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A New Cytotoxic Oxyneolignan from the Roots and Stems of Clausena lansium (Rutaceae)*

黄皮根和茎中一个新的木脂体



全 文 :黄皮根和茎中一个新的木脂体∗
宋卫武1ꎬ2ꎬ 曾广智1ꎬ 彭文文1ꎬ 谭宁华1∗∗
(1 中国科学院昆明植物研究所 植物化学与西部植物资源持续利用国家重点实验室ꎬ 云南 昆明  650201ꎻ
2 中国科学院大学ꎬ 北京  100049)
摘要: 从黄皮 (Clausena lansium) 根和茎中分离得到 8个化合物ꎬ 其中化合物 claulignan (1) 为一个新的
木脂体类化合物ꎬ 通过 1D ̄、 2D ̄NMR 和高分辨质谱确定其结构ꎮ 化合物 2-8 首次从黄皮属 (Clausena)
植物中分离得到ꎮ 活性筛选结果表明ꎬ 化合物 1和 6显示一定的细胞毒活性ꎬ 对人宫颈癌 HeLa细胞株的
IC50分别为 25􀆰 03和 53􀆰 99 μMꎻ 化合物 3具有一定的抗氧化活性ꎬ 在 DPPH实验中的 EC50为 268􀆰 96 g􀅰kg
-1ꎮ
关键词: 黄皮ꎻ 木脂体ꎻ 细胞毒ꎻ DPPHꎻ Claulignan
中图分类号: Q 946            文献标识码: A                文章编号: 2095-0845(2014)04-545-06
A New Cytotoxic Oxyneolignan from the Roots and
Stems of Clausena lansium (Rutaceae)∗
SONG Wei ̄Wu1ꎬ2ꎬ ZENG Guang ̄Zhi1ꎬ PENG Wen ̄Wen1ꎬ TAN Ning ̄Hua1∗∗
(1 State Key Laboratory of Phytochemistry and Plant Resources in West Chinaꎬ Kunming Institute of Botanyꎬ Chinese Academy
of Sciencesꎬ Kunming 650201ꎬ Chinaꎻ 2 University of Chinese Academy of Sciencesꎬ Beijing 100049ꎬ China)
Abstract: A new oxyneolignanꎬ claulignan (1)ꎬ together with seven known compounds (2-8)ꎬ were isolated from
the methanol extract of the roots and stems of Clausena lansium. Their structures were elucidated on the basis of 1D ̄
and 2D ̄NMR experiments and mass spectrometry. Compounds 2-8 were isolated from the genus Clausena for the first
time. Compounds 1 and 6 showed cytotoxicity against human cervical cancer HeLa cell line with the IC50 values of
25􀆰 03 and 53􀆰 99 μMꎬ respectively. Compound 3 exhibited antioxidant activity in the DPPH assay with the EC50
value of 268􀆰 96 g􀅰kg-1 .
Key words: Clausena lansiumꎻ Oxyneolignanꎻ Cytotoxicityꎻ DPPHꎻ Claulignan
  Clausena belongs to the family Rutaceae and
includes about 10 species in China. Clausena lansi ̄
um (Lour.) Skeels is a fruit tree and widely distrib ̄
utes in southern China (Huangꎬ 1997). Its fruits
have been used for treating indigestionꎬ coldꎬ
coughꎬ and stomach painꎻ its seeds for treating a ̄
cuteꎬ chronic gastrointestinal inflammationꎬ and ul ̄
cerꎻ and its leaves and roots for the treatment of
coughꎬ asthmaꎬ dermatological diseaseꎬ viral hepati ̄
tisꎬ and gastro ̄intestinal disease (Shen et al.ꎬ 2012).
Previous phytochemical investigation revealed that
C􀆰 lansium contained flavonoid glycosidesꎬ couma ̄
rinsꎬ cabazole alkaloidsꎬ amidesꎬ and terpenoids
with cytotoxic and neuroprotective activities ( Ito et
alꎬ 1998ꎻ Lakshmi et al.ꎬ 1989ꎻ Liu et al.ꎬ 2012ꎻ
Maneerat et al.ꎬ 2012ꎻ Yang et al.ꎬ 1988ꎻ Zhao et
植 物 分 类 与 资 源 学 报  2014ꎬ 36 (4): 545~550
Plant Diversity and Resources                                    DOI: 10.7677 / ynzwyj201413182

∗∗
Funding: Found of the National New Drug Innovation Major Project of China (2011ZX09307 ̄002 ̄02)ꎬ the National Natural Science Foun ̄
dation of China (U1032602 and 91013002)ꎬ the National Basic Research Program of China (2009CB522300)ꎬ the Fund of Chi ̄
nese Academy of Sciences (Hundred Talents Program)ꎬ and the Natural Science Foundation of Yunnan Province (2012GA003)
Author for correspondenceꎻ E ̄mail: nhtan@mail􀆰 kib􀆰 ac􀆰 cn
Received date: 2013-09-16ꎬ Accepted date: 2013-10-10
作者简介: 宋卫武 (1983-) 男ꎬ 在读博士研究生ꎬ 主要从事天然药物化学研究ꎮ
al.ꎬ 2010). In order to discover bioactive constitu ̄
entsꎬ the methanol extract of the roots and stems of
C􀆰 lansium was studied. Herein we report the isola ̄
tion and structural elucidation of one new oxyneolig ̄
nan and 7 known compounds from this extract with
cytotoxic and antioxidant activities.
1  Results and discussion
Claulignan (1) was obtained as a brown pow ̄
der and its molecular formula was deduced as C20H22
O7 on the basis of HR ̄EI ̄MS at m / z 374􀆰 1378
[M] + ( calcd for C20H22O7ꎬ 374􀆰 1366). The 13C ̄
NMR spectrum of 1 (Table 1) displayed ten carbon
signalsꎬ including six aromatic carbons ( δC 152􀆰 3ꎬ
148􀆰 3ꎬ 130􀆰 6ꎬ 124􀆰 0ꎬ 115􀆰 4 and 111􀆰 5)ꎬ two
methylene carbons ( δC 58􀆰 7 and 41􀆰 5)ꎬ one keto ̄
carbon ( δC 198􀆰 2) and one methoxy carbon ( δC
56􀆰 2). In the 1H ̄NMR spectrum of 1 (Table 1)ꎬ
three aromatic proton signals at δH 7􀆰 55 (dꎬ J= 1􀆰 5
Hz)ꎬ 6􀆰 92 ( dꎬ J = 8􀆰 2 Hz) and 7􀆰 59 ( ddꎬ J =
8􀆰 2ꎬ 1􀆰 5 Hz) were observed. These data indicated
that 1 might be an oxyneolignan with the same unit
of 3ꎬ 4 ̄disubstituted phenylpropanoid. Position of
the keto ̄group was determined by the HMBC corre ̄
lations from H ̄2ꎬ H ̄6ꎬ H ̄8 and H ̄9 to C ̄7ꎬ and
the COSY correlation between H ̄8 and H ̄9
(Fig􀆰 1). The HMBC correlation from OCH3 to C ̄3
and the ROESY correlation between H ̄2 and OCH3
suggested that the methoxy group linked at C ̄3 and
the two same phenylpropanoid units linked at C ̄4
(Fig􀆰 2). Thusꎬ the structure of 1 was established.
Seven known compounds were identified to be
clemaphenol A (2) (Song et al.ꎬ 2010)ꎬ syringa ̄
resinol (3) (Ouyang et al.ꎬ 2007)ꎬ ficusol ( 4)
(Li and Kuoꎬ 1998)ꎬ berfussinol (5) (Kim et al.ꎬ
2011)ꎬ 4 ̄hydroxy ̄2 ̄methoxycinnamaldehyde ( 6 )
(Xu and Wangꎬ 2011)ꎬ 3 ̄methoxy ̄4 ̄hydroxybenz ̄
aldehyde (7) (Yang et al.ꎬ 2007) and 4 ̄hydroxy ̄
benzaldehyde (8) ( Feng et al.ꎬ 2008) ( Fig􀆰 3).
Compounds 2-8 were isolated from the genus Clause ̄
na for the first time. Compounds 1 and 6 showed cy ̄
totoxicity against human cervical cancer HeLa cell
line with the IC50 values of 25􀆰 03 and 53􀆰 99 μMꎬ
respectively. Compound 3 exhibited antioxidant ac ̄
tivity in the DPPH assay with the EC50 value of
268􀆰 96 g􀅰kg-1 .
Table 1  1H and 13C NMR data for compound 1
in acetone ̄d6 (δ in ppmꎬ J in Hz)
NO. δC a)       δH b)
1ꎬ 1′ 130􀆰 6 (s)
2ꎬ 2′ 111􀆰 5 (d) 7􀆰 55 (dꎬ 1􀆰 5)
3ꎬ 3′ 148􀆰 3 (s)
4ꎬ 4′ 152􀆰 3 (s)
5ꎬ 5′ 115􀆰 4 (d) 6􀆰 92 (dꎬ 8􀆰 2)
6ꎬ 6′ 124􀆰 0 (d) 7􀆰 59 (ddꎬ 8􀆰 2ꎬ 1􀆰 5)
7ꎬ 7′ 198􀆰 2 (s)
8ꎬ 8′ 41􀆰 5 ( t) 3􀆰 16 (m)
9ꎬ 9′ 58􀆰 7 ( t) 3􀆰 91 (overlapped)
OH 8􀆰 70 (s)
OMe 56􀆰 2 (q) 3􀆰 90 (s)
a) 600 MHzꎻ b) 100 MHz
Fig􀆰 1  Structure of compound 1
Fig􀆰 2  The key HMBC ( ) and 1Hꎬ 1H ̄COSY
( ) correlations for compound 1
2  Experimental
General experimental procedures   Optical
rotation was measured on a Jasco P ̄1020 polarime ̄
ter. UV spectrum was obtained from shimadzu UV ̄
2401PC spectrophotometer. IR spectrum was recor ̄
ded on a BRUKER Tensor 27 FT ̄IR spectrometer
with KBr pellets. The 1D ̄ and 2D ̄NMR spectra were
645                                  植 物 分 类 与 资 源 学 报                            第 36卷
Fig􀆰 3  Structures of compounds 2-8
obtained from Bruker AM ̄400 ( 1H: 400 MHzꎬ 13C:
100 MHz) or Bruker DRX ̄500 (1H: 500 MHzꎬ 13C:
150 MHz) or Bruker AV ̄III 600 ( 1H: 600 MHzꎬ 13C:
150 MHz) spectrometer in acetone ̄d6ꎬ CD3OD or
pyridine ̄d5 at the room temperature. ESI ̄MS was de ̄
termined on a Waters Xevo TQ ̄S or Bruker HCT /
Esquire. EI ̄MS and HR ̄EI ̄MS were determined on
a Waters AutoSpec Premier P776. Silica gel (200-
300 meshꎻ Qingdao Yuminyuan Silica Reagent Fac ̄
toryꎬ Qingdaoꎬ P􀆰 R. China)ꎬ RP ̄18 (40-60 μmꎬ
Merckꎬ Darmstadtꎬ Germany)ꎬ and MCI gel (CHP ̄
20Pꎬ 70 - 150 μmꎬ Mitsubishi Chemical Corpora ̄
tionꎬ Japan) were used for column chromatography
(CC). Fractions were monitored by TLC (Silica gel
GF254 Platesꎬ Qingdao Yuminyuan Silica Reagent
Factoryꎬ Qingdaoꎬ P􀆰 R. China) and spots were vis ̄
ualized by UV light ( 254 and 365 nm)ꎬ and by
spraying with 5% aq. H2SO4 soln. followed by heat ̄
ing. MPLCꎬ equipped with a UV detector (EZ Puri ̄
fier 100 / 200ꎬ Lisure Science (Suzhou) Co.ꎬ LTD.
P􀆰 R. China) and a RP ̄18 column (3􀆰 5×25 or 4􀆰 5
×40 cm) or a MCI column (4􀆰 5×40 cm) at a flow
rate of 25 mL􀅰min-1ꎬ and preparative reversed ̄phase
HPLCꎬ Agilent 1100 apparatus equipped with a UV
detector and a SunFire OBD (Watersꎬ 1􀆰 9×25 cmꎬ
5 μm) column at a flow rate of 10 mL􀅰min-1 were
used for purification.
Plant material  The roots and stems of Clause ̄
na lansium were collected from Hekouꎬ Honghe Ha ̄
ni & Yi Autonomous Prefectureꎬ Yunnan provinceꎬ
P􀆰 R. Chinaꎬ in September 2010 and identified by
Prof. Yu ̄Min Shuiꎬ Kunming Institute of Botanyꎬ
Chinese Academy of Sciences. A voucher specimen
(No. 0599043) was deposited at the Herbarium of
Kunming Institute of Botanyꎬ Chinese Academy of
Sciences.
Extraction and isolation  Air driedꎬ powdered
roots and stems of C􀆰 lansium (27 kg) were extrac ̄
ted and refluxed with MeOH for 3 times each 4 hours
(MeOHꎬ 50 L×3). The extract was evaporated un ̄
der reduced pressure to yield a dark brown residue
(900 g). The residue was suspended in MeOH / H2O
(7∶ 3ꎬ 3 000 mL) and then partitioned with EtOAc
(3×2 000 mL). After removing solventꎬ the EtOAc ̄
soluble part (406 g) was fractionated by silica gel
(200-300 mesh) column chromatograph (CC) and
eluted with CHCl3 ̄MeOH (30 ∶ 1-4 ∶ 1) to afford 6
fractionsꎬ Fractions 1-6 (Fr􀆰 1-6)ꎬ on the basis of
7454期      SONG Wei ̄Wu et al.: A New Cytotoxic Oxyneolignan from the Roots and Stems of Clausena lansium       
TLC analysis. Fr􀆰 3 (25 g) was chromatographed o ̄
ver MPLC with MCI (MeOH / H2O 10 ∶ 90→100 ∶ 0)
and silica gel (petroleum ether / acetone 15∶1→7∶3)
successively to afford 6 (15 mg) and 7 (10 mg).
Fr􀆰 4 (17􀆰 9 g) was firstly subjected to MPLC with
MCI column (MeOH / H2O 10 ∶ 90→60 ∶ 40) to re ̄
move pigment and then to silica gel CC (petroleum
ether / acetone 9∶1) and preparative HPLC (acetoni ̄
trile / H2O 55∶45) to afford 2 (26 mg). MPLC chro ̄
matograph of Fr􀆰 5 (6􀆰 9 g) over MCI (MeOH / H2O
5 ∶ 95→60 ∶ 40) afforded 7 sub ̄fractions: Fr􀆰 5-1 ̄
Fr􀆰 5-7. Fr􀆰 5-1 was repeatedly chromatographed o ̄
ver silica gel ( petroleum ether / acetone 9 ∶ 1) and
then subjected to preparative HPLC (acetonitrile / H2
O 55∶45) to afford 1 (22 mg)ꎬ 3 (280 mg)ꎬ 4 (94
mg) and 5 (35 mg). Fr􀆰 6 (77 g) was subjected to
silica gel CC (CHCl3 / acetone 15 ∶ 1→7 ∶ 3)ꎬ silica
gel CC (petroleum ether / acetone 5∶1)ꎬ MPLC with
MCI (MeOH / H2 O 10 ∶ 90→60 ∶ 40)ꎬ MPLC with
RP ̄18 (MeOH / H2O 5 ∶ 95→70 ∶ 30) and silica gel
CC (petroleum ether / EtOAc 5 ∶ 1)ꎬ subsequently to
afford 8 (21 mg).
Claulignan (1) was obtained as brown pow ̄
derꎬ C20 H22 O7ꎻ [ α] 2 1􀆰 0D = - 12􀆰 93 ( c = 0􀆰 10ꎬ
MeOH)ꎻ UV (MeOH) λmax (log ε): 205 (4􀆰 48)ꎬ
228 (4􀆰 48)ꎬ 276 (4􀆰 30)ꎬ 303 (4􀆰 23) nmꎻ IR
(KBr) νmax ( cm
-1 ): 3423ꎬ 2960ꎬ 1665ꎬ 1591ꎬ
1517ꎬ 1464ꎬ 1427ꎬ 1385ꎬ 1279ꎬ 1197ꎬ 1167ꎬ
1136ꎬ 1032ꎻ 1H NMR (acetone ̄d6ꎬ 600 MHz) and
13C NMR ( acetone ̄d6ꎬ 100 MHz): see Table 1ꎻ
ESI ̄MS (positive) m / z: 397 [M+Na] +ꎬ 771 [2M
+Na] +ꎻ HR ̄EI ̄MS m / z: 374􀆰 1378 [M] + ( calcd.
for C20H22O7
+ꎬ 374􀆰 1366).
Clemaphenol A (2) was obtained as colorless
oilꎬ C20 H22 O6ꎻ [ α ] 23􀆰 7D = + 0􀆰 16 ( c = 0􀆰 42ꎬ
MeOH)ꎻ 1H NMR ( 400 MHzꎬ acetone ̄d6 ): δH
6􀆰 98 ( 2Hꎬ dꎬ J = 1􀆰 3 Hzꎬ H ̄2ꎬ H ̄2′)ꎬ 6􀆰 83
(1Hꎬ ddꎬ J = 8􀆰 1ꎬ 1􀆰 3 Hzꎬ H ̄6ꎬ H ̄6′)ꎬ 6􀆰 78
(2Hꎬ dꎬ J= 8􀆰 1 Hzꎬ H ̄5ꎬ H ̄5′)ꎬ 4􀆰 65 (2Hꎬ dꎬ J
= 3􀆰 8 Hzꎬ H ̄7ꎬ H ̄7′)ꎬ 4􀆰 20 (2Hꎬ mꎬ Ha ̄9ꎬ Ha ̄
9′)ꎬ 3􀆰 83 (6Hꎬ sꎬ OCH3)ꎬ 3􀆰 80 (2Hꎬ ddꎬ J =
9􀆰 2ꎬ 3􀆰 8 Hzꎬ Hb ̄9ꎬ Hb ̄9′)ꎬ 3􀆰 10 (2Hꎬ mꎬ H ̄8ꎬ
H ̄8′)ꎻ 13C NMR ( 100 MHzꎬ acetone ̄d6 ): δC
148􀆰 2 ( sꎬ C ̄4ꎬ C ̄4′)ꎬ 146􀆰 8 ( sꎬ C ̄3ꎬ C ̄3′)ꎬ
134􀆰 1 ( sꎬ C ̄1ꎬ C ̄1′)ꎬ 119􀆰 6 ( dꎬ C ̄6ꎬ C ̄6′)ꎬ
115􀆰 5 ( dꎬ C ̄5ꎬ C ̄5′)ꎬ 110􀆰 5 ( dꎬ C ̄2ꎬ C ̄2′)ꎬ
86􀆰 6 (dꎬ C ̄7ꎬ C ̄7′)ꎬ 72􀆰 1 (tꎬ C ̄9ꎬ C ̄9′)ꎬ 56􀆰 2
(qꎬ OCH3)ꎬ 55􀆰 2 (dꎬ C ̄8ꎬ C ̄8′)ꎻ ESI ̄MS (pos ̄
itive) m / z: 381 [M+Na] +ꎬ 739 [2M+Na] + . (Song
et al.ꎬ 2010)
Syringaresinol (3) was obtained as colorless
oilꎬ C22H26O8ꎻ [α] 21􀆰 5D = -6􀆰 7 (c= 0􀆰 20ꎬ MeOH)ꎻ
1H NMR (400 MHzꎬ CD3OD): δH 6􀆰 51 (4Hꎬ sꎬ
H ̄2ꎬ H ̄2′ꎬ H ̄6ꎬ H ̄6′)ꎬ 4􀆰 54 (2Hꎬ dꎬ J = 3􀆰 8
Hzꎬ H ̄7ꎬ H ̄7′)ꎬ 4􀆰 09 (2Hꎬ mꎬ Ha ̄9ꎬ Ha ̄9′)ꎬ
3􀆰 71 (2Hꎬ ddꎬ J = 9􀆰 3ꎬ 2􀆰 8 Hzꎬ Hb ̄9ꎬ Hb ̄9′)ꎬ
3􀆰 67 (12Hꎬ sꎬ OCH3)ꎬ 2􀆰 97 (2Hꎬ br. sꎬ H ̄8ꎬ
H ̄8′)ꎻ 13C NMR (100 MHzꎬ CD3OD): δC 149􀆰 1
(sꎬ C ̄3ꎬ C ̄3′ꎬ C ̄5ꎬ C ̄5′)ꎬ 135􀆰 9 ( sꎬ C ̄4ꎬ C ̄
4′)ꎬ 133􀆰 0 ( sꎬ C ̄1ꎬ C ̄1′)ꎬ 104􀆰 3 (dꎬ C ̄2ꎬ C ̄
2′ꎬ C ̄6ꎬ C ̄6′)ꎬ 87􀆰 4 (dꎬ C ̄7ꎬ C ̄7′)ꎬ 72􀆰 6 ( tꎬ
C ̄9ꎬ C ̄9′)ꎬ 56􀆰 7 (qꎬ OCH3)ꎬ 55􀆰 3 (dꎬ C ̄8ꎬ C ̄
8′)ꎻ ESI ̄MS (negative) m / z: 417 [M ̄H] -ꎬ 418
[M] - . (Ouyang et al.ꎬ 2007)
Ficusol (4) was obtained as colorless oilꎬ C11
H14O5ꎻ [ α] 21􀆰 7D = - 5􀆰 5 ( c = 0􀆰 38ꎬ MeOH)ꎻ 1H
NMR (400 MHzꎬ acetone ̄d6): δH 6􀆰 92 (1Hꎬ dꎬ J
= 0􀆰 8 Hzꎬ H ̄2′)ꎬ 6􀆰 77 (2Hꎬ overlappedꎬ H ̄5′ꎬ
H ̄6′)ꎬ 4􀆰 07 (1Hꎬ mꎬ Ha ̄3)ꎬ 3􀆰 82 (3Hꎬ sꎬ 3′ ̄
OCH3)ꎬ 3􀆰 74 ( 1Hꎬ overlappedꎬ Hb ̄3 )ꎬ 3􀆰 68
(1Hꎬ overlappedꎬ H ̄2)ꎬ 3􀆰 63 (3Hꎬ sꎬ 1 ̄OCH3)ꎻ
13C NMR (100 MHzꎬ acetone ̄d6): δC 174􀆰 1 ( sꎬ
C ̄1)ꎬ 148􀆰 3 (sꎬ C ̄3′)ꎬ 146􀆰 8 (sꎬ C ̄4′)ꎬ 128􀆰 6
(sꎬ C ̄1′)ꎬ 121􀆰 5 (dꎬ C ̄6′)ꎬ 115􀆰 8 (dꎬ C ̄5′)ꎬ
112􀆰 5 (dꎬ C ̄2′)ꎬ 65􀆰 2 ( tꎬ C ̄3)ꎬ 56􀆰 2 ( qꎬ 3′ ̄
OCH3)ꎬ 54􀆰 9 ( dꎬ C ̄2)ꎬ 52􀆰 0 ( qꎬ 1 ̄OCH3 )ꎻ
ESI ̄MS (positive) m / z: 249 [M+Na] + . ( Li and
Kuoꎬ 1998)
Berfussinol (5) was obtained as white solidꎬ
C12H16O6ꎻ [α] 21􀆰 7D = -5􀆰 8 ( c = 0􀆰 15ꎬ MeOH)ꎻ 1H
NMR (400 MHzꎬ acetone ̄d6 ): δH 6􀆰 60 (2Hꎬ sꎬ
H ̄2′ꎬ H ̄6′)ꎬ 4􀆰 07 (1Hꎬ mꎬ Ha ̄3)ꎬ 3􀆰 79 (6Hꎬ
sꎬ 3′ ̄OCH3ꎬ 5′ ̄OCH3 )ꎬ 3􀆰 71 ( 1Hꎬ overlappedꎬ
845                                  植 物 分 类 与 资 源 学 报                            第 36卷
Hb ̄3)ꎬ 3􀆰 70 (1Hꎬ overlappedꎬ H ̄2)ꎬ 3􀆰 63 (3Hꎬ
sꎬ 1 ̄OCH3)ꎻ 13C NMR (100 MHzꎬ acetone ̄d6): δC
174􀆰 0 ( sꎬ C ̄1)ꎬ 148􀆰 6 ( sꎬ C ̄3′ꎬ C ̄5′)ꎬ 136􀆰 2
(sꎬ C ̄4′)ꎬ 127􀆰 5 (sꎬ C ̄1′)ꎬ 106􀆰 5 (dꎬ C ̄2′ꎬ C ̄
6′)ꎬ 65􀆰 2 ( tꎬ C ̄3)ꎬ 56􀆰 6 ( qꎬ 3′ ̄OCH3ꎬ 5′ ̄
OCH3)ꎬ 55􀆰 1 (dꎬ C ̄2)ꎬ 51􀆰 9 (qꎬ 1 ̄OCH3). ESI ̄
MS ( positive) m / z: 279 [M +Na] +ꎬ 535 [2M +
Na] + . (Kim et al.ꎬ 2011)
4 ̄Hydroxy ̄2 ̄methoxycinnamaldehyde ( 6 )
was obtained as yellow oilꎬ C10 H10 O3ꎻ 1H NMR
(600 MHzꎬ acetone ̄d6): δH 9􀆰 63 (1Hꎬ dꎬ J = 7􀆰 8
Hzꎬ H ̄9)ꎬ 8􀆰 43 (1Hꎬ sꎬ OH)ꎬ 7􀆰 57 (1Hꎬ dꎬ J=
15􀆰 8 Hzꎬ H ̄7)ꎬ 7􀆰 39 (1Hꎬ dꎬ J= 2􀆰 0 Hzꎬ H ̄3)ꎬ
7􀆰 21 ( 1Hꎬ ddꎬ J = 8􀆰 0ꎬ 2􀆰 0 Hzꎬ H ̄5)ꎬ 6􀆰 91
(1Hꎬ dꎬ J = 8􀆰 0 Hzꎬ H ̄6)ꎬ 6􀆰 67 (1Hꎬ ddꎬ J =
15􀆰 8ꎬ 7􀆰 8 Hzꎬ H ̄8)ꎬ 3􀆰 92 (3Hꎬ sꎬ OCH3)ꎻ 13C
NMR (150 MHzꎬ acetone ̄d6 ): δC 194􀆰 0 ( dꎬ C ̄
9)ꎬ 154􀆰 2 (dꎬ C ̄7)ꎬ 150􀆰 9 (sꎬ C ̄4)ꎬ 148􀆰 9 (sꎬ
C ̄2)ꎬ 127􀆰 4 ( sꎬ C ̄1)ꎬ 127􀆰 0 (dꎬ C ̄8)ꎬ 124􀆰 9
(dꎬ C ̄6)ꎬ 116􀆰 2 ( dꎬ C ̄5)ꎬ 111􀆰 4 ( dꎬ C ̄3)ꎬ
56􀆰 3 ( qꎬ OCH3)ꎻ ESI ̄MS ( negative) m / z: 177
[M ̄H] -ꎬ 178 [ M] -ꎬ 355 [ 2M ̄H] - . ( Xu and
Wangꎬ 2011)
3 ̄Methoxy ̄4 ̄hydroxybenzaldehyde (7) was
obtained as white powderꎬ C8H8O3ꎻ 1H NMR (600
MHzꎬ acetone ̄d6): δH 9􀆰 83 (1Hꎬ sꎬ CHO)ꎬ 8􀆰 77
(1Hꎬ sꎬ OH)ꎬ 7􀆰 46 (1Hꎬ ddꎬ J = 7􀆰 9ꎬ 1􀆰 8 Hzꎬ
H ̄6)ꎬ 7􀆰 44 (1Hꎬ dꎬ J=1􀆰 8 Hzꎬ H ̄2)ꎬ 7􀆰 01 (1Hꎬ
dꎬ J = 7􀆰 9 Hzꎬ H ̄5)ꎬ 3􀆰 93 (3Hꎬ sꎬ OCH3)ꎻ 13C
NMR (150 MHzꎬ acetone ̄d6): δC 191􀆰 2 (dꎬ CHO)ꎬ
153􀆰 6 (sꎬ C ̄4)ꎬ 149􀆰 0 (sꎬ C ̄3)ꎬ 130􀆰 8 (sꎬ C ̄1)ꎬ
127􀆰 10 (dꎬ C ̄6)ꎬ 116􀆰 0 (dꎬ C ̄5)ꎬ 110􀆰 8 (dꎬ C ̄
2)ꎬ 56􀆰 3 (qꎬ OCH3)ꎻ ESI ̄MS (negative) m/ z: 151
[M ̄H] -ꎬ 152 [M] - . (Yang et al.ꎬ 2007)
4 ̄Hydroxybenzaldehyde (8) was obtained as
colorless crystalꎬ C7 H6 O2ꎻ 1H NMR (400 MHzꎬ
pyridine ̄d5): δH 10􀆰 00 (1Hꎬ sꎬ CHO)ꎬ 7􀆰 94 (2Hꎬ
dꎬ J=8􀆰 0 Hzꎬ H ̄2ꎬ H ̄6)ꎬ 7􀆰 21 (2Hꎬ dꎬ J = 8􀆰 0
Hzꎬ H ̄3ꎬ H ̄5)ꎻ 13 C NMR ( 100 MHzꎬ pyridine ̄
d5 ): δC 190􀆰 8 ( dꎬ CHO)ꎬ 165􀆰 0 ( sꎬ C ̄4)ꎬ
132􀆰 8 ( dꎬ C ̄2ꎬ C ̄6)ꎬ 129􀆰 9 ( sꎬ C ̄1)ꎬ 116􀆰 8
(dꎬ C ̄3ꎬ C ̄5)ꎻ ESI ̄MS (positive) m / z: 123 [M+
H] +ꎬ 145 [M+Na] + . (Feng et al.ꎬ 2008)
Cytotoxic Assay   All compounds were tested
for their cytotoxicity against A549ꎬ HeLa and BGC ̄
823 cancer cell lines with the sulforhodamine B as ̄
say (He et al.ꎬ 2011). Cells were cultured in RPMI
1640 medium (Gibco) supplemented with 10% FBS
(Tianjin Haoyang) at 37 ℃ and 5% CO2 . After see ̄
ded in 96 ̄well microtiter plates for 24 hꎬ cells were
treated with serial dilutions of the tested compounds
with the maximum concentration of 20 μg􀅰mL-1 .
Each compound was initially dissolved in DMSO and
further diluted by the medium to produce different
concentrations. After incubation for 48 or 72 hꎬ cells
were fixed with ice ̄cold 50% trichloroacetic acid at
4 ℃ for 1 h. With staining for 15 min by 0􀆰 4% SRB
(Sigma) in 1% glacial acetic acidꎬ excessive dye
was removed by washing with 1% glacial acetic acid.
Finallyꎬ SRB was re ̄suspended in 10 mmol􀅰L-1 Tris
buffer and the absorbance was measured at 560 nm
with a plate reader (Molecular Devicesꎬ SPECTRA
MAX 340). The result was expressed as IC50 valueꎬ
which is the concentration that causes 50% growth
inhibition. Taxol was used as a reference compound.
DPPH assay   The antioxidant activity of all
compounds was determined using 1ꎬ 1 ̄diphenyl ̄2 ̄
picrylhydrazyl (DPPH) (TCI) method according to
the procedure described by us ( Adebayo et al.ꎬ
2010). Each compound solution in dimethyl sulph ̄
oxide ( DMSOꎬ 5 μL) was added to 195 μL of
0􀆰 025 g􀅰L-1 DPPH methanol solution. Absorbance
at 515 nm was taken after 30 minutes by incubated
at room temperature in the microplate reader (Mo ̄
lecular Devicesꎬ SPECTRA MAX 340). If the ab ̄
sorbance decreased over 50% by compared with the
DMSO control at the concentration of 20 μg􀅰mL-1ꎬ
at least four serially diluted concentrations of tested
compounds were prepared to evaluate their an ̄
tioxidant activity using the above procedure. The
result was expressed as EC50 valueꎬ which is the
amount to decrease the initial DPPH concentration
by 50%. Vitamin C was used as a reference com ̄
pound.
9454期      SONG Wei ̄Wu et al.: A New Cytotoxic Oxyneolignan from the Roots and Stems of Clausena lansium       
Acknowledgement: The authors are grateful to the members
of the analytical group from the State Key Laboratory of Phy ̄
tochemistry and Plant Resources in West Chinaꎬ Kunming In ̄
stitute of Botanyꎬ Chinese Academy of Sciencesꎬ for measur ̄
ing the [α]ꎬ IRꎬ UVꎬ NMR and MS spectra.
References:
Adebayo AHꎬ Tan NHꎬ Akindahunsi AA et al.ꎬ 2010. Anticancer and
antiradical scavenging activity of Ageratum conyzoides L. (Aster ̄
aceae) [J] . Pharmacognosy Magazineꎬ 6: 62—66
Feng BM (冯宝民)ꎬ Yu ZJ (余正江)ꎬ Duan LX (段礼新) et
al.ꎬ 2008. Chemical constituents of roasted Sinapis seeds [ J] .
Chinese Traditional and Herbal Drugs (中草药)ꎬ 39: 331—
334
He WJꎬ Chu HBꎬ Zhang YM et al.ꎬ 2011. Antimicrobialꎬ cytotoxic
lignans and terpenoids from the twigs of Pseudolarix kaempferi
[J] . Planta Medicaꎬ 77: 1924—1931
Huang CJ (黄成就)ꎬ 1997. Rutaceae [ A]. In: Wu ZY ( ed.)ꎬ
Flora of China (中国植物志) [M]. Beijing: Science Pressꎬ
Vol. 43: 132
Ito Cꎬ Katsuno Sꎬ Furukawa Hꎬ 1998. Structures of lansiumarin ̄Aꎬ  ̄
Bꎬ  ̄Cꎬ three new furocoumarins from Clausena lansium [ J] .
Chemical & Pharmaceutical Bulletinꎬ 46: 341—343
Kim KHꎬ Moon Eꎬ Choi SU et al.ꎬ 2011. Biological evaluation of
phenolic constituents from the trunk of Berberis koreana [ J] .
Bioorganic & Medicinal Chemistry Lettersꎬ 21: 2270—2273
Lakshmi Vꎬ Raj Kꎬ Kapil RSꎬ 1989. A triterpene alcoholꎬ lansiolꎬ
from Clausena lansium [J] . Phytochemistryꎬ 28: 943—945
Li YCꎬ Kuo YHꎬ 1998. A monoterpenoid and two simple phenols from
heartwood of Ficus microcarpa [J] . Phytochemistryꎬ 49: 2417—
2419
Liu Hꎬ Li CJꎬ Yang JZ et al.ꎬ 2012. Carbazole alkaloids from the
stems of Clausena lansium [J] . Journal of Natural Productsꎬ 75:
677—682
Maneerat Wꎬ Ritthiwigrom Tꎬ Cheenpracha S et al.ꎬ 2012. Carbazole
alkaloids and coumarins from Clausena lansium roots [J] . Phy ̄
tochemistry Lettersꎬ 5: 26—28
Ouyang MAꎬ Wein YSꎬ Zhang ZK et al.ꎬ 2007. Inhibitory activity a ̄
gainst tobacco mosaic virus (TMV) replication of pinoresinol and
syringaresinol lignans and their glycosides from the root of Rhus
javanica var. roxburghiana [ J ] . Journal of Agricultural and
Food Chemistryꎬ 55: 6460—6465
Shen DYꎬ Chao CHꎬ Chan HH et al.ꎬ 2012. Bioactive constituents of
Clausena lansium and a method for discrimination of aldose en ̄
antiomers [J] . Phytochemistryꎬ 82: 110—117
Song WWꎬ Li Bꎬ Liu JKꎬ 2010. A new lignan from Elaeagnus lanceo ̄
lata (Elaeagnaceae) [J] . Acta Botanica Yunnanica (云南植物
研究)ꎬ 32: 455—462
Xu C (徐冲)ꎬ Wang ZT (王峥涛)ꎬ 2011. Chemical constituents
from roots of Andrographis paniculata [J] . Acta Pharmaceutica
Sinica (药学学报)ꎬ 46: 317—321
Yang MHꎬ Chen YYꎬ Huang Lꎬ 1988. Three novel cyclic amides from
Clausena lansium [J] . Phytochemistryꎬ 27: 445—450
Yang XW (杨秀伟)ꎬ Han MH (韩美华)ꎬ Jin YP (靳彦平)ꎬ
2007. Studies on the chemical constituents from herba anoec ̄
tochili [J] . Journal of Chinese Medicinal Materials (中药材)ꎬ
30: 797—800
Zhao Q (赵青)ꎬ Li C (李创)ꎬ Yang JZ (杨敬芝) et al.ꎬ 2010.
Chemical constituents of Clausena lansium [ J] . China Journal
of Chinese Materia Medica (中国中药杂志)ꎬ 35: 997—1000
055                                  植 物 分 类 与 资 源 学 报                            第 36卷