Original Papers 897

Blumeaenes A–J, Sesquiterpenoid Esters from

Blumea balsamifera with NO Inhibitory Activity

Authors Ming Chen 1, 3, Jiang-Jiang Qin 1, Jian-Jun Fu 1, Xiao-Jia Hu 1, Xiao-Hua Liu 2, Wei-Dong Zhang 1, 2, Hui-Zi Jin 1

1
Affiliations School of Pharmacy, Shanghai Jiao Tong University, Shanghai, P. R. China
2
Department of Phytochemistry, Second Military Medical University, Shanghai, P. R. China
3
Shanghai Institute for Food and Drug Control, Shanghai, P. R. China

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Key words Abstract of 1D and 2D NMR spectroscopic techniques. All
" Blumea balsamifera
l ! sesquiterpenoid esters were tested for their in-
l
" Asteraceae
Chemical examination of the ethanol extract of hibitory activity against LPS-induced NO produc-
l
" sesquiterpenoid esters
the aerial parts of Blumea balsamifera led to the tion in RAW264.7 macrophages. Compounds 1, 4
l
" blumeaenes

isolation of ten new sesquiterpenoid esters, blu- and 5 showed slight inhibitory effect on the pro-
l
" NO production

meaenes A–J (1–10), with 13 known flavonoids. duction of NO with IC50 values of 40.06, 46.35
Their structures were determined mainly by use and 57.80 µg/mL, respectively.

Introduction ters (1–10; l

" Fig. 1) were isolated, along with 13

! known flavonoids. In this article, we describe the

received Sept. 29, 2009
revised Dec. 14, 2009
accepted Dec. 18, 2009
Bibliography
DOI http://dx.doi.org/
10.1055/s-0029-1240800
Published online January 25,
2010
Planta Med 2010; 76: 897–902
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Dr. Hui Zi Jin and
Dr. Wei Dong Zhang
School of Pharmacy
Shanghai Jiao Tong University
Shanghai Dongchuan Rd 800
200240 Shanghai
Peopleʼs Republic of China
Phone: + 86 21 34 20 59 89
Fax: + 86 21 34 20 59 89
kimhz@sjtu.edu.cn
wdzhangy@hotmail.com
Blumea balsamifera DC (Asteraceae) is an aro-
matic hairy herb growing in Southeast Asia and
has been reported to possess stomachic, expecto-
rant, antispasmodic and sudorific properties. Its
leaves and roots have been used as a crude drug
for beriberi, menorrhagia, leukorrhea, lumbago,
rheumatism and a variety of ailments [1, 2]. Re-
cently, its leaves have attracted attention as a part
of the plant with various physiological activities,
including plasmin-inhibitory, antifungal, liver-
protective effects, and anticancer activities in he-
patocellular carcinoma cells [3–7]. Previous phy-
tochemical studies on B. balsamifera have re-
vealed that it is a rich source of essential oils [2].
However, only several flavonoids and two sesqui-
terpenes have been reported from chemical in-
vestigation [3, 8–10]. Nitric oxide (NO), synthe-
sized by nitric oxide synthase (NOS), is involved
in diverse physiological processes [11, 12]. Over-
expression of NO has been implicated in the
pathogenesis of a variety of inflammatory-medi-
ated disorders including septic shock, stroke, ar-
thritis, multiple sclerosis, and carcinogenesis
caused by mutagenesis [13]. Therefore, the
screening of bioactive compounds from natural
resources, which modulates NO production will
be useful for the treatment inflammatory dis-
eases.
As part of our ongoing investigation of bioactive
sesquiterpenes from natural sources, this plant
was investigated and ten new sesquiterpenoid es-
isolation and structural elucidation of these ses-
quiterpenes and inhibitory activities of all sesqui-
terpenes against LPS-induced NO production in
RAW264.7 macrophages.
Materials and Methods
!
General experimental procedures
Optical rotations were obtained with a Perkin-El-
mer 341 polarimeter. IR spectra were recorded
with a Bruker FTIR Vector 22 spectrometer. The
ESI‑MS were measured on an Agilent 1100 series
mass spectrometer. The TOF‑ESI mass spectra
were carried out on a Q‑Tof micro YA019 mass
spectrometer. NMR spectra were measured on a
Bruker DRX-500 instrument. All solvents used
were of analytical grade (Shanghai Chemical
Company, Ltd.). A Shimadzu PRC‑ODS EV0233
column was used for preparative HPLC (Shimadzu
LC-6AD). Silica gel was used for column chroma-
tography, and precoated silica GF254 plates were
used for TLC (Qingdao Haiyang Chemical Com-
pany, Ltd.).
Plant material
The aerial parts of B. balsamifera were collected
from Mengla, Yunnan, China in September 2006
and identified by Yang Hai-ou. A voucher speci-
men (ANX0601) was deposited in Shanghai Jiao
Tong University.

Chen M et al. Blumeaenes A–J, Sesquiterpenoid … Planta Med 2010; 76: 897–902
898 Original Papers

silica gel (200–300 mesh, 80 g) eluding with petroleum ether-

EtOAc (40 : 1) and preparative HPLC (MeOH‑H2O, 60 : 40, flow
rate: 8 mL/min) to obtain 8 (20 mg, tR: 30 min). Fr. 9 (7.0 g) was
separated over silica gel (140 g, 200–300 mesh, 6 × 40 cm) with
petroleum ether-EtOAc (10 : 1), Sephadex LH-20 column
(4 × 150 cm) and preparative HPLC (MeOH‑H2O, 75 : 25, flow rate:
8 mL/min) to yield 1 (39 mg, tR: 20 min) and 4 (25 mg, tR:
30 min); preparative HPLC (MeOH‑H2O, 70 : 30, flow rate: 8 mL/
min) to yield 2 (18 mg, tR: 30 min); preparative HPLC
(MeOH‑H2O, 65 : 35, flow rate: 8 mL/min) to yield 3 (15 mg, tR:
40 min). Fr. 12 (3.0 g) was subjected to Sephadex LH-20 column
(4 × 150 cm) and preparative HPLC (MeOH‑H2O, 70 : 30, flow rate:
8 mL/min) to yield 7 (100 mg, tR: 30 min). The purity of these
compounds were between 97.5 to 99.0 % as determined by HPLC.
D : − 29 (c 0.190, CH3OH); IR (KBr):
Blumeaene A (1): Yellow oil; [α]20
vmax = 3468, 2958, 2876, 2360, 1715, 1675, 1458, 1384, 1263,
1161, 975 cm−1; 1H- and 13C‑NMR spectroscopic data, see l " Table

1 and Table 2; HR‑ESI‑MS (negative): m/z = 385.1791 [M – H + Cl]−

(calcd. for C20H29O5Cl: 385.1781).

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D : − 26 (c 0.085, CH3OH); IR (KBr):
Blumeaene B (2): Yellow oil; [α]20
vmax = 3464, 2951, 2866, 2345, 1709, 1664, 1452, 1376, 1255,
1151, 975 cm−1; 1H- and 13C‑NMR spectroscopic data, see l " Table

1 and Table 2; HR‑ESI‑MS (positive): m/z = 373.2007 [M + Na]+

(calcd. for C20H30O5Na: 373.1991).
D : − 37 (c 0.205, CH3OH); IR (KBr):
Blumeaene C (3): Yellow oil; [α]20
vmax = 3496, 2960, 2874, 1727, 1674, 1469, 1207, 1161, 975,
878 cm−1; 1H- and 13C‑NMR spectroscopic data, see l " Table 1

and Table 2; HR‑ESI‑MS (positive): m/z = 361.1990 [M + Na]+

(calcd. for C19H30O5Na: 361.1991).
Blumeaene D (4): Yellow oil; [α]20 D : − 31 (c 0.120, CH3OH); IR

(KBr): vmax = 3464, 2949, 2871, 2355, 1708, 1664, 1450, 1374,
1263, 1156, 972 cm−1; 1H- and 13C‑NMR spectroscopic data, see
l" Table 1 and Table 2; HR‑ESI‑MS (positive): m/z = 387.2149 [M

+ Na]+ (calcd. for C21H32O5Na: 387.2147).

D : − 55 (c 0.055, CH3OH); IR (KBr):
Blumeaene E (5): Yellow oil; [α]20
vmax = 3477, 2959, 1742, 1674, 1610, 1267, 1164, 1112, 973,
877 cm−1; 1H- and 13C‑NMR spectroscopic data, see l " Table 1

and Table 2; HR‑ESI‑MS (positive): m/z = 389.1957 [M + Na]+

(calcd. for C20H30O6Na: 389.1940).
Blumeaene F (6): Yellow oil; [α]20 D : − 101 (c 0.065, CH3OH); IR

Fig. 1 The chemical structures of compounds 1–10. (KBr): vmax = 3464, 2937, 2866, 2342, 1724, 1641, 1370, 1224,
1135, 1059, 972, 851 cm−1; 1H- and 13C‑NMR spectroscopic data,
see l" Table 1 and Table 2; HR‑ESI‑MS (positive): m/z = 389.1959

[M + Na]+ (calcd. for C20H30O6Na: 389.1940).

Extraction and isolation Blumeaene G (7): Yellow oil; [α]20 D : + 19 (c 0.190, CH3OH); IR

The dried aerial parts of B. balsamifera (6.5 kg) were extracted at (KBr): vmax = 3479, 2958, 2876, 2358, 1713, 1654, 1378, 1229,
room temperature with 95 % EtOH under reflux three times. After 1149, 1075, 984, 850 cm−1; 1H- and 13C‑NMR spectroscopic data,
removal of solvent, the extract (335.0 g) was suspended in water see l" Table 1 and Table 2; HR‑ESI‑MS (positive): m/z = 373.2012

(2.0 L) and then partitioned with petroleum ether (60–90 °C), [M + Na]+ (calcd. for C20H30O5Na: 373.1990).
CH2Cl2, EtOAc, and n-BuOH (each 5 × 2.0 L), respectively. After Blumeaene H (8): Yellow oil; [α]20 D : + 43 (c 0.145, CH3OH); IR

evaporation of the solvent, the CH2Cl2 extract (90.0 g) was sub- (KBr): vmax = 3404, 2960, 2881, 2510, 1741, 1735, 1662, 1471,
jected to column chromatography over silica gel (1.0 kg, 100– 1339, 1278, 1162, 1061, 942, 852 cm−1; 1H- and 13C‑NMR spectro-
200 mesh, 10 × 70 cm) eluted using a gradient of CH2Cl2-MeOH scopic data, see l" Table 3; HR‑ESI‑MS (negative): m/z = 337.2082

(50 : 1–1 : 1, each 15 L) to obtain fractions 1–13. Fr. 1 (7.4 g) was [M – H]− (calcd. for C19H29O5: 337.2015).
separated over silica gel (150 g, 200–300 mesh, 6 × 40 cm) with Blumeaene I (9): Yellow oil; [α]20
D : + 21 (c 0.170, CH3OH); IR (KBr):

petroleum ether-EtOAc (20 : 1) and preparative HPLC vmax = 3396, 2961, 2880, 2508, 1736, 1731, 1649, 1451, 1330,
(MeOH‑H2O, 60 : 40, flow rate: 8 mL/min) to yield compounds 5 1268, 1157, 1049, 942, 843 cm−1; 1H- and 13C‑NMR spectroscopic
(35 mg, tR: 15 min) and 6 (11 mg, tR: 10 min). Fr. 3 (2.0 g) was data, see l " Table 3; HR‑ESI‑MS (positive): m/z = 389.1973 [M +

separated over preparative HPLC (MeOH‑H2O, 50 : 50, flow rate: Na]+ (calcd. for C20H30O6Na: 389.1940).
8 mL/min) to afford 10 (18 mg, tR: 30 min). Fr. 5 (2.0 g) was sub- Blumeaene J (10): Yellow oil; [α]20 D : + 38 (c 0.065, CH3OH); IR

jected to preparative HPLC (MeOH‑H2O, 50 : 50, flow rate: 8 mL/ (KBr): vmax = 3444, 2959, 2877, 2359, 1681, 1621, 1457, 1374,
min) to yield 9 (28 mg, tR: 20 min). Fr. 6 (1.6 g) was separated by 1160, 1078, 999 cm−1; 1H- and 13C‑NMR spectroscopic data, see

Chen M et al. Blumeaenes A–J, Sesquiterpenoid … Planta Med 2010; 76: 897–902
 Original Papers 899

1
Table 1 H‑NMR spectroscopic data of compounds 1–7 (in CD3OD)a,b.

Pos. 1 2 3 4 5 6 7
2 2.63 m; 1.79 m 2.64 m; 1.76 m 2.64 m; 1.74 m 2.63 m; 1.76 m 2.61 m; 1.75 m 2.47 m; 1.68 m 2.02 m; 1.79 m
3 2.51 m; 2.40 m 2.48 m; 2.39 m 2.49 m; 2.39 m 2.50 m; 2.37 m 2.48 m; 2.39 m 2.45 m; 2.41 m 2.61 m; 2.28 m
5 3.79 s
7 2.95 dt 2.93 dt 2.92 dt 2.92 m 2.91 m 2.50 m 2.37 dt
(11.5, 4.0) (11.0, 4.0) (12.0, 4.0) (12.5, 3.5)
8 (a) 1.88 m, (a) 1.85 m, (a) 1.81 m, (a) 1.87 m, (a) 1.75 m, (a) 2.58 m, (a) 1.97 m,
(b) 1.42 m (b) 1.39 m (b) 1.37 m (b) 1.37 m (b) 1.44 m (b) 1.50 m (b) 1.69 m
9 5.52 dd 5.46 dd 5.42 dd 5.47 dd 5.53 dd 4.90 dd 5.19 dd
(11.5, 3.5) (11.0, 3.0) (12.0, 3.0) (12.0, 3.5) (11.8, 3.4) (10.0, 2.0) (11.0, 1.5)
11 2.25 m 2.24 m 2.26 m 2.27 m 2.25 m 2.49 m 2.32 m
12 0.95 d (7.0) 0.94 d (7.0) 0.93 d (6.0) 0.95 d (7.0) 0.92 d (7.0) 0.91 d (7.0) 0.95 d (7.0)
13 0.84 d (7.0) 0.84 d (7.0) 0.84 d (6.0) 0.84 d (7.0) 0.82 d (7.0) 0.84 d (7.0) 0.84 d (7.0)
14 1.13 s 1.10 s 1.10 s 1.11 s 1.11 s 1.41 s 1.43 s
15 2.08 s 2.07 s 2.07 s 2.07 s 2.07 s 2.00 s 5.17 brs; 4.71 brs
17 5.76 s 2.60 m 5.74 s 5.78 brs
18 6.11 brq (7.0) 1.19 d (3.0) 3.11 q (6.0) 3.10 q (5.5)
19 2.00 brd (7.0) 2.18 s 1.18 d (3.0) 2.25 m; 2.22 m 1.31 d (6.0) 1.30 d (5.5) 2.18 s

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20 1.92 brs 1.92 s 1.08 m 1.56 s 1.56 s 1.95 s
21 2.17 s
a1
H‑NMR recorded at 500 MHz; b Chemical shifts in ppm; J values (Hz) in parentheses

Position 1 2 3 4 5 6 7 Table 2 13C‑NMR spectroscopic

1 91.3 91.2 91.2 91.5 91.5 90.7 87.8 data of compounds 1–7 (in
2 36.6 36.6 36.6 36.9 36.8 37.6 36.3 CD3OD)a, b.
3 37.7 37.7 37.7 38.0 37.9 38.3 32.1
4 161.0 160.9 160.9 161.1 161.4 162.1 149.6
5 139.2 139.2 139.2 139.5 139.3 140.9 61.4
6 204.0 204.1 203.9 204.3 203.8 206.2 212.5
7 52.9 52.9 52.8 53.2 53.0 57.1 56.4
8 29.3 29.4 29.1 29.7 29.5 27.0 28.3
9 79.3 78.5 79.1 78.9 81.0 82.9 78.3
10 79.3 79.3 79.3 79.6 79.5 79.2 78.9
11 28.0 28.0 27.9 28.3 28.1 30.4 33.1
12 21.2 21.2 21.2 21.5 21.4 21.2 21.2
13 18.5 18.5 18.5 18.8 18.7 18.9 18.8
14 16.6 16.5 16.3 16.8 16.5 19.6 17.4
15 17.8 17.7 17.8 18.0 18.0 18.2 111.4
16 169.2 167.9 178.5 168.5 171.6 171.4 168.3
17 129.7 117.4 35.5 116.2 61.6 61.3 117.6
18 138.6 158.0 19.3 163.3 61.3 61.1 158.5
19 16.1 20.4 19.4 34.9 14.1 13.8 20.7
20 20.8 27.4 12.7 19.6 19.3 27.7
21 19.3
a 13
C‑NMR recorded at 125 MHz; b Chemical shifts in ppm

l" Table 3; HR‑ESI‑MS (positive): m/z = 407.2047 [M + Na]+ (calcd. phosphoric acid solution. The absorbances at 570 nm were read
for C20H32O7Na: 407.2046). using a microtiter plate reader [14].

Determination of NO production
RAW264.7 macrophages (obtained from Shanghai Institute of
Life Science Cell Resource Center) were seeded in 96-well plates
(1 × 105 cells/well). The cells were co-incubated with tested com-
pounds and LPS (1 µg/mL) (LPS, Escherichia coli Serotype 055:B5;
Sigma) for 24 h. Aminoguanidine (Sigma; purity: > 98 %) was used
as a positive control. The amount of NO was assessed by deter-
mining the nitrite concentration in the cultured RAW264.7 mac-
rophage supernatants with Griess reagent. Aliquots of superna-
tants (100 µL) were incubated, in sequence, with 50 µL of 1% sul-
fanilamide and 50 µL of 0.1 % naphthylethylenediamine 2.5 %
Results and Discussion
!
The dried aerial parts of B. balsamifera were extracted with 95 %
EtOH under reflux three times. After removal of the solvent, the
extract was suspended in water and then partitioned with petro-
leum ether, CH2Cl2, EtOAc and n-BuOH successively. Separation
by repeated column chromatography (including SiO2 and Sepha-
dex LH-20) and preparative reverse-phase HPLC afforded ten
new sesquiterpenoid esters, blumeaenes A–J (1–10) from the
CH2Cl2 fraction, as well as 13 known flavonoids, 3,5,3′,4′-tetrahy-

Chen M et al. Blumeaenes A–J, Sesquiterpenoid … Planta Med 2010; 76: 897–902
900 Original Papers

1
Table 3 H- and 13C‑NMR spectroscopic data of compounds 8–10 (in CD3OD)a,b.

Position 8 9 10
δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz)
1 91.3 92.6 92.9
2 37.3 2.02 m; 1.73 m 38.8 2.04 m; 1.76 m 38.9 2.00 m; 1.73 m
3 32.8 2.53 m; 2.41 m 34.4 2.56 m; 2.40 m 34.4 2.50 m; 2.37 m
4 149.7 151.0 151.1
5 57.2 3.04 s 58.7 3.02 s 58.9 3.02 s
6 103.6 105.1 105.3
7 49.9 1.86 m 51.5 1.86 m 51.4 1.85 m
8 25.8 (a) 2.03 m, (b) 1.87 m 27.3 (a) 2.08 m, (b) 1.88 m 27.1 (a) 2.12 m, (b) 1.83 m
9 77.0 4.75 dd (11.0, 3.0) 79.9 4.80 dd (10.0, 5.0) 79.7 4.74 dd (10.5, 4.0)
10 79.6 80.9 81.1
11 27.6 1.92 m 29.1 1.91 m 29.1 1.90 m
12 22.0 1.06 d (6.5) 23.5 1.04 d (7.0) 23.5 1.02 d (6.5)
13 17.9 0.97 d (6.5) 19.4 0.93 d (7.0) 19.4 0.93 d (6.5)
14 17.0 1.18 s 18.6 1.17 s 18.8 1.20 s
15 108.8 5.21 brs; 4.89 brs 110.3 5.18 brs; 4.87 brs 110.6 5.19 brs; 4.89 brs
16 177.1 171.3 176.7

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17 33.8 2.61 m 61.3 79.7
18 17.8 1.20 d (2.5) 61.2 3.12 q (5.4) 72.9 3.90 q (6.5)
19 17.8 1.20 d (2.5) 13.8 1.17 d (5.4) 17.0 1.18 d (6.5)
20 19.3 1.54 s 22.7 1.33 s

H- and 13C‑NMR recorded at 500 MHz and 125 MHz, respectively; b Chemical shifts in ppm; J values (Hz) in parentheses
a1

droxy-7-methoxyflavone [15], 5,7-dihydroxy-3,3′,4′-trimethoxy-

Fig. 2 Key 1H-1H COSY
flavone [16], davidigenin [17], ayanin [18], davidioside [19], cate-
(−) and HMBC (H to C)
chin [20], dihydroquercetin 7,4′-dimethyl ether [21], blumeatin (→) correlations of blu-
[21], dihydroquercetin 4′-methyl ether [21], 3,5,3′-trihydroxy- meaene A (1).
7,4′-dimethoxyflavone [15], 5,7,3′,5′-tetrahydroxyflavanone
[21], luteolin [22], and quercetin [21], from the EtOAc fration.
The known flavonoids were determined by detailed NMR spec-
troscopic analysis and comparison with the literature data. The
structures of new sesquiterpenoid esters were elucidated mainly
by NMR spectroscopy, including 1D and 2D NMR experiments
(1H-1H COSY, HMQC, HMBC, NOESY), in combination with mass
spectrometry (MS).
Blumeaene A (1) was obtained as a yellow oil. The molecular for-
mula was determined as C20H30O5 by HR‑ESI‑MS ([M – H + Cl]− at δC = 14.2 and 11.9) [23]. The HMBC correlation between H-9 with
m/z = 385.1792). The 1H‑NMR spectroscopic data of 1 (l " Table 1) C-16 suggested that this angelic acid moiety was attached to the
showed a tertiary methyl (δH = 1.13, 3H, s), two secondary meth- C-9 position through an ester linkage. Therefore, the structure of
yls (δH = 0.85 and 0.95, each 3H, d, J = 7.0 Hz), three olefinic meth- 1 was as depicted in l" Fig. 2 and named as blumeaene A.

yls (δH = 2.08, 3H, s; δH = 2.00, 3H, br d, J = 7.0 Hz; δH = 1.92, 3H, br
s), and a vinylic proton (δH = 6.11, 1H, br q, J = 7.0 Hz). The
13
C‑NMR spectroscopic data of 1 (l " Table 2) displayed 20 carbon
signals, and a DEPT experiment confirmed the presence of one
Blumeaene B (2) was obtained as a yellow oil. The molecular for-
mula was determined as C20H30O5 by HR‑ESI‑MS ([M + Na]+ at m/
z = 373.2007). The assignments of the 1H- and 13C‑NMR data of 2
(l" Table 1 and Table 2), determined on the basis of extensive

carbonyl, one ester carbonyl, four olefinic carbons, two oxygen-
ated quaternary carbons, an oxymethine, two unoxygenated
methines, three methylenes, and six methyls. The connectivities
from H-2-H-3, H-7-H-8-H-9, and H-7-H-11-H-12/H-13 were es-
tablished from the 1H-1H COSY spectrum. The important HMBC
correlations of H-2/C-1, C-3, C-4, C-5; H-3/C-1, C-4, C-5; H‑7/C-5,
C-6, C-8, C-11; H-9/C-1, C-8, C-10 were also observed (l " Fig. 1).
This information led to a guaiane sesquiterpene skeleton for 1
which was similar to the compound isolated from the B. balsami-
2D NMR experiments, were in good agreement with those of 1,
except for the side chain (C-16 to C-20). Analysis of the 2D NMR
data (HMBC correlations of H3-19/C‑18; H3-20/C‑18; H‑17/C-16,
C-19, and C-20) finally revealed that a senecioyl moiety was con-
nected at the C-9 position (HMBC correlation of H‑9/C-16).
Blumeaene C (3) was obtained as a yellow oil. The molecular for-
mula was determined as C19H30O5 by HR‑ESI‑MS ([M + Na]+ at m/
z = 361.1990). The assignments of the 1H- and 13C‑NMR data of 3
(l" Table 1 and Table 2), determined on the basis of extensive

fera [3]. The 1H-1H COSY (H-18/H3-19) and the HMBC correla-
tions (H-18/C-19, C-20; H3-19/C-17 and C-18; H3-20/C-16, C-17
and C-18) also suggested the presence of a 2-methylbutenoic ac-
id. The 13C‑NMR chemical shift data of two vinylic methyl carbons
at δC = 20.8 and 16.1 suggested the geometry of this unsaturated
ester was Z form (angelic acid, δC = 20.8 and 16.0; tiglic acid,
2D NMR experiments, were in good agreement with those of 1,
except for the side chain (C-16 to C-19). Analysis of the 2D‑NMR
data (1H-1H COSY correlation of H-17/H3-18, H3-19; HMBC corre-
lations of H‑17/C-16, C-18, and C-19; H3-18/C‑17; H3-19/C‑17) fi-
nally revealed that an isobutyryl moiety was attached to the ses-

Chen M et al. Blumeaenes A–J, Sesquiterpenoid … Planta Med 2010; 76: 897–902

quiterpene nucleus at the C-9 position (HMBC correlation from
H‑9/C-16) through an ester linkage.
Blumeaene D (4) was obtained as a yellow oil. The molecular for-
mula was determined as C21H32O5 by HR‑ESI‑MS ([M + Na]+ at m/
z = 387.2149). The assignments of the 1H- and 13C‑NMR data of 4
(l" Table 1 and Table 2), determined on the basis of extensive
2D NMR experiments, were in good agreement with those of 1,
except for the side chain (C-16 to C-21). Analysis of the 2D NMR
data (1H-1H COSY correlation of H-19/H3-20; HMBC correlations
of H‑17/C-16, C-18; H‑19/C-18, C-20; H3-20/C-18, C-19; H3-21/C-
18) finally revealed that a methylsenecioyloxy moiety was at-
tached to the sesquiterpene nucleus at the C-9 position (HMBC
correlation from H‑9/C-16).
Blumeaene E (5) was obtained as a yellow oil. The molecular for-
mula was determined to be C20H30O6 by HR‑ESI‑MS ([M + Na]+ at
m/z = 389.1957). The assignments of the 1H- and 13C‑NMR data of
5 (l" Table 1 and Table 2), determined on the basis of extensive
2D NMR experiments, were in good agreement with those of 1,
except for the side chain (C-16 to C-20). The relatively high-field
resonances of the two oxygenated C-17 and C-18 (δC = 61.6 and
61.3) implied the presence of an epoxide ring, and this epoxide
moiety was inferred to be adjacent to two methyls and the ester
carbonyl group observed from the HMBC experiment (H‑18/C-17,
C-19, C-20; H3-19/C-18; H3-20/C-16, C-17) to give a 2,3-epoxy-2-
methylbutyryl moiety. This moiety was attached to the C-9 posi-
tion through an ester linkage, which was confirmed by the HMBC
correlation from H-9 to C-16. The strong NOESY correlation be-
tween H3-19 and H3-20 indicated the two methyls of 2,3-epoxy-
2-methylbutyryl moiety were cis-configured.
Blumeaene F (6) was obtained as a white power. The molecular
formula was determined to be C20H30O6 by HR‑ESI‑MS ([M + Na]
+
at m/z = 389.1959). The assignments of the 1H- and 13C‑NMR da-
ta of 6 (l
" Table 1 and Table 2), determined on the basis of exten-
sive 2D NMR experiments, were very similar with those of 5, ex-
cept for the shifts of C-7, C-11, C-14, H-7 and H-9, which were be-
lieved to be due to the relative configuration.
The relative configurations of compounds 1–6 were established
by the NOESY correlations and carbon chemical shifts at C-14.
The NOESY experiments of compounds 1–6 showed correlations
for H-7/H-9 in 1–6, and H3-14/H-8a in 6. At the same time, the
chemical shifts of C-14 (δC = 16.3–16.8) in 1–5 were at a much
higher field than that (δC = 19.6) in 6, suggesting the hydroxy
group C-14 has a β-orientation in 1–5 and an α-orientation in 6,
respectively. From the above evidence, it suggested that H3-14
was α-orientated and H-7, H-9 and OH-10 had the same β-orien-
tation in 1–5. Furthermore, H3-14 and H-8a had the β-orientation
and H-7, H-9 and OH-10 had the same α-orientation in 6. Energy-
minimized structures of 1 and 6 by MM2 molecular modeling
with the selected NOESY correlations are shown in l " Fig. 3.
Blumeaene G (7) was obtained as a yellow oil. The molecular for-
mula of C20H30O5 was determined by HR‑ESI‑MS ([M + Na]+ at m/
z = 373.2012). The 1D and 2D NMR data of 7 (l " Table 1 and Table
2) were in good agreement with those of 2, except for the chem-
ical shifts of C-4, C-5 and C-15. Analysis of the 1H‑NMR data
(δH = 5.17 and 4.71, each 1H, br s) combined with 13C‑NMR and
the DEPT experiment (δC = 111.4 and 149.6) revealed an exo-
methylene group in 7. The NOESY experiment of 7 showed corre-
lations for H-5/H3-14 and H-7/H-9, suggesting that H-5 and H3-
14 had the α-orientation and H-7, H-9 and OH-10 β-oriented.
Therefore, the structure and the relative configuration of 7 were
determined.
Chen M et al. Blumeaenes A–J, Sesquiterpenoid …
Original Papers 901
Fig. 3 Energy-minimized structures of 1 and 6 with selected NOESY
(H↔H) correlations.
Blumeaene H (8) was obtained as a yellow oil. The molecular for-
mula was determined as C19H30O5 by HR‑ESI‑MS ([M – H]− at m/
Downloaded by: Nanyang Technological University NTU. Copyrighted material.
z = 337.2082). The 1D and 2D NMR data of 8 (l " Table 3) were in
good agreement with those of 7, except for the C-6 and the side
chain (C-16 to C-19). Analysis of the 2D‑NMR data (1H-1H COSY
correlation of H-17/H3-18, H3-19; HMBC correlations of H‑17/C-
16, C-18, and C-19; H3-18/C‑17; H3-19/C‑17) revealed that an iso-
butyryl moiety was attached to the C-9 position (HMBC correla-
tion of H-9/C-16) through an ester linkage. The 13C NMR spectro-
scopic data showed four oxygenated carbons, and C-6 was ace-
talic (δC = 103.6). Since two of the oxygens had already been ac-
counted for by the isobutyryl side chain, C-6 and C-10 were linked
through an oxygen bridge. The lowfield shift of C-6 at δC = 103.6
also supported this deduction. The NOESY correlations of H-5/
H-8a and H-11, H-7/H-9, and H-8a/H3-14 suggested that H-5
was α-oriented and both H-7 and H-9 had the same β-orienta-
tion. Thus, the structure of 8 was as depicted in l " Fig. 1 and
named as blumeaene H.
Blumeaenes I and J (9 and 10) were obtained as yellow oils, and
the molecular formulas were determined as C20H30O6 and
C20H32O7 by HR‑ESI‑MS, respectively. Both of the 1D and 2D NMR
data of 9 and 10 (l " Table 3) were in good agreement with those
of 8, except for the side chain (C-16 to C-20). Comparison of the
data of 9 with 5 (C-16 to C-20) revealed that a 2,3-dimethyloxir-
ane-2-carboxylic acid moiety was attached to the sesquiterpene
nucleus at the C-9 position (HMBC correlation of H‑9/C-16)
through an ester linkage. For compound 10, analyzed the
2D‑NMR data (1H-1H COSY correlation of H-18/H3-19; HMBC cor-
relations of H-18/C-17, C-19, and C-20; H3-19/C-17, C-18; H3-20/
C-16, C-17 and C-18), the side chain was elucidated and it was at-
tached to the sesquiterpene nucleus at the C-9 position (HMBC
correlation from H‑9/C-16).
All isolates were tested for inhibitory activities against LPS-in-
duced NO production in RAW264.7 macrophages [14]. Com-
pounds 1, 4 and 5 showed a slight inhibitory effect on the NO pro-
duction with IC50 values of 40.06, 46.35, and 57.80 µg/mL, respec-
tively (other sesquiterpenes: IC50 > 100 µg/mL), whereas amino-
guanidine was used as the positive control (IC50 = 1.55 µg/mL).
The cell viability measured by the MTT assay showed that these
three sesquiterpenes had no significant cytotoxicity to the
RAW264.7 cells at their effective concentration for the inhibition
of NO production [24].
Planta Med 2010; 76: 897–902
902 Original Papers

Acknowledgements
! plants from genus Blumea. Chem Biodivers 2009; 6: 809–817
This work was supported by program NCET Foundation, NSFC
(30725045), the Special Program for New Drug Innovation of the
Ministry of Science and Technology, China (2009ZX09311–001,
2008ZX09101-Z‑029, 2009ZX09103–375), Shanghai Leading
Academic Discipline Project (B906), and in part by the Scientific
Foundation of Shanghai China (07DZ19728, 09DZ1975700,
09DZ1971500, 09DZ1972200, 08DZ1971302).
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Chen M et al. Blumeaenes A–J, Sesquiterpenoid … Planta Med 2010; 76: 897–902