全 文 ：水朝阳旋覆花中新的细胞毒活性麝香草酚类化合物?
黄火强1 , 2 , 谭宁华1
??
, 曾广智1 , 嵇长久1 ,2 ,
韩洪金1 , 徐俊驹1 ,2 , 张玉梅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 , 云南 昆明 650204;
2 中国科学院研究生院 , 北京 100049 )
摘要 : 从水朝阳旋覆花 ( Inula helianthus-aquatica) 的 95% 乙醇提取物中分离得到 4 个麝香草酚类化合物 ,
其中化合物 1 为新化合物。它们的化学结构通过波谱方法鉴定为 : 8-hydroxy-9 , 10-dioxyisopropylidene-thymol
( 1) , 10-hydroxy-8 , 9-dioxyisopropylidene-thymol (2) , 8-hydroxy-9 , 10-diisobutyryloxy-thymol ( 3) 和 8 , 10-dihydroxy-
9-isobutyryloxy-thymol (4)。肿瘤细胞毒试验结果表明它们在 6 种肿瘤细胞株上 (K562 , HT-29 , SGC-7901 ,
DU145 , MDA-MB-231 , U251 ) 显示一定的细胞毒活性 , 其中化合物 2 活性最强 , 它的 IC50 值为 4 .20～33 .12
μmol?L。具有细胞毒活性的麝香草酚类化合物是首次在该种植物中发现。
关键词 : 水朝阳旋覆花 ; 麝香草酚类化合物 ; 细胞毒活性
中图分类号 : Q 946 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 02 - 190 - 03
New Cytotoxic Thymol Derivatives from Inula
helianthus-aquatica ( Compositae) *
HUANG Huo-Qiang1 , 2 , TAN Ning-Hua1 * * , ZENG Guang-Zhi1 , J I Chang-J iu1 , 2 ,
HAN Hong-J in
1
, XU Jun-Ju
1 , 2
, ZHANG Yu-Mei
1
( 1 State Key Laboratory of Phytochemistry and Plant Resources in West China , Kunming Instituteof Botany,
Chinese Academy of Sciences, Kunming 650204 , China;
2 GraduateUniversity of Chinese Academy of Sciences, Beijing 100049 , China)
Abstract: A new thymol derivative, 8-hydroxy-9 , 10-dioxyisopropylidene-thymol (1 ) , together with three known thymol
derivatives 10-hydroxy-8 , 9-dioxyisopropylidene-thymol (2 ) , 8-hydroxy-9 , 10-diisobutyryloxy-thymol ( 3 ) and 8 , 10-di-
hydroxy-9-isobutyryloxy-thymol (4) , was isolated from95% EtOH extract of the flowers of Inula helianthus-aquatica . Their
structures wereelucidated on the basis of detailed spectroscopic analysis . These compounds showedcytotoxicities on six can-
cer cell lines (K562 , HT-29 , SGC-7901 , DU145 , MDA-MB-231 , U251) , and compound2 isthe strongest onewith IC50s of
4 .20 - 33.12μmol?L . This is thefirst time that the cytotoxic thymol derivativeswere isolated fromthis titled plant .
Key words: Inula helianthus-aquatica; Thymol derivatives; Cytotoxicity
Inula helianthus-aquatica C . Y . Wu ex Ling
( Compositae) has been used commonly to treat various
cancers in Yunnan, China ( Hu and Xuan, 1982 ) .
Some sesquiterpenes, particularly anti-cancer ones in
vitro and in vivo such as ergolide and bigelovin, were
isolated and determined from this herb ( Wang et al. ,
云 南 植 物 研 究 2009 , 31 (2) : 190～192
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.08199
?
?? ?Author for correspondence; Tel : + 86 - 871 - 5223800 ; E-mail : nhtan@ mail .kib. ac. cn
Received date: 2008 - 11 - 04 , Accepted date: 2009 - 01 - 05
作者简介 : 黄火强 (1981 - ) 男 , 在读硕博生 , 主要从事抗肿瘤活性成分研究。 ?
Foun ?dation items: The National Natural Science Foundation of China ( 30725048 ) , National Basic Research Program of China ( 2009CB522303 )
and the Foundation of Chinese Academy of Sciences ( West Light Program)
1996; Zhang et al. , 1998; Liang et al. , 1998; Zeng
et al. , 2008 ) . We describe herein the isolation and
structural elucidation of a new thymol derivative ( 1 )
and three known thymol derivatives 10-hydroxy-8 , 9-
dioxyisopropylidene-thymol (2) (Liang et al. , 2007 ) ,
8-hydroxy-9 , 10-diisobutyryloxy-thymol (3) (Mossa et
al. , 1997 ) and 8 , 10-dihydroxy-9-isobutyryloxy-thymol
(4) (Trang et al. , 1993 ) from 95% EtOH extract of
the flowers of I . helianthus-aquatica ( Fig. 1 ) . These
compounds were tested on six cancer cell lines (K562 ,
HT-29 , SGC-7901 , DU145 , MDA-MB-231 , U251 ) .
Compound 1 wasobtained as colorlessoil . Its mo-
lecular formulawas established as C13 H18 O4 by FAB
+
-
MS andDEPT spectra (Table 1) , which was confirmed
byHR-ESI-MS m?z261 .1099 ( [M + Na] + calcd . for
C13 H18 NaO4 , 261 .1102 ) . The IR spectrum displayed
absorption bonds of hydroxyl group ( 3424 cm- 1 ) and
benzene ring (1629 cm- 1 ) , the latter was confirmed by
the UV spectrum (191 , 204 , 276 , 281 nm) . The 13 C
and 1 H NMR spectra revealed the presence of one 1 ,
3 , 4-trisubstituted phenyl and isopropyl moieties . The
left four carbons areonemethyl , two oxygenated meth-
ylenes and one oxygenated quaternary carbon . The
HMQC , COSY and HMBC spectra, particularly HMBC
correlations of H - 2 with C - 4 , C - 6 and C - 7 , H - 5
with C - 1 , C - 3 and C - 8 , H - 6 with C - 4 and C -
7 , H - 7 with C - 1 , H - 9 and H - 10 with C - 4 , C - 8
and C - 1′, H - 2′and H - 3′with C - 1′, indicated
that 1 is one 8, 9, 10-trioxygenated derivativeof thymol
(Trang et al. , 1993; Mossa et al. , 1997; Liang et al. ,
2007) .Thereforethestructureof 1 was determined to be
8-hydroxy-9, 10-dioxyisopropylidene-thymol .
Results fromcell growth inhibition assay indicated
that these four compounds showed cytotoxicities on a
panel of six cancer cell lines ( K562 , HT-29, SGC-
7901 , DU145 , MDA-MB-231, U251) (Table 2) . Par-
ticularly compound 2 inhibited cell proliferation of all
tested cancer cell lines, in which K562 was the most
sensitiveonewith IC50 of 4 .20μmol?L , while compound
1, 3 , 4 selectively affected K562 with IC50s of 92 .89 ,
20 .69 , 30 .38μmol?L . That means thymol derivatives
Table 1 1 H and 13 C NMR spectral data of compound 1 in
CDCl3 (1 H : 400 MHz, 13 C : 100 MHz)
position δC δH ( J ) HMBC
1 ?140 . 1s
2 ?118 .7d 6 . 73s C-4 E, C-6 , C-7
3 ?156 . 6s
4 ?118 . 4s
5 ?124 .9d 6 . 84d (7 !. 9) C-1 E, C-3 , C-8
6 ?120 .5d 6 . 66br d (7 |. 9 ) C-4 E, C-7
7 ?21 .0q 2 . 28s C-1 E, C-2 , C-6
8 ?72 .1s
9 ?67 .9t 3 ?.84d (11 (.9) , 4. 22 (11.9) C-4 E, C-8 , C-1′
10 ?67 .9t 3 ?.84d (11 (.9) , 4. 22 (11.9) C-4 E, C-8 , C-1′
1 ?′ 98 .7s
2 ?′ 17 .7q 1 . 57s C-1 E′
3 ?′ 29 .1q 1 . 52s C-1 E′
Table 2 Cytotoxicities of compounds 1 - 4 against cancer cell lines
Cell lines
IC50 e(μmol?L )
1 }2 ?3 4 K
K562 ?92 X. 89 4 .20 20 .69 30 &. 38
HT-29 2— 19 ?. 12 — —
SGC-7901 ?— 4 .43 — —
DU145 B— 15 ?. 74 — —
MDA-MB-231 ?— 19 .41 — —
U251 ?— 33 .12 — —
“—”, no activity .
Fig . 1 Structures of compounds 1 - 4
1912 期 HUANG Huo-Qiang et al . : NewCytotoxic Thymol Derivatives from Inula helianthus-aquatica ( Compositae)
are another kind of anti-cancer active constituents for
this herb . This is the first time that the cytotoxic thy-
mol derivatives were isolated fromthis titled plant .
Experimental
General IR spectrumwas recorded using a Bruker Ten-
sor27 spectrometer . UV spectrumwas taken on a Shimadzu UV-
2401PC spectrometer . MS was carried out on a VG Autospec-
3000 mass spectrometer and API QSTAR Pulsar i spectrometer .
NMR spectrumwas run inCDCl3 on aBruker AM-400 and DRX-
500 spectrometer with TMS as the internal standard . Column
chromatographic silica gel and TLC silica gel plates were from
Qingdao Marine Chemical Group Corporation, Qingdao, P . R .
China . Fractions were monitored by TLC, in which spots were
detected with 5% H2 SO4 in ethanol .
Material The flowers of I . helianthus-aquatica were col-
lected in 2004 in Dali of Yunnan province, P . R . China . Itwas
identified byProfessor Li Xi-WenfromKunmingInstituteof Bota-
ny, Chinese Academy of Sciences . Human cancer cell lines
K562 ( leukemia cell line) , HT-29 ( colon cancer cell line) ,
SGC-7901 (gastric cancer cell line) , DU-145 ( prostate cancer
cell line) , MDA-MB-231 ( breast cancer cell line) and U-251
(glioma) werepurchasedfromthe Cell CultureCentre of Institute
of Basic Medical Sciences, Chinese Academyof Medical Scienc-
es (Perking, China) .
Extraction and Isolation Dried flowers of I . helianthus-
aquatica ( 20 .0 kg) were powdered and extracted with 95%
EtOH under reflux for threetimes . The 95% EtOH extract (1 .0
kg) was dissolved in water and partitioned with ethyl acetateand
n-butanol successively . The ethyl acetate extracts ( 300 .0 g)
were subjected to column chromatography over silica gel eluted
with petroleumether?acetone (15∶1 to 0∶1 ) to afford four frac-
tions (1 - 4 ) . Fraction 1 ( 100 .0 g) was chromatographed over
silica gel (200 - 300 mesh) , eluted with petroleum ether?acetone
(20∶1 to1∶1) to afford fractions A-F . Fraction C ( 6 .5 g) was
isolated by silicagel column (400 - 600 mesh) , eluted with pe-
troleum ether?acetone ( 10∶1 to3∶1) togivecompound3 (110 .0
mg) . Fraction 4 ( 40.0 g) was chromatographed over silica gel
(200 - 300 mesh) , elutedwithchloroform?acetone (10∶1 to0∶1)
to afford4 fractionsG-J . Fraction H (6 . 3 g) was isolated by sili-
ca gel column chromatography [ 400 - 600 mesh, chloroform?ace-
tone (10∶1 to 2∶1) ] to give compounds 1 (4 .0 mg) , 2 ( 23 .0
mg) , and 4 (4 .0 mg) .
8-Hydroxy-9 , 10-dioxyisopropylidene-thymol ( 1 ) , col-
orless oil , C13 H18 O4 ; UV λmax CH3 OH nm ( logε) : 191
(3 .70) , 204 ( 4 .31 ) , 276 ( 3 .35 ) , 281 ( 3 .35 ) ; IR νKBrmax
cm
- 1
: 3424 , 2926 , 1629 , 1057; 1 H- and 13 C-NMR: seeTable
1 ; FAB-MS ( pos .) m?z: 238 [M] + ( 14 ) , 221 ( 12 ) , 207
(100) ; HR-ESI-MS ( pos .) : 261.1099 ( [M + Na] + , C13 H18
NaO4 , calcd . 261.1102) .
Cell Growth Inhibition Assay All cancer cells were cul-
tured in RPMI-1640 containing10% fetal bovine serumand 5 CC
Pen-Strep . Compound cytotoxicity was assessed by the sulfurho-
damineB (SRB) assay described before (Skehan et al. , 1990) .
Firstly, 3000 - 7000 cells?well were plated in 96-well plates .
Twenty-four hours later, compoundswereadded to a final concen-
tration of 10μg?mL . After incubated for 48 h, cellswere fixed by
the addition of 50% (or 80% for K562 cells) ice-cold trichloro-
acetic acid and left at 4℃ for 1 h . Then Plates were washed 5
times in water, air-dried, and stained for 15 min with 100μL
0 .4% SRB in1% glacial acetic acid . Excessive dyewas removed
by washing 5 times in 1% glacial acetic acid . After plates were
air-dried, SRB was resuspended in100μL 10 mmol?L Tris andthe
OD valueswereread at 560 nmon a96-well plate reader (Molec-
ular Devices, SPECTRA MAX 340 ) . Inhibition data were ex-
pressed as IC50 values, which were calculated by dose-response
curveswith at least four concentrations ( dilution ratio= 1?10) .
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