全 文 ：Studies on the Chemical Constituents of
Euphorbia hirta
Yi WANG, Jinhe JIANG, Yegao CHEN, Yong ZHAO*
College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, China
Supported by the National Natural Science Foundation of China (21162044); Yunnan
Natural Science Foundation (2009CD051); Middle-aged and Young Talents Program of
Yunnan Province (2010CI040) .
*Corresponding author. E-mail: zhaooy@126.com
Received: November 22, 2011 Accepted: December 12, 2011A
Abstract [Objective] The aim was to study the chemical constituents of Euphorbia
hirta. [Method] The constituents were isolated and purified by various chromatogra-
phy methods including normal phase and reverse phase silica gel as well as
Sephadex LH-20 gel column, and then elucidated by spectroscopic analysis such as
MS and NMR etc. Compounds 1, 4, 5 and 6 were evaluated for their immunostimu-
latory activity against splenic cell of mouse by FCM. [Result] Six compounds were
isolated from Euphorbia hirt, and identified as diisobutyl-O-phthalate (compond 1), di-
ethylhexyl phthalate (compond 2), hispidulin (compond 3), acetyl peroxide (compond
4), quercetein (compond 5) and gallic acid (compond 6). All compounds were inac-
tive against splenic cell of mouse. [Conclusion] Compounds 1, 2 and 3 were isolated
from this plant for the first time, and compound 4 was the first naturally occurring
compound. This study provided reference for isolating bioactive substances from
Euphorbia hirta.
Key words Euphorbiaceae; Euphorbia hirta; constituents; Immunological activities
E uphorbia plants have the effi-cacies of anti-anaphylaxis, anti-oxidation, anti-proliferation, anti-
inflammatory, antibacterial and cyto-
toxic activities[1-5]. Euphorbia hirta L., a
traditional Chinese medicine, is mainly
distributed in south and southwest Chi-
na[6], and can be used for treating gas-
trointestinal diseases (such as diar-
rhea, dysentery and intestinal para-
sites, etc.), bronchitis, conjunctivitis
and respiratory diseases (like asthma,
hay fever, etc .) [7-8]. Previous studies
have shown that Euphorbia hirta con-
tains hydrocarbons, diterpenes, triter-
penes, phytocholesterol, tannin, polyp-
henols and flavonoids[9-12].
In order to search for bioactive
substances from Euphorbia hirta, its
chemical constituents were investigat-
ed, and six compounds were obtained
in this study. The extraction and sepa-
ration of the compounds, as well as
the structure identification and the im-
munological activity of some com-
pounds were illustrated in detail in this
study.
Materials and Methods
Materials
Plant materials
Plant sample was collected from
Yunnan Xishuangbanna in September
2009, and identified as Euphorbia hirta
by Professor Yaowen YANG from
Yunnan University of TCM; the speci-
men (YTCM 20090909) was stored in
Yunnan University of TCM.
Instruments and reagents
WZZ-2B-type optical rotation de-
tector; Bio-Rad FTS-135 infrared
spectrometer; Bruker DRX-500 su-
perconducting NMR; VG Auto Spec-
3000 mass spectrometer; column
chromatography silica gel (100 -200
mesh and 200 -300 mesh) was pro-
duced by Qingdao Marine Chemical
Factory; reverse phase silica gel RP-
18 (40-63 μm) was produced by Mer-
ck company in Germany; MCI gel CHP
20P (75-150 μm) was purchased from
the Mitsubishi Corporation in Japan;
Sephadex LH-20 column was pro-
duced by the Swedish Amersham
Biotechnology Company; thin-layer
chromatography material GF254 silica
gel plate was produced by Qingdao
Marine Chemical Factory.
Methods
Extraction and isolation of the
compounds Firstly, 4.0 kg of the
dried above-ground part of Euphorbia
hirta was ground, extracted with 30 L
of 95% ethanol at room temperature
for three times, followed by filtration
and evaporation. The crude extract
was dispersed in water and extracted
with ethyl acetate. After that, 280 g of
the extract was obtained and decol-
ored with MCI gel (90% CH3OH-H2O).
The 90% CH3OH fraction (173 g) was
chromatographed over silica gel (200-
300 mesh), eluted with chloroform-
acetone gradient (from 1∶0 to 0∶1), to
give six fractions (A, B, C, D, E and F).
Fractions A (6 g) was chromatogr-
aphed over silica gel again and eluted
with petroleum ether-acetone gradient
(from 99 ∶1 to 2 ∶1), to afford four frac-
tions (A1, A2, A3 and A4). Fraction A2
was chromatographed over silica gel
column repeatedly, eluted with petrol-
eum ether-acetone (49:1) to give 3 mg
of compound 1 and 2 mg of compound
2. Fraction A3 was chromatographed
over silica gel column and eluted with
petroleum ether-acetone (20∶1) to af-
ford 5 mg of compound 4.
Fraction C (5 g) was chro-
matographed over silica gel column,
eluted with petroleum ether-acetone
gradient (from 9∶1 to 1∶1) to abtain five
fractions (C1, C2, C3, C4 and C5).
Fraction C3 was chromatographed
over reverse phase silica gel RP-18
column and eluted with 45% methanol-
water, chromatographed again over
Agricultural Science & Technology, 2012, 13(2): 248-250
Copyright訫 2012, Information Institute of HAAS. All rights reserved Agronomy and Forestry
DOI:10.16175/j.cnki.1009-4229.2012.02.028
Agricultural Science & Technology
Vol.13, No.2, 2012 Agricultural Science & Technology
2012
Fig.1 Structures of the six compounds
Sephadex LH-20 gel column and elut-
ed with 100% methanol to give com-
pound 3(2 mg). Fraction C4 was chro-
matographed over reverse phase sili-
ca gel RP-18 column and eluted with
30% methanol-water, followed by
Sephadex LH-20 gel column and elut-
ed with 100% methanol to get com-
pound 5(20 mg).
The fraction D was chromatogra-
phed over silica gel column, eluted
with petroleum ether-acetone gradient
(from 4:1 to 0:1) to give four fractions
(D1, D2, D3 and D4). Then fraction D1
was chromatographed over reverse
phase silica gel RP-18 column and
eluted with 37% methanol-water to
obtain 3 mg of compound 6.
Activity test The immunostimulatory
activity of compounds 1, 4, 5 and 6
against splenic cell of mouse was
tested by FCM[13], with rapamycin (Ra-
pa) as positive control.
Results and Analysis
Structure identification
Compound 1 It was colorless oil
(ethyl acetate), and turned scarlet on
TLC with sulfuric acid-ethanol solution
as color regent, IR (KBr) νmax ( cm-1)
2 963, 2 876, 1 730, 1 581, 1 470,
1 377, 1 287, 1 123, 1 073, 981, 745;
1H-NMR (300 MHz, CDCl3) δ (ppm):
7.70 (dd, J= 8.2, 3.1 Hz, 2H, H-2, 5),
7.50 (m, 2H, H-3, 4), 4.06 (d, J = 6.7
Hz, 4H, H-1’, 1”), 2.02 (m, 2H, H-2’,
2”), 0.96 (d, J = 6.7 Hz, 12H, H-3’, 3”,
4’, 4”); 13C-NMR (75 MHz, CDCl3) δ
(ppm): 167.62 (C-7, 8), 132.38 (C-1,
6), 130.88 (C-2, 5), 128.81 (C-3, 4),
71.74 (C-1’,1”), 27.70 (C-2’, 2”),
19.13 (C-3’, 3”, 4’, 4”); ESI-MS m/z
(rel. int): 579 [2M + Na]+ (10), 301 [M +
Na]+ (100), 279 [M + H]+ ( 8). These
data agreed well with the literature val-
ues[14]. Compound 1 was identified as
diisobutyl-O-phthalate.
Compound 2 It was colorless oil
(ethyl acetate) and turned scarlet on
TLC with sulfuric acid-ethanol solution
as color reagent, [α]22.5D-7.97 (c 0.15,
MeOH); IR ( KBr) νmax (cm -1) 2 960,
2 929, 2 859, 1 730, 1 464, 1 381, 1 287,
1 275, 1 124, 1 073, 743; 1H-NMR (300
MHz, CDCl3) δ (ppm ): 7.70 (dd, J=
8.1, 3.0 Hz, 2H, H-2, 5), 7.53 (m, 2H,
H-3, 4), 4.22 (m, 4H, H-1’, 1”), 1.68
(m, 2H, H-2’, 2”), 0.92 (overlap, 6H,
H-6’, 6”), 0.89 (overlap, 6H, H-8’,
8”); 13C -NMR (75 MHz, CDCl3) δ
(ppm): 168.51 (C-7), 167.74 (C-8),
132.39 (C -1, 6), 130.85 (C -2, 5),
128.76 (C -3, 4), 68.12 (C -1’,1”),
38.68 (C-2’, 2”), 30.31 (C-3’, 3”),
28.88 (C-4’, 4”), 23.69 (C-7’, 7”),
22.95 (C-5’, 5”), 14.02 (C-6’, 6”),
10.92 (C-8’, 8”); ESI-MS m/ z (rel.
int): 803 [2M + Na]+ (100), 413 [M +
Na]+ (5), 391 [ M + H]+ (4). These data
agreed well with the literature values[15].
Compound 2 was identified as diethyl-
hexyl phthalate.
Compound 3 It was yellow needle-
like crystals (methanol), mp. 284-286
℃ , turned red in hydrochloric acid-Mg
power, IR ( KBr) νmax (cm-1) 3 408, 1 652,
1 610, 1 493, 1 369, 1 295, 1 251, 1 178,
1 095, 829; 1H-NMR (400MHz, CD3OD)
δ (ppm ): 7.83 (d, J= 8.8 Hz, 2H, H-2’,
6’), 6.91 (d, J= 8.8 Hz, 2H, H-3’, 5’),
6.58 ( br s, 1H, H-8), 6.56 (br s, 1H,
H -3), 3.87 (s, 3H, OCH3); 13C-NMR
(100 MHz, CD3OD) δ (ppm): 184.26
(C-4, C), 166.38 (C-2, C), 162.76 (C-
4’, C), 158.88 (C-7, C ), 154.67 (C-5,
C), 153.79 (C-9, C), 132.70 (C-6, C),
129.44 (C-2’,6’, CH), 123.25 (C-1’,
C), 117.00 (C-3’,5’, CH), 105.32 (C-
10, C), 103.37 (C-3, CH), 95.32 (C-8,
CH), 60.92 (OCH3). The above data
was in agreement with previous litera-
ture[16]. Compound 3 was identified as
hispidulin.
Compound 4 It was colorless flaky
crystals (methanol), mp. 30-32℃, and
colorless on TLC with case of sulfuric
acid-ethanol solution as color regent ,
mp. 30-35℃, IR (KBr) νmax (cm-1) 1 696,
1 416, 1 309, 1 201, 921, 803, 638;1H-
NMR (400 MHz, CDCl3) δ (ppm): 2.55
(s, 6H, H-1, 1’); 13C-NMR (100 MHz,
CDCl3) δ (ppm): 176.25 (C-2, 2’, C) ,
29.85 (C-1, 1’, CH3), ESI-MS m/z (rel.
int): 141 [M + Na]+ (100). The above
data agreed well with the literature re-
ported [17]. Compound 4 was identified
as acetyl peroxide.
Compound 5 It was yellow powder
(methanol), and turned red in hy-
drochloric acid-Mg power, IR (KBr) νmax
(cm-1) 3 265, 1 657, 1 604, 1 499, 1 358,
1 271, 1 200, 1 069, 997, 964, 917, 822;
1H-NMR (400 MHz, CD3OD) δ (ppm ):
7.34 (dd, J = 8.2, 1.9 Hz, 1H, H-6’),
7.30 (d, J = 1.9 Hz, 1H, H-2’), 6.92 (d,
J = 8.2 Hz, 1H, H-5’), 6.37 (d, J = 1.8
Hz, 1H, H-6), 6.20 ( d, J = 1.8 Hz, 1H,
H-8), 5.36 (br s, 1H, H-1”), 4.24 (d, J =
2.4 Hz, 1H, H-2”), 3.73-3.80 (m, 2H,
H-3”, 4”), 3.33 ( m, 1H, H-5”), 1.14
(d, J = 6.3 Hz, 3H, H-6”); 13C -NMR
(100 MHz, CD3OD) δ (ppm): 179.65 (C-
4), 165.86 (C-7), 163.21 (C-5), 158.52
(C-9), 149.80 (C-4’), 146.41 (C-3’),
136.27 ( C-2), 122.91 (C-3), 122.90
(C-1’), 116.99 (C-6’), 116.40 (C-5’),
105.81 (C-10), 103.57 (C-2’), 102.03
(C-1”), 99.85 (C-6), 94.76 (C-8),
73.30 (C-4”), 72.05 (C-3”), 72.03 (C-
2”), 71.93 (C-5”), 17.69 (C-6”). The
above data was consistent with the lit-
erature reported [18]. Compound 5 was
identified as quercetin.
Compound 6 It was white powder
(methanol), mp. 253-255 ℃ , IR (KBr)
νmax (cm-1) 3 496, 3 284, 1 667, 1 612,
1 541, 1 425, 1 386, 1 320, 1 267, 1 221,
1 028, 867, 735; 1H-NMR (400 MHz,
(CD3)2CO) δ (ppm): 7.14 (s, 2H, H-2,
6); 13C-NMR [100 MHz, (CD3)2CO] δ
(ppm): 167.62 (COOH), 145.84 (C-3,
5), 138.43 (C-4), 121.97 (C-1), 109.95
(C-2, 6). The above data was consis-
tent with the literature reported [19]. Co-
mpound 6 was identified as gallic acid.
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Agricultural Science & Technology
Agricultural Science & Technology Vol.13, No.2, 2012
2012
飞扬草化学成分研究
王 壹，蒋金和，陈业高，赵 勇 * （云南师范大学化学化工学院，云南昆明 650500）
摘 要 [目的]对飞扬草地上部分进行化学成分研究。[方法]利用正反相硅胶和 Sephadex LH-20对飞扬草的化学成分进行分离、纯化，并利用
MS,NMR等光谱技术鉴定化合物结构，采用流式细胞术对化合物 1,4,5,6进行了小鼠脾细胞免疫刺激活性测试。[结果]从飞扬草地上部分分离得到
6个化合物，经鉴定分别为邻苯二甲酸二异丁基酯(化合物 1)、邻苯二甲酸二乙基己基酯(化合物 2)、高车前素(化合物 3)、过氧化乙酰(化合物 4)、槲
皮素(化合物 5) 和没食子酸(化合物 6)，所有测试化合物均未发现有明显的免疫刺激活性。[结论]成分 1~3为首次从飞扬草中分离得到，4为首次
从天然产物中得到。该研究为从飞扬草中寻找生物活性物质提供了参考依据。
关键词 大戟科；飞扬草；化学成分；免疫活性
基金项目 国家自然科学基金（21162044）；云南省自然科学基金（2009CD051）；云南省中青年学术后备人才项目（2010CI040）。
作者简介 王壹（1986-），男，辽宁朝阳人，硕士，主要从事药用植物化学的研究，E-mail：wyioioioioio@163.com。*通讯作者，教授，硕士生导师，主要
从药用植物化学的研究，E-mail: zhaooy@126.com。
收稿日期 2011-11-22 修回日期 2011-12-12
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
Responsible editor: Qingqing YIN Responsible proofreader: Xiaoyan WU
Biological activity
As the amount of compounds 2
and 3 was limited, their activity was not
detected. The compounds 1, 4, 5 and
6 were detected for their immunostim-
ulatory activity against splenic cell of
mouse by FCM; the growth rates of
compounds 1, 4, 5 and 6 at 5.6 μmol/L
were 108% , 100% , 104% and 100%
respectively; while the growth rate of
control Rapa at 0.02 μmol/L was 57%.
The results proved that all the com-
ponds tested were inactive against
splenic cell of mouse.
Conclusion
Six compounds were isolated
from Euphorbia hirta, and identified as
diisobutyl-O-phthalate (compond 1),
diethylhexyl phthalate (compond 2),
hispidulin (compond 3), acetyl perox-
ide (compond 4), quercetein (compond
5) and gallic acid (compond 6). Com-
pounds 1, 2 and 3 were isolated from
this plant for the first time, and com-
pound 4 was the first naturally occur-
ring compound. The compounds 1, 4,
5 and 6 did not exhibit any immunos-
timulatory activity against splenic cell
of mouse.
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