全 文 ：鹅不食草中具有抗菌活性的三萜类成分?
梁恒兴1 , 2 , 宝福凯3 , 董晓萍2 , 吕 青1 , 程永现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 成都中医药大学药学院 , 四川 成都 610075; 3 昆明医学院微生物与免疫学系 , 云南 昆明 650031)
摘要 : 从鹅不食草 ( Centipeda minima) 全草的乙醇提取物中分离得到 3 个乌苏烷型三萜 , 其中一个新化合
物用波谱学方法鉴定为 ursane-20 (30 )-en-3β, 16β, 21α-triol (1 ) , 二个已知化合物的结构分别为 taraxasterol
acetate (2) , taraxasterol (3)。抗菌试验表明化合物 2 和 3 具有较强的抗菌活性。
关键词 : 鹅不食草 ; 菊科 ; 三萜 ; 抗菌活性
中图分类号 : Q 946 文献标识码 : A 文章编号 : 0253 - 2700 (2007) 04 - 479 - 04
Antibacterial Triterpenes from Centipeda minima (Compositae)
LIANG Heng-Xing
1 , 2
, BAO Fu-Kai
3
, DONG Xiao-Ping
2
,
LU Qing1 , CHENG Yong-Xian1 *
( 1 State Key Laboratory of Phytochemistry and Plant Resources in West China , Kunming Instituteof Botany, ChineseAcademy of Sciences,
Kunming 650204 , China; 2 Collegeof Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 610075 , China;
3 Department of Microbiology and Immunology, Kunming Medical College, Kunming 650031 , China)
Abstract: A newtriterpene, ursane-20 (30 ) -en-3β, 16β, 21α-triol ( 1) , togetherwith twoknowncompounds, taraxaster-
ol acetate (2) and taraxasterol (3) , was isolatedfromthewholeplants of Centipeda minima . Their structureswere identi-
fied by spectroscopic analysis . Antibacterial properties of compounds 2 and 3 wereevaluated against eight disease-associat-
ed microorganisms by the agar dilution method, Both of them displayed potential antibacterial activities .
Key words: Centipeda minima; Compositae; Triterpenes; Antibacterial activity
Centipeda minima ( L .) is a Compositae plant
distributingover south Asia and Oceania (Shi and Fu,
1983) . It has been using as an important medicinal
herb for the treatment of cold, nasal allergy, diarrhea,
malaria, and asthma in China ( J iangsu New Medical
College, 1992 ) . Previous studies revealed that fla-
vonoids, sesquiterpenes are the main bioactive constit-
uents (Wu et al , 1985 , 1991; Iwakami et al , 1992;
Taylor and Towers, 1998) . During our search for new
antimicrobial agents fromtraditional Chinesemedicine,
C. minima was investigated . Fromthe ethanol extracts
of the whole plants, three usane-type triterpenes, in-
cluding anew naturally occurring compound, were pu-
rified and structurally characterized . In this paper, we
describe the isolation and structural identificationof the
new compound, and antibacterial properties of com-
pounds 2 and 3 .
Compound 1 was obtained as a colorless crystal .
The HREIMS at m?z481 .3665 [M + Na] + (calcd for
C30 H50 O3 Na 481 .3657) established the molecular for-
mula of 1 asC30 H50 O3 . Besides adoublebond, the
13
C
NMR andDEPT spectraof 1 (Table1 ) is characteristic
of an ursane-type triterpene . The molecular of 1 is 32
Da larger than that of 3 , indicating thepresenceof two
云 南 植 物 研 究 2007 , 29 (4) : 479～482
Acta Botanica Yunnanica

?
?? ?Author for correspondence; E-mail : yxcheng@mail .kib. ac. cn; Tel?Fax . + 86 - 871 - 5223048
Received date: 2007 - 02 - 02 , Accepted date: 2007 - 04 - 16
作者简介 : 梁恒兴 (1978 - ) 男 , 博士 , 主要从事中药化学研究。 ?
Foundation item: Supported by theNatural Science Foundation of Yunnan (2006B0041Q) and“Xi-Bu-Zhi-Guang”Project fromthe Chinese Acade-
my of Sciences
more hydroxyl groups in 1 , which are attributed to C -
16 atδ76 .1 and C - 21 atδ70 .4 , respectively, by
thefollowingHMBC interactions: H - 15 (δ1 .97 ) with
C - 14 (δ42 .6) , C - 16 and C - 27 (δ16 .6) , H - 16
(δ3 .83) with C - 22 (δ47 .1) and C - 28 (δ13 .0 ) ,
H - 28 (δ1 .19) with C - 17 (δ40 .5 ) , C - 22 and C
- 16 , H - 22 (δ3 .08 , 2 .01) with C - 16 , C - 17 , C -
18 (δ47 .8 ) , C - 28 , C - 20 (δ157 .7 ) and C - 21 (δ
70 .4) , H - 21 (δ4 .82 ) with C - 22 , C - 17 , C - 19
(δ38.7 ) , C - 20 and C - 30 (δ111 .7 ) . The stereo-
chemistry of C16 -OH and C21 -OH was determined to be
β, α, respectively, on the basis of the followingNOE-
SY interactions: H - 28 with H - 21 (δ4 .82) and H -
22β (δ3 .08 ) , H - 16 with H - 22α (δ2 .01) and H -
29 (δ1 .55) . Theβconfiguration of C3 -OH was deter-
minedby comparison of the chemical shift of C - 3 (δ
78 .1) with those reported data, and further supported
by the observation of HMBC correlation of H - 3 (δ
3 .47) with H - 24 (δ0 .89 ) . Therefore, the structure
of 1 was assigned as 20 ( 30 )-taraxasten-3β, 16β,
21α-triol . It was noted that the ester forms of 1 have
been previously isolated from Arnica lonchophylla
(Schmidt et al , 2004) . During the structural elucida-
tion of 20 ( 30 )-taraxastene-3β, 16β, 21α-triol 3-
laurate, myristate, -palmitate, and -stearate, Schmidt
and coworker speculated these esters having a same
unit by MS fragment at m?z458 , and thus named this
unit as arnitriol A (Schmidt et al , 2004 ) . However,
1 , as a natural product, was not previously isolated .
Two known triterpeneswere identified as taraxasterol
acetate (2) (Reynolds et al , 1986) , and taraxasterol (3)
(Reynolds et al , 1986) , respectively, by comparison of
their spectroscopic data with literature values . They were
isolated from C. minima for the first time .
Bioassay revealed that compounds2 and 3 exhibit-
ed antimicrobial effects against some bacteria investi-
gated (Table 2) . Compound 2 was found to bemost ef-
fective against Salmonella typhimurium and S. pa-
ratyphi-A with the MIC of 6 .25μg?mL comparable to
that of cefradine and gentamycin with the MIC of 7 .5
μg?mL and 3 .25 μg?mL , respectively . Compound 3
could inhibit thegrowth of Staphylococcus aureus, Es-
cherichia coli , and S. typhimuriumwith the MIC value
of 50μg?mL . However, theother microorganisms were
not sensitive to these two compounds even at the con-
centration of 100μg?mL .
Experimental
General Experimental Procedures Melting points was
obtained on an XRC-1 micromelting apparatus . Optical rotations
was determined on a JASCO-20C digital polarimeter . UV spectra
was recorded on a Shimadzu UV-2401PC spectrophotometer . IR
spectra was obtained with a Bruker Tensor 27 FT-IR spectropho-
tometerwith KBr pellets . 1 H NMR (400 MHz) and 13 C NMR
(100 MHz) spectra were recorded on a Bruker AM-400 spec-
trometer with TMS as an internal reference . 2D NMR spectra
were measured with a DRX-500 spectrometer . EIMS ( 70 eV )
were recorded on a VG Auto Spec-3000 spectrometer . ESIMS
and HRESIMS were carried our with an API QSTAR Pulsar 1
spectrometer . Silica gel ( 200 - 300 mesh and 10 - 40μm) for
column chromatography and GF254 for TLC were obtained from
Qingdao Marine Chemical Factory, Qingdao, People’s Republic
of China . Sephadex LH-20 was obtained from Amersham Phar-
macia Biotech, Sweden . RP-18 silica gel (40 - 63μm) used for
open column chromatographywas purchased fromDaisoCo ., Ja-
pan . Diaion HP20 and MCI gel CHP 20P ( 75 - 150μm) were
obtained from Mitsubishikasei , Tokyo, Japan . Fractions were
monitored by TLC and spots were visualized after spraying with
10% H2 SO4 inethanol or anisaldehyde reagent followed by heat-
ing .
Plant Material Thewhole plants of C. minima were pur-
chased from Yunnan Corporation of Materia Medica, Yunnan
province, People’s Republic of China, and identifiedbyMr . H .
Y . Sun at Yunnan Corporation of Materia Medica . A voucher
specimen ( CHYX0159 ) was depositedat the State Key Laborato-
ryof Phytochemistry and Plant Resources in West China, Kun-
ming Institute of Botany, Chinese Academy of Sciences .
084 云 南 植 物 研 究 29 卷
Table 1 1 H and 13 C NMR data for compound 1 in C5 D5 N (δ in ppm, J in Hz)a
No . 1 ?H 13 C No . 1 H 13 C
1 ?0 ?. 97 ( m) 39 .5 16 3 .83 ( dd, 11 .4 , 4 L. 0) 76 .1
2 ?1 ?. 88 ( m) 28 .3 17 40 .5
3 ?3 ?. 47 ( br t, 8 . 0 ) 78 .1 18 1 .59 ( m) b 47 .8
4 ?0 ?. 80 ( m) 39 .2 19 2 .41 ( m) 38 .7
5 ?55 ?. 9 20 157 .7
6 ?a 1 ?. 56 ( m)b 18 .7 21 4 .82 ( dd, 8 t. 8 , 4 . 4) 70 .4
6 ?b 1 ?. 40 ( m)c 22a 3 .08 ( dd, 13 .6 , 8 L. 8) 47 .1
7 ?1 ?. 43 ( m)c 34 .5 22b 2 .01 ( dd, 13 .6 , 4 L. 4)
8 ?41 ?. 3 23 1 .22 (s) 28 .6
9 ?1 ?. 37 ( m) 50 .3 24 0 .89 (s) 16 .6
10 ?36 ?. 9 25 1 .05 (s) 16 .1
11 ?1 ?. 58 ( m)b 21 .7 26 1 .05 (s) 16 .4
12 ?1 ?. 77 ( m) 26 .6 27 0 .89 (s) 16 .6
13 ?1 ?. 72 ( m) 39 .1 28 1 .19 (s) 13 .0
14 ?42 ?. 6 29 1 .55 ( d, 6 O. 8) b 28 .1
15 ?1 ?. 97 ( m) 37 .3 30 7 .56 ( br s) , 7 . 19 ( br s) 111 .7
a: The spectra were obtained at 400 MHz for 1 H and 100 MHz for 13 C . b, c: Signals with the same superscripts are overlapped .
Table 2 Antibacterial activities of compounds 2 and 3 ( MIC values, μg?mL)
Pathogen 2 ?3 cefradine gentamycin
Reference strains
S ?. aureus CMCC26001 > 100 b50 ?15 7 .5
E ?. coli CMCC44103 > 100 b50 ?7 .5 7 .5
S ?. typhimuriumCMCC80087 6 .25 50 ?7 .5 7 .5
S ?. flexneri CMCC51335 > 100 b> 100 D3 .25 3 .25
Clinically isolated strains
S ?. epidermidis > 100 b> 100 D3 .25 7 .5
B ?. subtilis > 100 b> 100 D3 .25 3 .25
S ?. paratyphi-A 6 .25 > 100 D3 .25 3 .25
S ?. paratyphi-B > 100 b> 100 D3 .25 3 .25
Extraction and Isolation Dried and powdered whole
plant materials of C. minima (10 kg) were extracted with 95%
EtOH under reflux for three times, the extracts were evaporated
and suspended into water followed by successive partition with
petroleumether, EtOAc and n-BuOH, respectively . The EtOAc
extracts (170 g) were subjected to column chromatography (CC)
over silica gel ( 200 - 300 mesh) and eluted with CHCl2 -MeOH
(8∶1) to givefractions 1 - 4 . Fraction2 ( 30 g) was chromatogr-
amphed on Diaion HP20 ( 95% EtOH ) to decolor, the eluents
were then subjected to CC on silica gel ( 200 - 300 mesh) , elut-
ing with CHCl3 -MeOH (1∶0 - 5∶1) to give fractions 2 .1 - 2 .8 .
Fraction 2.2 ( 10 g) was passed through MCI gel CHP 20P
(MeOH-H2 O, 9∶1) to decolor, and then subjected to Sephadex
LH-20 (MeOH) , RP-18 (MeOH-H2 O 1∶1→1∶0 ) and repeated
vacuumliquid chromatography (VLC) to yield 1 ( 11 mg) . The
petroleumether extracts (197 g) were subjectedtoCC over silica
gel ( 200 - 300 mesh) and elutedwith petroleumether-EtOAc (3∶
1 ) togive fractionsA-E . Fraction A (35 g) was subjectedtore-
peated CC on silicagel , eluting with petroleumether-EtOAc (1∶
0 - 0∶1) to afford acolorless crystal 2 (570 mg) . Fraction C ( 26
g) was repeatedly chromatographed on silica gel ( 200 - 300
mesh) with petroleum ether-Me2 CO (50∶1 - 10∶1 ) as eluent to
afford six fractions C1 - C6 . Fraction C2 ( 4 g) was passed
through MCI gel CHP 20P (MeOH-H2 O, 9∶1) to decolor , fol-
lowed by Sephadex LH-20 chromatography (CHCl3 -MeOH, 6∶4)
and repeated VLC to yield 3 (36 mg) .
Compound 1: colorless crystal; mp: 239 - 240℃ ; [α]21D
+ 65 .0 ( c 0 .1 , CHCl3?MeOH 2∶1) ; UVλCHCl3max ( logε) nm:
248 ( 2 .51 ) ; IR νKBrmax cm- 1 : 1639 , 1444 , 1388; 1 H NMR
(C5 D5 N, 400 MHz) and 13 C NMR ( C5 D5 N, 100 MHz) , see
Table 1; EIMS m?z 458 [M] + ( 4) , 440 (4 ) , 422 (3 ) , 299
(54) , 207 (55) , 189 (100) , 175 (30) , 161 (32) , 149 (42) ,
135 ( 88 ) , 121 (98) , 107 (98) , 95 (93) , 81 (78) , 69 (76) ,
55 (81) ; HRESIMS m?z 481.3665 [M+ Na] + ( calcd for C30
H50 O3 Na 481 .3657) .
Bioassay Antibacterial activity was tested by agar dilution
method ( Baker et al, 1994 ) . The bacterial strains employed
were Staphylococcus aureus CMCC26001 ( CMCC , National Cen-
ter for Medical Culture Collections, Beijing, China ) , Es-
cherichia coli CMCC44103 , Salmonella typhimurium CM-
1844 期 LIANG Heng-Xing et al .: Antibacterial Triterpenes from Centipeda minima (Compositae)
CC80087 , and Shigella flexneri CMCC51335 , and the following
clinically isolated strains: Staphylococcus epidermidis, Bacillus
subtilis, Salmonella paratyphi-A , Salmonella paratyphi-B . The
bacterial strainswereremovedfromstorage kept in - 70℃ refrig-
erator and streaked onto tryptone soy agar (TSA , Oxoid) plates,
and then incubated for 18 to24 hour at 37℃ . The inoculumwas
prepared by culturing each isolated bacterial colony in brain heart
infusion broth (Oxoid) at 37℃ to a turbidity equivalent to Mc-
Farland 0 .5 standard (1 . 0×108 CFU?mL) and subsequently di-
luting the organism to 1 .0×106 CFU?mL for susceptibility test-
ing . For agar dilution tests, compounds ( 2 mg each) were first
dissolved in 0 .2 mL DMSO, serial dilutions of test compounds
(ten serial two-fold dilutions per compound) were prepared as
described by CLSI ( Clinical and Laboratory Standards Institute,
Wayne, PA , USA) ( formerly NCCLS, National Committee for
Clinical Laboratory Standards) ( Clinical and Laboratory Stan-
dards Institute, 2006 ) . Nine mililiters of molten ( 48℃) Muel-
ler-Hinton agar was addedto eachmilliliter of dilutedcompound,
mixed completely, and put into plates . With a Steers replicator,
an organism density of 104 CFU?spot was inoculated onto the ap-
propriate plate with various concentrations of test compounds
(range of final concentrations: 0 .195 - 100μg?mL) . The plates
were incubated overnight in ambient air at 37℃ for 24 hours .
The minimum inhibition concentration was taken as the lowest
concentration that inhibited visible growth after incubation at
37℃ for 24 h . Cefradine and gentamycin were used as reference
standards in order to control the sensitivity of the test strains .
Plates containingonlyMHA andMHA and1% DMSO inmedium
served as negativeand solvent controls . Tests were performed in
triplicate and repeated once .
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