, and luciferase assays. Compound 1
demonstrated significant inhibition against COX-2, with an IC50
value of 0.37 µM, whereas compounds 2 and 3 were inactive.
However, none of these compounds exhibited activity toward COX-
(
film) νmax 3498 (br), 2924, 2853, 1714, 1465, 1376, 1181, 1117, 1098
-
1 1 13
cm ; H and C NMR data, see Table 1; FABMS m/z 750 [M +
Na] (9), 734 (1), 710 (1), 622 (1), 465 (1), 439 (1), 425 (6), 341 (2),
3
2
+
29 (2), 315 (2), 299 (3), 289 (1), 269 (1), 257 (2), 245 (2), 229 (3),
17 (5), 203 (11), 176 (41), 161 (16), 149 (25), 135 (34), 121 (43),
1. Bioassays for cancer chemopreventive activity, namely, the
inhibition of TPA-activated NFκB luciferase and the induction of
ARE luciferase, were also employed. The petroleum ether extract
and bruguierin A (1) were found to inhibit activation, with IC50
values of 19.7 µg/mL and 1.4 µM, respectively. For induction of
ARE luciferase, the petroleum ether extract of B. gymnorrhiza
showed activity with an EC50 value of 0.7 µg/mL, whereas
compounds 1-3 were active with EC50 values of 7.8, 9.4, and 15.7
µM, respectively.
48 86 4
109 (100); HRFABMS m/z 749.6438 (calcd for C H NaO , 749.6424).
20
Bruguierin B (2): white needles; [R]
D
3
+23.6 (c 0.13, CHCl ); IR
(film) νmax 3466 (br), 3341 (br), 2920, 2861, 1722, 1485, 1367, 1249,
-
1
1
13
1
7
(
(
(
104, 1025 cm ; H and C NMR data, see Table 1; FABMS m/z
+
50 [M + Na] (4), 722 (2), 694 (1), 666 (1), 622 (1), 553 (1), 502
1), 479 (1), 465 (1), 459 (1), 441 (1), 425 (2), 407 (2), 330 (6), 329
12), 199 (10), 176 (100), 154 (38), 136 (25), 115 (14), 109 (20), 81
18), 69 (51), 55 (30); HRFABMS m/z 749.6396 (calcd for C48
, 749.6424).
3
Bruguierin C (3): colorless gum; [R] +26.9 (c 0.58, CHCl ); IR
86
H -
NaO
4
20
D
Experimental Section
(
film) νmax 3414 (br), 2924, 2853, 1709, 1466, 1377, 1264, 1181, 1117
-
1 1 13
Gerneral Experimental Procedures. Optical rotations were mea-
sured on a Perkin-Elmer model 241 polarimeter. IR spectra were run
on a JASCO FT/IR-410 spectrometer, equipped with a Specac Silver
Gate ATR system by applying a film on a germanium plate. 1D and
cm ; H and C NMR data, see Table 1; FABMS m/z 782 [M +
+
Na] (100), 754 (43), 687 (22), 681 (9), 597 (7), 515 (15), 510 (7),
86
413 (14), 408 (11), 361 (8); HRFABMS m/z 781.6322 (calcd for C48H -
6
NaO , 781.6322).
Basic Hydrolysis15 of 1-3. Compounds 1 (4.0 mg), 2 (4.0 mg),
and 3 (1.4 mg) were hydrolyzed by 5% KOH in MeOH (2 mL) and
stirred at 80 °C for 3 h. Each reaction mixture was neutralized with
2
D NMR spectra were recorded on a Bruker DPX 300 or a 400 MHz
spectrometer. Chemical shifts (δ) are expressed in ppm with reference
to the solvent signals. FABMS were recorded on a JEOL GC Mate II
spectrometer. GC-MS was carried out on an Agilent 6890N, with a
fused silica capillary column (HP-INNOWAX, 0.32 mm (i.d.) × 30 m
2
1
% HCl in MeOH (pH 4-5) and filtered. The filtrate was poured into
0 mL of water and then extracted with CHCl
(5 × 5 mL). The
SO and filtered.
3
combined organic layers were dried over anhydrous Na
2
4
×
0.25 µm). The column temperature was first set at 140 °C and then
After evaporation, the residues 1a (3.0 mg), 2a (2.5 mg), and 3a (1.0
programmed from 140 °C to 240 °C at a rate of 4 °C/min. The column
was held at the final temperature for 15 min. The inlet temperature
was kept at 250 °C. Split injections were performed with a 20:1 split
ratio. Helium carrier gas was used at a constant flow rate of 1 mL/min
with a constant pressure of 2.01 psi. The mass spectrometric detector
was operated in the electron-impact ionization mode (EI, 70 eV).
Column chromatography was carried out on silica gel G60 (0.063-0.2
mm, E. Merck). Thin-layer chromatography (TLC) was performed on
TLC aluminum sheets coated with 0.25 mm layers of silica gel 60.
Fractions were monitored by TLC, and spots were visualized by heating
mg) were identified by GC-MS analysis. Compounds 1a-3a: colorless
+
gum, t
R
14.06 min; EIMS m/z [M] 298 (16), 267 (5), 255 (14), 213
(
(
3), 199 (8), 185 (4), 157 (1), 143 (19), 129 (7), 111 (3), 97 (7), 87
69), 74 (100), 55 (28), 43 (37), 28 (12). In comparison with standard
compounds, the methylated products 1a-3a were found to correspond
+
to octadecanoic acid methyl ester ([M] m/z 298).
COX Assays. The effect of test compounds on cyclooxygenase-1
2
and -2 (COX-1 and -2) was determined by measuring PGE production
as previously described.10 Indomethacin was used as a positive control,
yielding IC50 values between 0.05-0.1 and 1-5 µM observed with
COX-1 and COX-2, respectively.
2 4
silica gel plates sprayed with 30% H SO in MeOH.
Plant Material. The flowers of Bruguiera gymnorrhiza were
collected in Samutsongkram Province, Thailand, in September 2003.
Luciferase Assays. Luciferase assays were conducted as previously
described.10 Data for ARE induction (EC50 values) were expressed as

4
24 Journal of Natural Products, 2006, Vol. 69, No. 3
Notes
the concentration of compound that provoked an activation halfway
between baseline (DMSO control) and maximum response at a
concentration of 20 µg/mL. Data for NFκB constructs were expressed
as IC50 values (the concentration required to inhibit TPA-activated NFκB
activity by 50%). For ARE induction, sulforaphane (EC50 4-6 µM),
(4) Othman, S. Bruguiera Lamk. In Prosea, Plant Resources of South-
east Asia 5. (3) Timber Trees: Lesser-Known Timbers; Sosef, M. S.
M., Hong, L. T., Prawirohatmodjo, S., Eds.; Bogor Indonesia Press:
Bogor, Indonesia, 1998; pp 122-125.
(5) Achmadi, S.; Syahbirin, G.; Choong, E. T.; Hemingway, R. W.
Phytochemistry 1994, 35, 217-219.
6) Raiham, S. M. A. ACGC Chem. Res. Commun. 1994, 2, 21-24.
7) Misra, S.; Choudhury, A.; Datta, A. K.; Ghosh, A. Phytochemistry
4
′-bromoflavone (EC50 30 µM), and â-naphthoflavone (EC50 8 µM)
(
(
were used as standard inducer. With the experimental conditions
employed, no signs of overt cellular toxicity were observed with the
test compounds or extracts.
1984, 23, 2823-2827.
(
(
8) Kato, A.; Numata, M. Tetrahedron Lett. 1972, 203-206.
9) Han, L.; Huang, X.; Sattler, I.; Dahse, H.-M.; Fu, H.; Lin, W.;
Grabley, S. J. Nat. Prod. 2004, 67, 1620-1623.
Acknowledgment. This study was supported by the Thailand
Research Fund through the Royal Golden Jubilee Ph.D. program (grant
No. PHD/0191/2545) to S.H. and N.B. and in part by program project
P01-CA48112 funded by the National Cancer Institute, NIH, Bethesda,
MD. The authors are grateful to the NMR Laboratory of the Department
of Medicinal Chemistry and Pharmacognosy, and the Research
Resources Center (RRC), University of Illinois at Chicago, for access
to the Bruker DPX 300 and 400 MHz instruments.
(
10) Homhual, S.; Zhang, H. J.; Bunyapraphatsara, N.; Kondratyuk, T.
P.; Santasiero, B. D.; Mesecar, A. D.; Herunsalee, A.; Chaukul, W.;
Pezzuto, J. M.; Fong, H. H. S. Planta Med. 2006, in press.
(11) Ding, S.-L.; Zhu, Z.-Y. Planta Med. 1993, 59, 373-375.
(12) Asakawa, J.; Kasai, R.; Yamasaki, K.; Tanaka, O. Tetrahedron 1977,
33, 1935-1939.
(
(
(
13) Fuchino, H.; Satoh, T.; Tanaka, N. Chem. Pharm. Bull. 1995, 43,
1937-1942.
14) Jiang, Z.-H.; Fukuoka, R.; Aoki, F.; Tanaka, T.; Kouno, I. Chem.
Pharm. Bull. 1999, 47, 257-262.
References and Notes
(
1) Hou, D. Rhizophoraceae. In Flora of Thailand; Smitinand, T., Larsen,
K., Eds.; ASRCT Press: Bangkok, Thailand, 1970; Vol. 2, pp 5-15.
2) Bunyapraphatsara, N. J. Trop. Med. Plants 2000, 1, 138-142.
3) Bamroongrugsa, N. Songklanakarin J. Sci. Technol. 1999, 21, 377-
15) Torres, P.; Ayala, J.; Grande, C.; Macias, M. J.; Grande, M.
Phytochemistry 1998, 47, 57-61.
(
(
386.
NP058112X