全 文 ：Constituents from Ranunculus sieboldii Miq.
PAN Yun-xue1 , 2 , ZHOU Chang-xin1 , ZHANG Shui-li1 , ZHENG Xiao-xiang2 , and ZHAO Yu1＊
(1 .Department of Traditional Chinese Medicine and Natural Drug Research , College of Pharmaceutical Sciences ,
Zhejiang University , Hangzhou 310031 , China ;2 .Department of Biomedical Engineering , College of Biomedical Engineering
and Instrument Sciences , Zhejiang University , Hangzhou 310006 , China)
Abstract:Aim　To investigate the chemical composition of Ranunculus sieboldii Miq..Methods　Repeated column
chromatography over silica gel , polyamide and RP-18 followed by gel filtration on sephadex LH-20 were used to isolate
chemical constituents , and their structures were elucidated by extensive spectroscopic methods (UV , IR , MS , 1H NMR ,
13C NMR)including 2D NMR(COSY , HMQC , HMBC , NOESY)techniques and by direct comparing spectral data with
those reported in literature.Results　Five flavonoid glycosides named apigenin-4′-O-α-L-rhamnopyranoside(1), apigenin-
7-O-β-D-glucopy ranosyl-4′-O-α-L-rhamnopyranoside(2), apigenin-8-C-α-L-arabinopy ranoside(3), apigenin-8-C-β-D-ga-
lactopyranoside(4), tricin-7-O-β-D-glucopyranoside(5), together with tricin(6), luteolin(7), scopoletin(8), esculetin
(9), scoparone(10), ferulic acid(11), protocatechuic acid(12), and ternatolide(13)were isolated from the 95% etha-
nolic extract of its whole plant , and their cytotoxic activities were preliminarily tested.Conclusion　Compounds 1-12 were
obtained from this genus and compound 13 from this species for the first time.Furthermore , compound 1 was for the first
time isolated from nature while the 13C NMR data of compounds 2 and 3 are reported for the first time.The bioassay revealed
that compound 1 was active against BEL-7407 and A549 cell lines(IC50 43 , 77μg·mL-1), 8 and 10 showed inhibitory ac-
tivities on KB cell lines(IC50 78 , 44μg·mL-1)and HL-60 cell lines(IC50 85 , 85μg·mL-1), while 7 exerted moderate
cytotoxic activities on KB , BEL-7407 , A549 and HL-60 cell lines with their IC50 being 51 , 55 , 44 and 10 μg·mL-1 , re-
spectively.
Key words:Ranunculus sieboldii Miq.;flavonoid glycosides;apigenin-4′-O-α-L-rhamnopyranoside;apigenin-8-C-α-
L-arabinopy ranoside;apigenin-7-O-β-D-glucopyranosyl-4′-O-α-L-rhamnopyranoside;cytotoxicity
CLC number:R284.1;R284.2;R965　　　Document code:A　　　Article ID:1003-1057(2004)2-092-05
Introduction
Ranunculus sieboldii Miq.belonging to the Ranun-
culaceae family is distributed widely in China and tradi-
tionally used in the treatment of various diseases such as
jaundice , hepatitis , tuberculosis , sarcoma , stomachache
and toothache in local people
[ 1] .Pharmacologic tests re-
vealed that the plant has antibacterial and antihistaminic
activities
[ 1] .So far , little is known about its chemical
constituents.In a search for its biologically active com-
pounds , our phytochemical investigation on the title plant
led to the characterization of thirteen compounds including
five flavonoid glycosides , apigenin-4′-O-α-L-rhamnopyr-
anoside(1), apigenin-7-O-β-D-glucopyranosyl-4′-O-α-L-
Received date:2003-11-21.
＊Corresponding author:Tel 0571-87217313 , Fax 0571-87217313 ,
E-mail dryuzhao@zju.edu.cn
rhamnopyranoside (2), apigenin-8-C-α-L-arabinopyr-
anoside (3), apigenin-8-C-β-D-galactopyranoside (4)[ 2] ,
and tricin-7-O-β-D-glucopyranoside (5)[ 3] , two flavones
tricin(6)[ 3] and luteolin(7)[ 4] , three coumarins , scopo-
letin (8)[ 5] , esculetin (9)[ 5] , and scoparone (10)[ 5] ,
two phenolic acids , ferulic acid (11)[ 6] and protocate-
chuic acid(12)[ 7] , and one lactone , ternatolide(13)[ 8] .
Compounds 1 -12 were obtained from this genus and
compound 13 from this species for the first time.Among
them compound 1 was for the first time isolated from na-
ture , and the 13 C NMR data of compounds 2 and 3 are
firstly reported.This paper describes the isolation and
characterization of the three flavonoid glycosides , and the
preliminary tests of in vitro anticancer activities of com-
pounds 1-13.
Results and Discussion
The 95% ethanolic extract of R.sieboldii Miq.was
92 Journal of Chinese Pharmaceutical Sciences 2004 , 13(2)
partitioned successively between petroleum ether , ethyl
acetate , saturated aqueous n-butanol and water.The
EtOAc and BuOH soluble fractions were subjected to re-
peated column chromatography to afford thirteen com-
pounds.
Compound 1 was isolated as pale yellow amorphous
powder.The positive Mg-HCl color and Molish tests indi-
cated 1 to be a flavonoid glycoside.In the 1H NMR spec-
trum , the typical signal atδ12.90 (1H , brs)indicated
the presence of a chelated hydroxyl in the 5-position of ring
A.A pair of doublets atδ6.20(1H , d , J =2.0Hz)and
δ6.50(1H , d , J =2.0 Hz), characteristic of H-6 and
H-8 of 5 ,7-dihydroxyflavonoid[ 9] , as well as a pair of dou-
blets atδ8.04(2H , d , J =8.8Hz)andδ7.21(2H , d ,
J =8.8 Hz)ascribed to H-2′, 6′and H-3′, 5′in ring B ,
and a singlet atδ6.87(1H , s)assigned to H-3 in ring C ,
disclosed the aglycon to be 5 ,7 ,4′-trihydroxyflavone , i.e.
apigenin.A quasimolecular ion peak at m z 417 in its
positive ESI-MS spectrum and a base peak at m z 271 in
the ESI-MS-MS spectrum suggested the presence of a rh-
amnosyl moiety.This functional group was shown to beα-
L-rhamnopyranose by comparing its 13 C NMR chemical
shifts with those reported in the literature
[ 10] .The attach-
ment of the rhamnopyranosyl moiety was deduced to be at
C-4′according to glycosydation rule[ 11] .The conclusion
was further confirmed by the HMBC spectrum in which the
anomeric proton of the rhamnopyranosyl moiety at δ5.53
showed long range correlation with C-4′(δ159.5).From
the above evidence , 1 was established as 4′-O-α-L-rham-
nopyranosyl-apigenin[ 12] .
Compound 2 was obtained as pale yellow amorphous
powder.The positive ion ESI-MS spectrum displayed a
quasimolecular ion peak at m z 579 , which was 162 amu
higher than that of 1 , suggesting the presence of an addit-
ional glucosyl moiety.The 1H NMR , 13C NMR in associ-
ation with 2D NMR spectral analysis was consistent with
the structure of 7-O-β-D-glucopyranosyl-4′-O-α-L-rham-
nopyranosyl-apigenin[ 13] .This was the first report of its
13
C NMR data.
Compound 3 was obtained as yellow crystals , with
the molecular formula determined to be C20H18O 9 by its
positive ESI-MS spectrum exhibiting a protonated ion peak
at m z 403.The similarity of its UV spectrum to that of
1 , along with its 13C NMR spectrum in which typical car-
bon signals for α-L-arabinose resonated at δ74.8 , 68.6 ,
75.0 , 69.3 , and 71.2[ 14] suggested that 3 is most likely
an α-L-arabinopyranoside of apigenin.Furthermore , a
singlet atδ6.27(1H , H-6)and the characteristic ano-
meric proton signal atδ4.63(1H , d , J =9.2Hz)in its
1
H NMR spectrum revealed that compound 3 is probably
an 8-C-α-L-arabinopyranoside of apigenin , in accordance
with its property that acid hydrolysis of 3 failed to give
sugar molecule.This supposition was finally confirmed by
the HMBC spectra in which the arabinosyl H-1 signal
(δ=4.63)showed long range correlation with C-8 (δ=
104.7).Therefore , the structure of 3 was identified as
apigenin-8-C-α-L-arabinopyranoside[ 15] .Its 13C NMR da-
ta is reported for the first time.
Compounds 1-13 were preliminarily tested in vitro
by MTT assay
[ 16]
on four different human tumor cell lines ,
KB , BEL-7407 , A549 and HL-60 cell lines , with diam-
minedichloroplatinum(DDP)as the positive control.The
results showed that some of them exhibited cytotoxic activ-
ities to a certain extent(Table 1).It seemed that the pr-
esence of phenyl-methoxyl group of coumarins (Com-
pounds 8-10)might enhance the cytotoxicity to KB cell
lines , while the decrease of free hydroxyl group of f la-
vonoids(Compounds 6 , 7)could lower the inhibitory ef-
fects on the four tested cell lines.
Table 1　Cytotoxicity data of compounds 1-13 in vitro (IC50μg·mL-1)
Tumor cells 1 2 3 4 5 6 7 8 9 10 11 12 13 DDP
KB ─ ─ ─ ─ ─ ─ 51 78 ─ 44 ─ ─ ─ 2.7
BEL-7407 43 ─ ─ ─ ─ ─ 55 ─ ─ ─ ─ ─ ─ 9.8
A549 77 ─ ─ ─ ─ ─ 44 ─ ─ ─ ─ ─ ─ 8.7
HL-60 ─ ─ ─ ─ ─ ─ 10 85 ─ 85 ─ ─ ─ 0.2
─:IC50 >100μg·mL -1
93Journal of Chinese Pharmaceutical Sciences 2004 , 13(2)
Experimental
General
Melting points were determined on X-4 digital melt-
ing point instrument.Optical rotations were measured on
a Polax-2L polarimeter.UV spectra were recorded with
UV-1600 spectrophotometer and IR spectra with Bruker
Vector-22 spectrometer.MS spectra were examined on
Esquire-LC-00075 mass spectrometer.NMR were mea-
sured on Inova NMR spectrometers using TMS as an inter-
nal standard with
1
H and
13
C-nuclei observed at 400 and
100 MHz , respectively.
The following experimental conditions were used for
chromatography:silica gel (Qingdao Marine Chemical
Factory);polyamide (Shanghai Chemical Reagent Com-
pany);sephadex LH-20 (Amersham Pharmacia Biotech
AB);RP-18 (Merck Company);macroporous resin D
101(Tianjin Resin Co.Ltd.).TLC was carried out on
silica gel GF254 and polyamide film.
Plant material
The whole plant of R .sieboldii was collected from
Hangzhou , Zhejiang Province , China in June 2001.It
was taxonomically authenticated by lecturer Zhang Shui-li
from the Department of Pharmacognosy , College of Phar-
maceutical Sciences , Zhejiang University and a herbarium
specimen was deposited there.
Extraction and isolation
The air dried and pulverized whole plant of
R .sieboldii (17 kg)was percolated with 95% ethanol
(50 L×4)at room temperature.Evaporation of the sol-
vent under reduced pressure gave the crude extract(2.60
kg), which was then suspended in warm water and parti-
tioned successively with petroleum ether , ethyl acetate ,
and saturated aqueous n-butanol.The n-BuOH soluble
fraction(360 g)was subjected to silica gel column (4.0
kg), gradient elution with CHCl3-MeOH (0%※100%
MeOH), and afforded ten fractions according to TLC re-
sults.Fraction 3 containing compounds 1 and 3 was sepa-
rated by polyamide column using a gradient of MeOH in
H2O whereas compound 1 was obtained from the 50%
MeOH elution and compound 3 from the 30%MeOH elu-
tion.Then , compounds 1 and 3 were applied to a sepha-
dex LH-20 column and eluted with MeOH , separately.
Finally , compound 1 was purified by recrystallization and
3 using preparative TLC plates.Fraction 8 was further
subjected to a sephadex LH-20 column eluted withMeOH
to afford compound 2.Fraction 5 and fraction 6 were re-
peatedly chromatographed on polyamide , RP-18 columns
and sephadex LH-20 column to give compounds 4 and 5 ,
respectively.The other eight compounds were all obtained
from EtOAc soluble fraction by repeated chromatography
on silica gel and finally purified by recrystallization.
Structure identification
Compound 1 　Pale yellow amorphous powder
(MeOH), mp 247 -249℃;[ α] 28D -222°(C 0.54 ,
DMSO);UV (MeOH )nm:271 , 323 ;IR (KBr)
cm
-1:3270(OH), 1656 (C=O), 1607 , 1502 (C=
C);ESI-MS (Pos)m z:417 [M+H] + , 271 [M+H-
rham] +;1H NMR(DMSO-d6)δ:1.10(3H , d , J =
6.1Hz , CH3 of rhamnose), 5.53(1H , brs , H-1 of rh-
amnose), 6.20(1H , d , J =2.0Hz , H-6), 6.50(1H ,
d , J =2.0 Hz , H-8), 6.87(1H , s , H-3), 7.21(2H ,
d , J =8.8 Hz , H-3′, 5′), 8.04(2H , d , J =8.8 Hz ,
H-2′, 6′), 12.90(1H , brs , 5-OH);13C NMR(DMSO-
d6)δ:163.6 (C-2), 104.3 (C-3), 182.3 (C-4),
162.0(C-5), 98.6(C-6), 164.7(C-7), 94.5(C-8),
157.8 (C-9), 104.4 (C-10), 124.4 (C-1′), 128.8
(C-2′, 6′), 117.2 (C-3′, 5′), 159.5 (C-4′), 99.4 ,
70.5 , 70.9 , 72.2 , 70.3 , 18.4 (C-1′′-C-6′′, rham-
nose).
Compound 2 　Pale yellow amorphous powder
(MeOH), mp 212-214℃;[ α] 28D -185°(C 0.73 ,
DMSO);UV (MeOH )nm:271 , 318;IR (KBr)
cm
-1:3418(OH), 1664 (C=O), 1607 , 1496 (C=
C);ESI-MS (Pos)m z:579 [M+H] + , 433 [M+H-
rham] + , 417 [M+H-glc] + , 271 [M+H-glc-rham] +;
1
H NMR(DMSO-d6)δ:1.10 (3H , d , J =6.1 Hz ,
CH3 of rhamnose), 5.08(1H , d , J =7.9 Hz , H-1 of
glucose), 5.54 (1H , brs , H-1 of rhamnose), 6.46
(1H , d , J =2.0 Hz , H-6), 6.86 (1H , d , J =2.0
Hz , H-8), 6.96(1H , s , H-3), 7.22(2H , d , J =8.9
Hz , H-3′, 5′), 8.07(2H , d , J =8.9 Hz , H-2′, 6′),
12.89 (1H , brs , 5-OH);13 C NMR (DMSO-d6)δ:
164.2(C-2), 104.7(C-3), 182.6 (C-4), 161.6 (C-
5), 100.1(C-6), 163.6 (C-7), 95.4 (C-8), 157.5
(C-9), 105.9 (C-10), 124.3 (C-1′), 128.9 (C-2′,
6′), 117.2(C-3′, 5′), 159.6 (C-4′), 100.4 , 73.6 ,
94 Journal of Chinese Pharmaceutical Sciences 2004 , 13(2)
77.0 , 70.1 , 77.7 , 61.1 (C-1′-C-6′, glucose);
98.7 , 70.5 , 70.9 , 72.2 , 70.3 , 18.4 (C-1″′-C-6″′,
rhamnose).
Compound 3　Yellow crystals(MeOH), mp 202-
204℃;[α] 28D -90°(C 0.50 , DMSO);UV (MeOH)
nm:269 , 330;IR (KBr)cm-1:3170 (OH), 1656
(C=O), 1613 , 1506 (C =C);ESI-MS (Pos)m z:
403 [ M +H] + , 385 [ M +H-H2O ] + , 367 [M +H-
2H2O] + , 284 [M-B1] +;1HNMR(DMSO-d6)δ:4.63
(1H , d , J =9.2 Hz , H-1 of arabinose), 6.27(1H , s ,
H-6), 6.79(1H , s , H-3), 6.90(2H , d , J =8.8Hz ,
H-3′, 5′), 8.21 (2H , d , J =8.8 Hz , H-2′, 6′),
13.30(1H , brs , 5-OH );13 C NMR (DMSO-d6)δ:
164.3(C-2), 102.3(C-3), 182.3(C-4), 160.7(C-
5), 98.7(C-6), 162.9(C-7), 104.7 (C-8), 156.0
(C-9), 104.1 (C-10), 121.4 (C-1′), 129.6 (C-2′,
6′), 116.2 (C-3′, 5′), 161.3 (C-4′), 74.8 , 68.6 ,
75.0 , 69.3 , 71.2(C-1′′-C-5′′, arabinose).
Figure 1　Structures of compounds 1-3
Acknowledgements
The authors wish to thank the Post-doctoral Founda-
tion Committee of China for the grant.One of the au-
thors , Prof.Zhao Yu , would also express his gratefulness
to Chinese Ministry of Education and Mr.Li Ka-shing for
the Cheung Kong Scholars Chair Professorship at Zhejiang
University , for the financial support in the work.Dr.Li
Hai-bo and Yang Lei-xiang , Laboratory of Biotechnology ,
Zhejiang University , are gratefully acknowledged for the
in vitro anticancer activity assay.
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扬子毛茛中的化学成分研究
潘云雪1 ,2 , 周长新1 , 张水利1 , 郑筱祥2 , 赵　昱1
(1.浙江大学药学院中药与天然药物研究室 , 杭州 310031;
2.浙江大学生物医学工程与仪器科学学院 , 杭州 310006)
摘要:目的　研究扬子毛茛的化学成分 。方法　应用多种色谱方法和色谱材料进行提取 、分离和纯化 , 用各种现代光谱
方法并结合文献对分得的化合物进行结构解析。结果　从扬子毛茛的 95%乙醇提取物中分到十三个化合物 , 其中五个为黄
酮苷类化合物 ,分别命名为芹菜素-4′-O-α-L-鼠李糖苷(1), 芹菜素-7-O-β-D-葡萄糖苷-4′-O-α-L-鼠李糖苷(2), 芹菜素-8-C-α-L-阿
拉伯糖苷(3),芹菜素-8-C-β-D-半乳糖苷(4), 小麦黄素-7-O-β-D-葡萄糖苷(5);其余八个分别为小麦黄素(6), 木犀草素(7),东莨
菪内酯(8),秦皮乙素(9), 滨蒿内酯(10),阿魏酸(11), 原儿茶酸(12)和小毛茛内酯(13)。此外还对这些化合物的体外抗癌活性
进行了初步的测试。结论　化合物 1-12为首次从该属植物中分到 , 化合物 13 为首次从该种植物中分到 , 其中化合物 1为新
的天然产物 ,化合物 2 和 3首次报道了其碳谱数据。生物活性测试结果表明化合物 1 对 BEL-7407 及 A549 细胞的 IC50值分别
为 43及 77μg·mL-1 , 化合物8 和 10对 KB细胞的 IC50分别为 78和 44μg·mL-1 , 对 HL-60 细胞的 IC50均为85 μg·mL-1 。化合物
7 对所测试的四种肿瘤细胞株均显示一定的细胞毒活性 , 其对 KB , BEL-7407 , A549 及 HL-60 细胞的 IC50分别为 51 , 55 , 44 和
10 μg·mL-1 。
关键词:毛茛;黄酮苷;芹菜素-4′-O-α-L-鼠李糖苷;芹菜素-8-C-α-L-阿拉伯糖苷;芹菜素-7-O-β-D-葡萄糖苷-4′-O-α-L-鼠
李糖苷;细胞毒性
96 Journal of Chinese Pharmaceutical Sciences 2004 , 13(2)