全 文 ：104 Chin J Nat Med Mar. 2010 Vol. 8 No. 2 2010 年 3 月 第 8 卷 第 2 期








A Germacrane Sesquiterpenoid from Vernonia patula

LIANG Qiao-Li1*, JIANG Ji-Hong2, MIN Zhi-Da3
1Department of Chinese Medicinal Chemistry, Nanjing University of Chinese Medicine, Nanjing 210046;
2Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, Xuzhou Normal University, Xuzhou 221116, China
3Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
[ABSTRACT] AIM: To elucidate the chemical constituents of the whole plant of Vernonia patula and provide
samples for activity screening. METHODS: Column chromatography was used and the structures were determined by
IR, NMR and MS spectral analysis, X-ray analysis and comparison of physicochemical properties. RESULTS: A ger-
macrane sesquiterpenoid, incaspitolide D (1), along with (S)-N-benzoylphenylalanine-(S)-2-benzamido-3-phenyl- propyl
ester (2), indole-3-carboxylic acid (3), apigenin (4), diosmetin (5) and luteolin (6) was isolated. CONCLUSION: Com-
pounds 1-5 were isolated for the first time from Vernonia patula. The 1H and 13C NMR spectra of 1 were fully assigned
by 1H, 1H-COSY, HMQC and HMBC for the first time and its relative stereochemistry was determined by X-ray analy-
sis.
[KEY WORDS] Vernonia patula; Compositae; Sesquiterpenoid; Incaspitolide D; Diosmetin
[CLC Number] R284.1 [Document code] A [Article ID] 1672-3651(2010)02-0104-03
doi: 10.3724/SP. J. 1009.2010.00104
1 Introduction
Vernonia patula Merr. (Compositae) has been used in
traditional medicine for the treatment of acute gastroenteri-
tis, headache, rheum and malaria [1]. Previous phytochemi-
cal investigation of the whole plant resulted in the isolation
of flavones and caffeoylquinates [2]. In our continuing
search for cytotoxic antitumor sesquiterpenes from the
Compositae species [3], we have investigated the title plant
and resulted in the identification of incaspitolide D (1) (Fig.
1), along with (S)-N-benzoyphenylalanine-(S)-2-benzamido
-3-phenylpropyl ester (2), indole-3-carboxylic acid (3), api-
genin (4), diosmetin (5) and luteolin (6). Compound 1 (Fig.
1) is a highly oxidized germacrane sesquiterpenoid and was
only isolated from the aerial parts of Allagapappus viscosi-
ssimus, Inula caspidata and seeds of Carpesium triste [4-6].
It exhibited significant cytotoxicity against human SMMC-
7721 liver cancer cells in vitro (IC50 14.3 μg⋅mL-1) [6].
However, to our best knowledge, there was only one report
concerning its structural elucidation by 1H-NMR data com-
pared with those of the analogies [4, 7] and its stereo- chem-
istry is still ambiguous. We report herein, the complete 1H
and 13C NMR assignments of compound 1 conducted by the
combined use of one- and two- dimensional NMR tech-
niques with its relative configuration established through
X-ray analysis.

[Received on] 04-Jun-2009
[*Corresponding author] Liang Qiao-Li, E-mail: liangqiaoli2008
@163.com, Fax: 86-25-85811524

Fig. 1 Chemical structure of compound 1
2 Results and Discussion
Compound 1 was obtained as white needles, mp 221-
223°, [α] 20D -193° (c 0.02, CHCl3). Its positive HR-ESI-MS
showed [M + H]+ at m/z 455.2295 (Calcd. 455.2275), sug-
gesting the molecular formula C23H34O9. The DEPT and 13C
NMR spectra of compound 1 displayed 21 signals corre-
sponding to five methyl, three methylene (two sp3 and one
sp2), seven methine (all sp3) and six quaternary (five sp2
and one sp3) carbon atoms. The observed carbons were
fewer by two than the molecular formula, suggesting that
there are overlapping signals. In the HMQC spectrum, the
carbon signal at δ 78.7 was correlated with two hydrogens
at δ 5.04 and 5.35, while the carbon at δ 18.9 with six hy-
drogens at δ 1.21(3H) and 1.23(3H). Therefore the
methines C-5, C-9 at δ 78.7 and methyls C-3’, C-4’ at δ
18.9 overlapped in 13C NMR spectrum. In addition, the
two-dimensional NMR experiments [1H-1H COSY, 1H-13C
HMQC and 1H- 13C HMBC nJ (C, H) (n = 2 and 3 long-
range coupling)] allowed the assignment of all carbons and
hydrogens of compound 1.
LIANG Qiao-Li, et al. /Chinese Journal of Natural Medicines 2010, 8(1): 104−106
2010 年 3 月 第 8 卷 第 2 期 Chin J Nat Med Mar. 2010 Vol. 8 No. 2 105

Table 1 1H and 13C NMR assignments for 1 in CDCl3
No δC δH (JHz) (HMQC) 1H-1H COSY HMBC
1 214.5
2a 34.1 2.26 m C-1, 3, 4
2b 3.70 m H-3
3 25.4 1.85 m (2H) H-2b, 4 C-1, 2,4, 5, 15
4 30.2 2.22 m H-3, 5, 15 C-3, 15
5 78.7 5.04 dd (J4, 5 = 1.5 Hz, J5, 6 = 9.5 Hz) H-6 C-3, 4, 6, 5’
6 72.1 4.27 t (J5, 6 = 9.5 Hz, J6, OH = 9.5 Hz) H-5, 7, i
7 42.3 2.99 ddd (J7, 8 = 6.4 Hz, J7, 13a= 2.7 Hz, J7, 13b = 3 Hz) H-6, 8, 13a, 13b C-8, 11
8 78.9 4.69 dd (J7, 8 = 6.4 Hz, J8, 9 = 10 Hz) H-7, 9 C-6, 7
9 78.7 5.35 d H-8
10 81.4
11 132.8
12 168.1
13a 124.3 5.62 d (J7, 13a = 2.7 Hz) H-7
13b 6.43 d (J7, 13b = 3 Hz) H-7 C-7, 11, 12
14 25.6 1.29 s C-1, 9, 10
15 20.7 1.00 d (J4, 15 = 6.7 Hz) H-4 C-3, 4, 5
1’ 176.1
2’ 34.2 2.68 qq H-3’, 4’ C-1’
3’ 18.9 1.21d H-2’
4’ 18.9 1.23 d H-2’ C-1’
5’ 178.0
6’ 34.3 2.68 qq H-7’, 8’ C-5’
7’ 19.0 1.24 d H-6’
8’ 19. 1.25 d H-8’ C-5’, 6’
i 2 1.91 d (J = 9.6 Hz) C-4, 5
j 2.98 br s

Regarding the stereochemistry, the 3J (H, H) values
provided clues to the relative stereochemistry of H-5, 6, 7, 8
and 9. In the 1H NMR spectrum, H-8 (δ 4.69) was dis-
played as a well-defined one-proton doublet of doublet with
H-7 and H-9 (J8, 7 = 6.4 Hz, J8, 9 = 9.6 Hz), which indicated a
trans-axial relationship between H-7, H-8 and H-9, i.e.
H-7α, H-8β, and H-9β-oriented. These assignments were
based on the fact that H-7 has been assumed to be α-orien-
ted as in all other naturally occurring germacranolides [8].
Moreover, consideration of the relationship between the J
value and the dihedral angle indicated that no coupling
between H-6 and H-7 was suggestive of the angle of 90°
between them. This allowed the assignment of H-6 as 6β. In
addition, J5, 6 (9.5 Hz) was large, indicating a trans-axial
relationship between H-5 and H-6, i.e. H-5β-oriented.
However, the stereochemistry at C-4 and C-9 could not be
determined with certainty. In order to remove the ambiguity
concerning the stereochemistry, the compound was sub-
jected to single-crystal X-ray analysis. A perspec- tive view
of the solid-state conformation is shown in Fig. 2.
The other compounds, (S)-N-benzoylphenylalanine-
(S)-2-benzamido-3-phenylpropyl ester (2) [9], indole-
3-carboxylic acid (3) [10], apigenin (4), diosmetin (5) and
luteolin (6) [11] were identified on the basis of their spectral
data and literature evidence.

Fig. 2 Chemical structure of compound 1
3 Experimental
3.1 General experimental procedures
Melting points were determined on an X4 micromel-
ting point apparatus and are uncorrected. Optical rotations
were measured on a Perkin-Elmer model 241 polarimeter.
IR spectra were obtained on a Nicolet Impact infrared spec-
trophotometer with KBr pellets. ESI-MS were obtained on
an Agilent 110 MSD mass spectrometer. NMR spectra were
recorded on a Bruker DRX-400 spectrometer.
Column chromatography was made on silica gel (100-
200 mesh and 200-300 mesh, Qingdao Marine Chemical
Factory); TLC was performed on precoated silica gel plates
(HSG and HSF254, Yantai Chemical Factory).
3.2 Plant material
The whole plant of Vernonia patula was collected in
LIANG Qiao-Li, et al. /Chinese Journal of Natural Medicines 2010, 8(2): 104−106
106 Chin J Nat Med Mar. 2010 Vol. 8 No. 2 2010 年 3 月 第 8 卷 第 2 期

September 2000 in Guangxi Province, China, and identi-
fied by Prof. NAI Mao-Xiang at the Institute of Chinese
Medicine in Guangxi.
3.3 Extraction and isolation
The air-dried plant material (5.0 kg) was extracted
with 90% ethanol and the extracts were concentrated to
give the dark green solution (5 L). Diatomite (1.5 kg) was
added to the solution and stirred evenly, then dried in 60℃
and powdered. The dried powder was extracted in a Soxhlet
apparatus with ethyl acetate for 24 h, which yielded 240 g
of a residue on removal of ethyl acetate in vacuo. A portion
(190 g) of the residue was chromatographed on a silica gel
column eluted with petrol-EtOAc, EtOAc-MeOH by step-
wise addition of EtOAc or MeOH to yield 11 fractions.
After evaporation of Fr. 4 eluted with petroleum ether-
EtOAc (10∶1) and Fr. 5 eluted with petrol-EtOAc (5∶1),
the residues (7 g and 9 g) were further isolated on silica gel
CC eluted with petrol-EtOAc, respectively. 2 (18 mg) was
obtained from the elution of petroleum ether-EtOAc (97∶3)
and 3 (15 mg) and 4 (21 mg) were isolated from the elution
of petrol-EtOAc (90∶10 and 88∶12, respectively). Frs. 6
eluted with petrol-EtOAc (1∶1), was rechromatographed
on a silica gel column using CHCl3-MeOH mixtures as
eluate, yielding 5 (23 mg, CHCl3-MeOH, 99∶1), 6 (29mg,
CHCl3-MeOH, 98∶2) and 1 (32 mg, CHCl3-MeOH, 95∶5).
X-ray anaylsis
Single crystals of compound 1 were prepared from
acetone-hexane. Colorless piece crystals, Monoclinic Cry-
stal system, space group P21, with a = 13.693(1)Ǻ, b =
9.581(1)Ǻ, c = 19.032(2)Ǻ, β = 74.576 (6)°, V = 2 406.9
(4)Ǻ3, and Dcald = 1.252g·cm−3 for Z = 4 (C23H34O9, MW
454.51). Crystal dimensions: 0.15 mm × 0.30 mm × 0.50
mm. The diffraction maxima were collected on a MAC
DIP-2030K image plate detector, using graphite–mono-
chrimated MoKα radiation. A total of 3858 reflections were
measured, of which 3836 with |F|2≥3σ|F|2 were used for
structural determination and refinement. The final credible
factors are Rf = 0.076 and Rw = 0.076 with weighting
scheme W = σ −1│F│2.
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咸虾花中的牻牛烷型倍半萜内酯
梁侨丽 1*, 蒋继宏 2, 闵知大 3
1 南京中医药大学中药化学教研室, 南京 210046;
2 徐州师范大学江苏省药用植物和生物技术重点实验室，徐州 221116；
3 中国药科大学天然药化教研室, 南京 210009
【摘 要】 目的：分离鉴定斑鸠菊属植物咸虾花中的化学成分, 为活性筛选提供样品。方法：90%乙醇提取, 硅胶柱色谱
分离, 用 IR, NMR, MS 和 X-衍射等方法确定结构。结果：分离到 1 个牻牛烷型倍半萜内酯, 1 个苯丙氨酸衍生物、1 个生
物碱和 3 个黄酮化合物, 根据波谱数据和 X-衍射分析分别鉴定为：incaspitolide D (1), (S)-N-苯甲酰基苯基丙氨酸– (S)-2-苯
甲酰氨基–3-苯基丙酯((S)-N-benzoylalanine-(S)–2-benzamido-3-phenylpropyl ester, 2); 吲哚-3-羧酸(indole-3-carboxylic acid,
3); 香叶木素(diosmetin, 4), 芹菜素(apigenin, 5)和木犀草素(luteolin, 6)。结论：化合物 1-5 是首次从咸虾花中得到, 且首次
通过二维 NMR 技术对化合物 1 的波谱数据进行了全归属, 用 X-单晶衍射确证 1 的立体构型。
【关键词】 咸虾花; 菊科; 倍半萜内酯; incaspitolide D; 香叶木素