Cytotoxic germacrane-type sesquiterpenes, pimarane-type diterpenes, and a naphthalene derivative from Wollastonia biflora

Article abstract of DOI:10.1021/np060515p

Phytochemical investigation of the whole plants of Wollastonia biflora led to the isolation and identification of three new germacrane-type sesquiterpenes (1-3), two new pimarane-type diterpenes (4, 5), and a new naphthalene glycoside (6), along with 11 known compounds. Their structures were characterized on the basis of spectroscopic analyses and chemical methods. Compounds 1, 2, and 3 showed significant cytotoxic activity against me growth of hepatocellular carcinoma BEL-7402 cells in vitro.

Full text of DOI:10.1021/np060515p

J. Nat. Prod. 2007, 70, 567-570
567
Cytotoxic Germacrane-Type Sesquiterpenes, Pimarane-Type Diterpenes, and a Naphthalene
Derivative from Wollastonia biflora
Wenliang Chen, Weidong Tang, Rujun Zhang, Liguang Lou, and Weimin Zhao*
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 201203, People’s Republic of China
ReceiVed October 17, 2006
Phytochemical investigation of the whole plants of Wollastonia biflora led to the isolation and identification of three
new germacrane-type sesquiterpenes (1-3), two new pimarane-type diterpenes (4, 5), and a new naphthalene glycoside
(6), along with 11 known compounds. Their structures were characterized on the basis of spectroscopic analyses and
chemical methods. Compounds 1, 2, and 3 showed significant cytotoxic activity against the growth of hepatocellular
carcinoma BEL-7402 cells in Vitro.
The family Asteraceae, comprising about 1000 genera and over
5 000 species, ranks first among the dicotyledons and second
2
among the angiosperms. About 200 genera and 2000 species are
found in China. Many Asteraceae species have been used for
1
medicinal purposes since ancient times, and many bioactive
2
compounds with diverse skeletons have been identified. Wollas-
tonia biflora (L.) DC. [syn. Melanthera biflora (L.) Wild. or
Wedelia biflora (L.) DC.], an Asteraceae species (tribe Heliantheae)
widely distributed in the south of Asia and Oceania, has been used
as a folk medicine for the treatment of rheumatism, bone aches,
sore galls, and injuries in some provinces of China.3 Previous
,4
5
phytochemical studies of this species have yielded phenols,
6
6,7
steroids, and kaurane-type diterpenoids. In our continuing efforts
to find new bioactive compounds from plants used in Chinese
traditional and folk medicine,8 a systematic investigation into the
chemical constituents of W. biflora (L.) DC. was undertaken. As a
result, six new compounds (1-6), together with 11 known
,9
compounds, darutoside,1 spathulenol, darutigenol, 3,3′,4′,7′-
0,11
12
10
13
tetramethoxy-5-hyhroxyflavone, 3,7-dimethoxy-3′,4′,5-trihydroxy-
flavone,13 3-methoxy-3′,4′,5,7-tetrahydroxyflavone,14 pubeside B,
15
16
2
-carbomethoxy-3-prenyl-1,4-naphthohydroquinone diglucoside,
17
18
19
scandoside, 6-O-acetylscandoside, and asperuloside, have been
isolated from the whole plants of W. biflora (L.) DC. The new
compounds (1-3) showed significant cytoxicity against carcinoma
BEL-7402 cells.
Results and Discussion
The whole plants of W. biflora (L.) DC. collected in Wenshan
County of Yunnan Province and Jinxiu County of Guangxi
Province, People’s Republic of China, (each 2.5 kg), were
respectively percolated at room temperature with 95% ethanol three
times. After evaporation of ethanol in Vacuo and filtration of the
precipitated chlorophyll in 20% ethanol, the aqueous residue was
extracted with chloroform and n-butanol, successively, yielding
chloroform and n-butanol fractions, respectively. These fractions
were subjected to chromatography, including silica gel and Sepha-
dex LH-20 columns, HPLC, and PTLC to afford six new com-
pounds (1-6) and 11 known compounds.
Compound 1 was obtained as a colorless gum with the molecular
formula C20
H
28
O
6
as deduced by HRESIMS and NMR analyses. Its
H NMR spectrum revealed the existence of two terminal olefinic
protons (δ 6.25, 1H, s and δ 5.66, 1H, s), one doublet methyl
1.94, d, 7.2 Hz), and one singlet methyl at δ
1.78. The 13
1
H
H
and two for oxygenated methylenes), six methines (two for sp2
methines and two for oxygenated methines), and five quaternary
(δ
H
H
C
NMR spectrum displayed 20 signals separated by DEPT experi-
ments into two methyls, seven methylenes (one for sp methylene
2
2
carbons (two for carboxylate carbons and three for sp carbons).
Analysis of its HSQC and 1H- H COSY spectra led to the
deduction of the fragment C-3-C-2-C-1-C-10-(C-14)-C-9-
C-8-C-7-C-6-C-5. The NMR data suggested that 1 was similar
1
*
Corresponding author. Tel and Fax: +86-21-50806052. E-mail:
wmzhao@mail.shcnc.ac.cn.
1
0.1021/np060515p CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/10/2007

5
68 Journal of Natural Products, 2007, Vol. 70, No. 4
Chen et al.
Figure 1. H- H COSY ()) and key H- C long-range correlation signals ( H c 13C) in the HMBC spectra of 1 and 6.
1
1
1
13
1
in structure to orientin,20 a compound that differs from 1 only by
its acyl moiety and the position of the double bond. The planar
structure of 1 was confirmed by its HMBC spectrum (Figure 1). In
the NOESY spectrum of 1, NOE correlation signals were found at
H-3/H-15, H-15/H-5R, H-5R/H-6, H-5â/H-8, H-5â/H-2, H-7/H-8,
H-7/H-2, H-8/H-10, H-14/H-1R, H-14/H-9R, H-14/H-10, H-1R/H-
methyls. The 13C NMR spectrum of 6 exhibited 27 signals
constituted by 12 aromatic/olefinic signals (five for sp methines)
2
in the low-field region, 12 signals typical of sugar moieties, and
two methyls and one methylene in the high-field region. The NMR
data were similar to those of 2-carbomethoxy-3-prenyl-1,4-naph-
thohydroquinone diglucoside,16 a known compound isolated with
6, except that signals for the carbomethoxy group of the former
compound were missing in 6, which, in addition to the variance of
their molecular weights, suggested that 6 had the carbomethoxy
group displaced by -OH at C-2. The hypothesis was confirmed
by the HMBC spectrum (Figure 1). To determine its sugar moieties,
enzymatic and acid hydrolysis experiments were performed and
glucose was identified by co-TLC of the hydrolyzed samples with
authentic sugar samples. However, the “aglycons” were character-
3
, H-3′/H-4′, and H-3′/H-5′. Therefore, 1 was characterized as 8R-
angeloxy-14,15-dihydroxy-3(4),11(13)-germacradien-6,12-olide.
Compound 2 had the molecular formula C19 as deduced
24 6
H O
1
13
by HRESIMS and NMR analyses. Its H and C NMR spectra were
similar to those of 1, except for the emergence of an aldehyde group
(
δ
H
9.44, 1H, s; δ
protons (δ 5.98, 1H, s and δ
of the oxygenated methylene (δ
the olefinic hydrogen (δ 6.05, 1H, dd, J ) 1.5 and 7.2 Hz, H-3′),
and the doublet methyl (CH
C
194.3) and two additional terminal olefinic
5.52, 1H, s) and the disappearance
4.14, 2H, s; δ 67.6, CH -15),
H
H
H
C
2
2
1
H
ized as lapachol (due to the instability of its aglycon) and R- and
1
1
22
3
-5′). Analysis of the HSQC, H- H
â-lapachone (due to the instability of its chain under acidic
COSY, HMBC, and NOESY spectra of 2 enabled the establishment
of its structure. Therefore, compound 2 was characterized as 8R-
methylacryloxy-14-hydroxy-15-al-3(4),11(13)-germacradien-6,12-
olide.
conditions), respectively. The two glucose moieties were both
1
determined as â-D-configured on the basis of the H NMR and
optical rotation values. Thus, 6 was determined to be lapachol-
1,4-di-O-â-D-glucopyranoside.
The NMR spectra of 3 were nearly superimposable with those
Although the two collections of plant material from Wenshan
County of Yunnan Province and Jinxiu County of Guangxi Province
shared the same name and common appearance, there were
significant differences in the chemical constituents found in their
n-butanol extracts. The main components of the former were
darutoside and its derivatives, while the latter contained mainly
scandoside, 6-O-acetylscandoside, and asperuloside. However, their
chloroform extracts showed very similar patterns on the co-TLC
plates. New compounds 1-5 were isolated from the former
collection of plant material, while 6 was isolated from the latter
collection.
of 2, except for the emergence of an oxygenated methylene (δ
H
4
9
.19, s; δ
.44, s; δ
C
67.4) and the disappearance of the aldehyde group (δ
H
C
194.3, C-15) in 2. Meanwhile, the NMR spectra of 3
indicated that it differed from 1 only by its acyl moiety. Therefore,
compound 3 was characterized as 8R-methylacryloxy-14,15-dihy-
droxy-3(4),11(13)-germacradien-6,12-olide.
46 9
Compound 4 possessed the elemental composition C28H O as
1
13
determined by HRESIMS and NMR analyses. Its H and C NMR
spectra were nearly superimposable with those of darutoside except
for an additional acetyl moiety. The acetyl group was unambigu-
ously attached to 16-OH, which was concluded from the variances
of the chemical shifts of C-16 (+3.1 ppm) and C-15 (-3.4 ppm),
with the chemical shifts of other carbons almost unchanged in the
In preliminary pharmacological tests, all of the isolated com-
pounds were evaluated for inhibitory activity against the growth
of hepatocellular carcinoma BEL-7402 cells in Vitro. The three new
germacrane-type sesquiterpenes 1, 2, and 3 exhibited significant
inhibitory activity against the growth of hepatocellular carcinoma
BEL-7402 cells in Vitro, with IC50 values of 3.00 ( 0.84, 1.50 (
0.14, and 1.72 ( 0.24 µM, respectively. Germacrane-type sesquit-
erpenes having the R-methylene-δ-butanolide moiety have been
13
10,11
C NMR spectrum compared to those of darutoside.
compound 4 is 16-O-acetyldarutoside.
Therefore,
1
Compound 5 showed the same molecular formula as 4. Its H
and 13C NMR spectra were again similar to those of 4 and
darutoside. The C NMR spectrum showed chemical shifts of C-15
+2.1 ppm) and C-16 (-2.0 ppm) compared to darutoside,
13
10,11
23,24
(
reported previously as cytotoxic components.
which suggested attachment of the acetyl group to the -OH at C-15.
Therefore, compound 5 was determined to be 15-O-acetyldaruto-
side. The structures of 4 and 5 were confirmed by their HMBC
spectra and the following chemical transformation: acetylation of
the aglycones of 4, 5, and darutoside led to the same product, 3,-
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured with a Perkin-Elmer 241MC polarimeter or Perkin-Elmer 341
polarimeters. UV spectra were recorded with a Beckman DU-7
spectrometer. IR spectra were recorded using a Perkin-Elmer 577
spectrometer. LR-ESIMS were measured using a Finnigan LCQ-DECA
instrument, and HR-ESIMS data were obtained on a Mariner spec-
trometer. LR-EIMS were obtained on a MAT-95 spectrometer, and HR-
EIMS were obtained on a Kratos 1H spectrometer. NMR spectra were
run on a Bruker AM 400 or INOVA-600 spectrometer with TMS as
15,16-tri-O-acetyldarutigenol. The coexistence of 4 and 5, and their
similar content found in the plant, suggested that they may be
equilibrated by molecular transesterification.
Compound 6 was obtained as a pale yellow powder with the
1
molecular formula C27
H
36
O
13. Its H NMR spectrum displayed a
pattern typical of one ortho-substituted phenyl group and two singlet

Cytotoxic Sesquiterpenes from Wollastonia biflora
Journal of Natural Products, 2007, Vol. 70, No. 4 569
, 600 MHz) δ 1.15 (1H, m, H-1R), 2.00 (1H, m, H-1â),
internal standard. Preparative HPLC was carried out using a Varian
(in CDCl
3
H
SD-1 instrument, equipped with NW25 C18 column (10 µm, 20 mm ×
2.30 (2H, m, H-2), 5.64 (1H, dd, J ) 6.2 and 10.8 Hz, H-3), 2.58 (1H,
dd, J ) 5.3 and 13.5 Hz, H-5R), 2.70 (1H, dd, J ) 10.3 and 13.5 Hz,
H-5â), 5.38 (1H, m, H-6), 3.15 (1H, br s, H-7), 4.85 (1H, m, H-8),
2
50 mm, Merck) and a Prostar 320 UV/vis detector. Column chro-
matographic separations were carried out using a LiChroprep RP-18
1
.97 (1H, m, H-9R), 1.54 (1H, m, H-9â), 1.88 (1H, m, H-10), 5.66
Lobar column (40-63 µm, Merck) and using silica gel H60 (300-
and 6.25 (each 1H, s, H-13), 3.41 (2H, ddd, J ) 6.3, 10.4 and 16.9
4
00 mesh) (Qingdao Haiyang Chemical Group Corporation, Qingdao,
Hz, H-14), 4.14 (2H, s, H-15), 6.05 (1H, q, J ) 7.2 Hz, H-3′), 1.78
People’s Republic of China) and Sephadex LH-20 (Pharmcia Biotech
AB, Uppsala, Sweden) as packing materials. HSGF254 silica gel TLC
plates (Yantai Chemical Industrial Institute, Yantai, People’s Republic
of China) and RP-18 WF254 TLC plates (Merck) were used for analytical
TLC. â-Cellulase was manufactured by Lizhu Dongfeng BioTech Co.
Ltd., Shanghai, People’s Republic of China.
Plant Material. Whole plants of Wollastonia biflora (L.) DC. were
collected in Wenshan County of Yunnan Province and Jinxiu County
of Guangxi Province, People’s Republic of China (each 2.5 kg), in
May 2005. They were identified by Professor Jingui Shen of Shanghai
Institute of Materia Medica, Chinese Academy of Sciences. Voucher
specimens (No. 050015SIMM and No. 050016SIMM, respectively) are
deposited in the herbarium of Shanghai Institute of Materia Medica.
Extraction and Isolation. Powdered air-dried whole plants of W.
biflora collected in Wenshan County of Yunnan Province, People’s
Republic of China (2.5 kg) were percolated at room temperature with
1
3
(
1
3
3H, s, H-4′), 1.94 (3H, d, J ) 7.2 Hz, H-5′); C NMR (in CDCl ,
C
00 MHz) δ 30.2 (t, C-1), 26.5 (t, C-2), 131.4 (d, C-3), 133.3 (s,
C-4), 30.9 (t, C-5), 76.2 (d, C-6), 47.7 (d, C-7), 79.2 (d, C-8), 40.4 (t,
C-9), 42.2 (d, C-10), 136.8 (s, C-11), 169.8 (s, C-12), 123.7 (t, C-13),
6
7.8 (t, C-14), 67.6 (t, C-15), 167.0 (s, C-1′), 127.6 (s, C-2′), 139.8 (d,
C-3′), 20.3 (q, C-4′), 15.7 (q, C-5′); HRESIMS m/z 387.1790 [M +
+
28 6
Na] (calcd for C20H O Na, 387.1784).
23
Compound 2: colorless gum; [R]
D
-129.0 (c 0.23, DMSO); UV
(
1
methanol) λ max 220 nm; IR (KBr) νmax 3435, 2928, 1759, 1720, 1682,
-1
1
637, 1454, 1406, 1275, 1163, 1014, 818, 731 cm ; H NMR (in
, 600 MHz) δ 1.38 (1H, m, H-1R) 2.19 (1H, m, H-1â), 2.64
2H, m, H-2), 6.70 (1H, t, J ) 8.1 Hz, H-3), 2.64 (1H, dd, J ) 5.1 and
CDCl
3
H
(
1
3.6 Hz, H-5R), 2.94 (1H, dd, J ) 10.5 and 13.6 Hz, H-5â), 5.50 (1H,
m, H-6), 2.78 (1H, br s, H-7), 4.75 (1H, m, H-8), 1.96 (1H, m, H-9R),
1
.42 (1H, m, H-9â), 1.94 (1H, m, H-10), 5.65 and 6.24 (each 1H, s,
H-13), 3.48 (2H, ddd, J ) 6.5, 10.7 and 16.6 Hz, H-14), 9.44 (1H. s,
9
5% ethanol (15 L × 3). The filtrate was concentrated to dryness in
13
H-15), 5.52 and 5.98 (each 1H, s, H-3′), 1.80 (3H, s, H-4′); C NMR
Vacuo and then suspended in 20% ethanol overnight. After filtration
of the precipitated chlorophyll and evaporation of ethanol in the filtrate,
the aqueous residue (1 L) was extracted with chloroform and n-butanol
(
1
4
in CDCl
3
, 100 MHz) δ
38.7 (s, C-4), 27.3 (t, C-5), 74.9 (d, C-6), 48.5 (d, C-7), 79.0 (d, C-8),
0.4 (t, C-9), 41.9 (d, C-10), 135.9 (s, C-11), 169.4 (s, C-12), 124.3 (t,
C
30.1 (t, C-1), 27.8 (t, C-2), 157.1 (d, C-3),
(
500 mL × 3 each), successively, yielding chloroform (5 g) and
C-13), 67.5 (t, C-14), 194.3 (d, C-15), 166.0 (s, C-1′), 135.6 (s, C-2′),
n-butanol fractions (40 g), respectively. The chloroform fraction was
chromatographed on silica gel eluted with a gradient of petroleum-
acetone (6:1 to 0:1) to yield fractions C1 (2.1 g), C2 (130 mg), C3
+
1
(
26.4 (t, C-3′), 18.1 (q, C-4′); HRESIMS m/z 371.1494 [M + Na]
calcd for C19 Na, 371.1471).
Compound 3: colorless gum; [R]
methanol) λmax 220 nm; IR (KBr) νmax 3398, 2928, 2872, 1757, 1716,
26 6
H O
23
D
-94.0 (c 0.26, DMSO); UV
(1.2 g), C4 (302 mg), and C5 (1.1 g). Fraction C2 was subjected to a
(
1
6
silica gel column eluted with petroleum-acetone (20:1) to afford
spathulenol (18.6 mg) and darutigenol (25.3 mg). Fraction C3 was
separated by HPLC eluted with a methanol-water gradient (40% to
-1 1
664, 1456, 1387, 1296, 1163, 1013, 820 cm ; H NMR (in CDCl
00 MHz) δ
3
,
H
1.20 (1H, m, H-1R), 2.06 (1H, m, H-1â), 2.36 (2H, m,
H-2), 5.74 (1H, dd, J ) 5.4 and 10.7 Hz, H-3), 2.66 (1H, dd, J ) 4.9
and 13.7 Hz, H-5R), 2.79 (1H, dd, J ) 10.7 and 13.7 Hz, H-5â), 5.39
1
00% methanol) to give fractions C3A (540 mg), C3B (240 mg), C3C
(50 mg), and C3D (320 mg). Fraction C3B was subjected to a silica
(
1H, m, H-6), 3.22 (1H, br s, H-7), 5.00 (1H, m, H-8), 2.06 (1H, m,
gel column eluted with chloroform-methanol (30:1), affording 3 (83.3
mg) and 2 (18.7 mg). Fraction C3C was purified by a silica gel column
eluted with chloroform-methanol (30:1) to give 1 (15.1 mg). Fraction
C4 was separated by a silica gel column eluted with chloroform-
methanol (20:1) to afford fractions C4A (130 mg), C4B (65 mg), and
C4C (80 mg), which were further purified by a Sephadex LH20 column
eluted with 95% ethanol to yield 3,3′,4′,7′-tetramethoxy-5-hyhroxy-
flavone (10.1 mg), 3,7-dimethoxy-3′,4′,5-trihydroxyflavone (7.5 mg),
and 3-methoxy-3′,4′,5,7-tetrahydroxyflavone (8.4 mg), respectively. The
n-butanol fraction was separated by a silica gel column eluted with
chloroform-methanol (10:1 to 1:1), affording fractions B1 (708 mg),
B2 (5.4 g), and B3 (32 g). Fraction B1 was subjected to HPLC eluted
with methanol-water (30% to 100% methanol) to give fractions B1A
H-9R), 1.60 (1H, m, H-9â), 1.95 (1H, m, H-10), 5.99 and 6.34 (each
H, s, H-13), 3.44 (1H, dd, J ) 6.4 and 10.3 Hz, H-14a), 3.51 (1H,
dd, J ) 6.0 and 10.3 Hz, H-14b), 4.19 (2H, s, H-15), 5.26 and 6.10
1
13
(each 1H, s, H-3′), 1.93 (3H, s, C-4′); C NMR (in CDCl
3
, 100 MHz)
δ
C
30.1 (t, C-1), 26.4 (t, C-2), 131.3 (d, C-3), 133.2 (s, C-4), 30.7 (t,
C-5), 77.4 (d, C-6), 47.6 (d, C-7), 79.2 (d, C-8), 40.3 (t, C-9), 41.9 (d,
C-10), 136.8 (s, C-11), 170.0 (s, C-12), 123.9 (t, C-13), 67.6 (t, C-14),
6
7.4 (t, C-15), 166.7 (s, C-1′), 135.5 (s, C-2′), 126.8 (t, C-3′), 18.0 (q,
+
C-4′); HRESIMS m/z 373.1614 [M + Na] (calcd for C19
3
H O
26 6
Na,
73.1627).
Compound 4: colorless gum; [R]25
D
-23.6 (c 0.23, MeOH); IR
(KBr) νmax 3396, 2941, 2877, 1720, 1647, 1456, 1369, 1259, 1086,
-1 13
1
2
3
041, 883 cm
;
C NMR (in CD
3 C
OD, 100 MHz) δ 38.5 (t, C-1),
(360 mg), B1B (102 mg), and B1C (300 mg). Fraction B1B was
4.8 (t, C-2), 86.4 (d, C-3), 39.8 (s, C-4), 56.6 (d, C-5), 23.9 (t, C-6),
7.6 (t, C-7), 140.9 (s, C-8), 52.4 (d, C-9), 39.4 (s, C-10), 19.8 (t,
separated by PTLC (developed with chloroform-methanol 7:1) to
afford 4 (30 mg) and 5 (40 mg). Fraction B2 was subjected to HPLC
eluted with methanol-water (20% to 100% methanol within 60 min)
to give fraction B2A (2.2 g), darutoside (2.5 g), and pubeside B (50
mg).
C-11), 33.6 (t, C-12), 39.0 (s, C-13), 129.5 (d, C-14), 74.6 (d, C-15),
7.9 (t, C-16), 23.3 (q, C-17), 29.7 (q, C-18), 15.7 (q, C-19), 17.8 (q,
C-20), 102.3 (d, C-1′), 75.5 (d, C-2′), 78.1 (d, C-3′), 72.3 (d, C-4′),
8.6 (d, C-5′), 63.4 (t, C-6′), 21.4 (q, 16-OCOCH ), 173.5 (s,
6
7
3
+
Powdered air-dried whole plants of W. biflora collected in Jinxiu
County of Guangxi Province, People’s Republic of China (2.5 kg), were
extracted using the same procedure as mentioned above to give
chloroform (3 g) and n-butanol fractions (34 g). The n-butanol fraction
was separated by a silica gel column eluted with chloroform-methanol
16-OCOCH ); HRESIMS m/z 549.3052 [M + Na] (calcd for C H O -
Na, 549.3040).
3
28 46
9
Compound 5: colorless gum; [R]23 -28.0 (c 0.07, DMSO); IR
D
(KBr) ν
max
3415, 2941, 2874, 1720, 1647, 1454, 1371, 1238, 1078,
1027 cm-1; C NMR (in CD OD, 100 MHz) δ 38.5 (t, C-1), 24.9 (t,
13
3
C
(10:1 to 1:1), affording fractions B′1 (1.2 g), B′2 (2.5 g), B′3 (4.4 g),
C-2), 86.6 (d, C-3), 39.9 (s, C-4), 56.6 (d, C-5), 24.2 (t, C-6), 37.6 (t,
C-7), 141.8 (s, C-8), 52.4 (d, C-9), 39.6 (s, C-10), 19.9 (t, C-11), 34.1
(t, C-12), 38.5 (s, C-13), 128.5 (d, C-14), 80.1 (d, C-15), 62.8 (t, C-16),
24.2 (q, C-17), 29.7 (q, C-18), 15.8 (q, C-19), 17.8 (q, C-20), 102.4
(d, C-1′), 75.6 (d, C-2′), 78.2 (d, C-3′), 72.4 (d, C-4′), 78.7 (d, C-5′),
63.5 (t, C-6′), 21.6 (q, 15-OCOCH ), 173.5 (s, 15-OCOCH ); HRESIMS
and B′4 (25 g). Fraction B′2 was subjected to HPLC eluted with
methanol-water (20% to 100% methanol) to give fractions B′2A (1.8
g), B′2B (407 mg), and B′2C (250 mg). Fraction B′2B was separated
by PTLC (developed with chloroform-methanol-water, 7:3:0.1) to
afford 6 (230 mg) and 2-carbomethoxy-3-prenyl-1,4-naphthohydro-
quinone diglucoside (150 mg). A portion of B′3 (500 mg) was separated
by PTLC to give scandoside (30 mg) and 6-O-acetylscandoside (17
mg) (developed with chloroform-methanol-water 3:2:0.1) and aspe-
ruloside (14 mg) (developed with chloroform-methanol-water, 7:3:
3
3
+
m/z 549.3041 [M + Na] (calcd for C H O Na, 549.3040).
28
46
9
Compound 6: pale yellow powder; [R]23 -1.0 (c 0.08, MeOH);
D
UV (methanol) λ max 235 nm, 285 nm, 295 nm, 333 nm; IR (KBr) ν
max
3415, 2918, 1630, 1605, 1452, 1402, 1365, 1068, 1026, 771 cm ; 1H
-1
0
.1), respectively.
NMR (in DMSO-d , 300 MHz) δ 8.40 (1H, d, J ) 8.3 Hz, H-5), 7.26
6
H
Compound 1: colorless gum; [R]25
(1H, t, J ) 8.3 Hz, H-6), 7.36 (1H, t, J ) 8.3 Hz, H-7), 8.30 (1H, d,
J ) 8.3 Hz, H-8), 3.90 (1H, dd, J ) 8.0 and 14.1 Hz, H-9a), 3.30 (1H,
m, H-9b), 5.20 (1H, dd, J ) 7.6 and 6.5 Hz, H-10), 1.59 (3H, s, H-12),
D
3
-84.8 (c 0.11, CHCl ); UV
(methanol) λ max 220 nm; IR (KBr) νmax 3419, 2928, 2872, 1755, 1716,
1
647, 1456, 1385, 1269, 1229, 1151, 1043, 997, 754 cm-1; 1H NMR

5
70 Journal of Natural Products, 2007, Vol. 70, No. 4
Chen et al.
1
3
3
.73 (3H, s, H-13), 4.53 (1H, d, J ) 7.8 Hz, H-1′), 3.43 (1H, m, H-2′),
.29 (1H, m, H-3′), 3.26 (1H, m, H-4′), 3.20 (1H, m, H-5′), 3.65 and
.52 (each 1H, m, H-6′), 5.69 (1H, d, J ) 7.6 Hz, H-1′′), 3.43 (1H, m,
References and Notes
(1) Chinese Traditional Medicinal Materials Company. Records of
Traditional Chinese Medicine Resources; Science Press: Beijing,
H-2′′), 3.25 (1H, m, H-3′′), 3.15 (1H, m, H-4′′), 2.95 (1H, m, H-5′′),
1
994; pp 1238-1357.
2) Xu, R. S. Chemistry of the Natural Products; Science Press: Beijing,
997; pp 252-259.
3) Wu, D. L.; Hu, Q. M.; Xia, N. H. Index of the Plants in Hong Kong;
Guangdong Dongsheng Press: Guangdong, 2001; p 229.
4) Guangxi Institute of Traditional Chinese Medicines. Index of the
Medicinal Plants in Guangxi; Guangxi People Press: Guangxi, 1986;
p 437.
1
3
3
δ
(
1
(
6
.61 and 3.41 (each 1H, m, H-6′′); C NMR (in DMSO-d
135.4 (s, C-1), 145.3 (s, C-2), 124.7 (s, C-3), 147.1 (s, C-4), 122.2
s, C-4a), 122.5 (d, C-5), 123.3 (d, C-6), 125.8 (d, C-7), 121.8 (d, C-8),
27.9 (s, C-8a), 24.3 (t, C-9), 123.4 (d, C-10), 130.4 (s, C-11), 25.7
q, C-12), 18.2 (q, C-13), 107.0 (d, C-1′), 72.4 (d, C-2′), 76.1 (d, C-3′),
9.6 (d, C-4′), 77.3 (1H, C-5′), 60.7 (t, C-6′), 105.0 (d, C-1′′), 74.3 (d,
C-2′′), 76.5 (d, C-3′′), 70.0 (d, C-4′′), 77.1 (d, C-5′′), 61.2 (t, C-6′′);
6
, 100 MHz)
(
(
(
C
1
+
HRESIMS m/z 591.2051 [M + Na] (calcd for C27
36
H O13Na, 591.2054).
(
(
(
(
(
5) Miles, D. H.; Chittawong, V.; Hedin, P. A.; Kokpol, U. Phytochem-
istry 1993, 32, 1427-1429.
6) Macleod, J. K.; Gaul, K. L.; Oelrichs, P. B. Austr. J. Chem. 1990,
Acidic Hydrolysis of 6. Compound 6 (100 mg) dissolved in 50%
MeOH (10 mL) containing 7% HCl was heated in boiling water for 2
h. After cooling, the reaction mixture was neutralized and extracted
with chloroform. Glucose was identified as the sugar moiety by co-
TLC of the aqueous solution compared with an authentic glucose
sample. The organic residue was subjected to PTLC (developed with
chloroform-acetone, 20:1) to afford R-lapachone (16.7 mg) and
â-lapachone (22.3 mg). The aqueous layer was subjected to HPLC
4
3, 1533-1539.
7) Miles, D. H.; Chittawong, V.; Payne, A. M. J. Agric. Food Chem.
990, 38, 1591-1594.
1
8) Zou, J.; Jin, D. Z.; Chen, W. L.; Wang, J.; Liu, Q. F.; Zhu, X. Z.;
Zhao, W. M. J. Nat. Prod. 2005, 68, 1514-1518.
9) Chen, W. L.; Liu, Q. F.; Wang, J.; Zou, J.; Meng, D. H.; Zuo, J. P.;
Zhu, X. Z.; Zhao, W. M. Planta Med. 2006, 72, 143-150.
2
1
eluted with water to afford glucose {36 mg, [R]
D
2
+50 (c 0.2, H O)}.
Enzymatic Hydrolysis of Compounds 4, 5, Darutoside, and 6.
Compounds 4, 5, darutoside, and 6 (2 mg for both 4 and 5, 100 mg for
both darutoside and 6) and â-cellulase (2 mg for both 4 and 5, 100 mg
(
(
(
(
(
10) Kim, J. H.; Han, K. D.; Yamasaki, K.; Tanaka, O. Phytochemistry
1
979, 18, 894-895.
11) Wenkert, E.; Ceccherelli, P.; Raju, M. S.; Polonsky, J.; Tingoli, M.
J. Org. Chem. 1979, 44, 146-148.
for both darutoside and 6) were dissolved in H
4
days. The aqueous solutions were then extracted with chloroform, and
the aglycons of 4, 5, darutoside, and lapachol (0.9, 1.1, 34.3, and 25.2
mg, respectively) were obtained from the organic layer.
2
O (2 mL for 4 and 5,
0 mL for darutoside and 6), respectively, and kept at 37 °C for 2
12) Ulubelen, A.; Topcu, G.; Eris, C.; Sonmez, U.; Kartal, M.; Kurucu,
S.; Bozok-Johansson. C. Phytochemistry 1994, 36, 971-974.
13) Guerrero, M. F.; Puebla, P.; Carron, R.; Martin, M. L.; San Roman,
L. J. Pharm. Pharmacol. 2002, 54, 1373-1378.
3
,15,16-Tri-O-acetyldarutigenol. The aglycons of 4, 5, and daru-
14) Bouktaib, M.; Lebrun, S.; Atmani, A.; Rolando, C. Tetrahedron 2002,
toside (obtained from the above enzymatic hydrolysis) were dissolved
in pyridine (0.5 mL for the former two compounds and 3 mL for the
last one), each followed by the addition of acetyl chloride (one drop
for the former two and three drops for the last one). These mixtures
were then stirred at 80 °C for 2 h, and a common spot was observed
on their co-TLC plate. After evaporation of pyridine, the residues were
subjected to PTLC (developed by petroleum-acetone, 15:1) to give
58, 10001-10009.
(
(
15) Xiong, J.; Jin, Q. D.; Xu, Y. L. Chin. Chem. Lett. 2001, 12, 51-54.
16) Inoue, K.; Shiobara, Y.; Nayeshiro, H.; Inouye, H.; Wilson, G.; Zenk,
M. H. Phytochemistry 1984, 23, 307-311.
(
(
(
17) Chaudhuri, R. K.; Afifi-Yazar, F. U¨ .; Sticher, O. HelV. Chim. Acta
1
979, 62, 1603-1604.
18) Kanchanapoom, T.; Kasai, R.; Yamasaki, K. Phytochemistry 2002,
9, 551-556.
19) El-Emary, N. A.; Backheet, E. Y. Phytochemistry 1998, 49, 277-
79.
2
3
3
,15,16-tri-O-acetyldarutigenol (21 mg): [R]
D
-29.5 (c 0.2, CHCl
ESIMS m/z 471 [M + Na] ; H NMR (in CDCl , 300 MHz) δ 5.12
1H, dd, J ) 2.4 and 9.2 Hz, H-15), 5.10 (1H, s, H-14), 4.50 (1H, dd,
3
);
5
+
1
3
H
(
2
J ) 4.5 and 11.4 Hz, H-3), 4.36 (1H, dd, J ) 2.4 and 11.5 Hz, H-16),
4
(
(
20) Rybalko, O. Chem. Nat. Compd. (Engl. Transl.) 1976, 12, 346.
21) Park, B. S.; Kim, J. R.; Lee, S. E.; Kim, K. S.; Takeoka, G. R.; Ahn,
Y. J.; Kim, J. H. J. Agric. Food Chem. 2005, 53, 1152-1157.
22) Fu, N. Y.; Yuan, Y. F.; Cao, Z.; Wang, S. W.; Wang, J. T.; Peppe,
C. Tetrahedron 2002, 58, 4801-4807.
.03 (1H, dd, J ) 9.2 and 11.5 Hz, H-16), 2.00, 2.01, and 2.03 (each
H, s, 3 × -COCH ), 0.82, 0.83, 0.84, and 0.87 (each 3H, s, 4 ×
3
3
methyl).
(
(
(
In Vitro Cytotoxicity Assay. The cytotoxicity of compounds 1-3
against human hepatoma BEL-7402 cells was evaluated by the
sulforhodamine B (SRB) assay as described previously. Briefly, BEL-
7
1
of incubation, SRB was added and the absorbance of the SRB solution
was measured at 560 nm. The IC50 values were calculated on the basis
of percentage inhibition using the Logit linear regression method.
23) Jisaka, M.; Ohigashi, H.; Takegawa, K.; Huffman, M.; and Ko-
shimizu, K. Biosci., Biotechnol., Biochem. 1993, 57, 833-834.
24) Gu, J.; Gills, J.; Park, E.; Mata-Greenwood, E.; Hawthorne, M.;
Axelrod, F.; Chavez, P.; Fong, H.; Mehta, R.; Pezzuto, J.; Kinghorn,
A. J. Nat. Prod. 2002, 65, 532-536.
25
402 cells were incubated with different concentrations of compounds
-3 (5, 0.5, and 0.05 µM, respectively) at 37 °C for 72 h. At the end
(
25) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J.; Bokesch, H.; Kenny, S.; Boyd, M. J. Natl.
Cancer Inst. 1990, 82, 1107-1112.
Acknowledgment. The authors are grateful to the Shanghai
Committee of Science and Technology for partial financial support (No.
0
3DZ19228).
NP060515P