Cytotoxic ent-Abietane-type diterpenoids from the roots of Euphorbia ebracteolata

Article abstract of DOI:10.1016/j.bioorg.2018.07.032

Euphoroids A–C (1–3), three new ent-abietane-type diterpenoids, together with ten known analogues (4–13) were obtained from the roots of Euphorbia ebracteolata. The structures of these compounds were determined by extensive spectroscopic data analysis, including UV, HRESIMS, 1D-, and 2D-NMR data. The inhibitory effects of compounds 1–13 on human cancer cells were determined using the MTT assay. The results revealed that new compounds 2 and 3 showed moderate cytotoxic activities against human cancer cell lines. Especially, compound 3 displayed selective cytotoxic effect agains cancer cell lines.

Full text of DOI:10.1016/j.bioorg.2018.07.032

Accepted Manuscript
Cytotoxic ent-Abietane-type Diterpenoids from the Roots of Euphorbia ebrac-
teolata
Chunhui Han, Yulin Peng, Yifei Wang, Xiaokui Huo, Baojing Zhang, Dawei
Li, Aijing Leng, Houli Zhang, Xiaochi Ma, Chao Wang
PII:
S0045-2068(18)30638-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.032
YBIOO 2451
Reference:
Bioorganic Chemistry
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28 July 2018
30 July 2018
Please cite this article as: C. Han, Y. Peng, Y. Wang, X. Huo, B. Zhang, D. Li, A. Leng, H. Zhang, X. Ma, C. Wang,
Cytotoxic ent-Abietane-type Diterpenoids from the Roots of Euphorbia ebracteolata, Bioorganic Chemistry (2018),
doi: https://doi.org/10.1016/j.bioorg.2018.07.032
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Cytotoxic ent-Abietane-type Diterpenoids from the Roots of Euphorbia
ebracteolata
Chunhui Han,†a Yulin Peng,†b Yifei Wang,†b Xiaokui Huo,b Baojing Zhang,b Dawei
Li,*a Aijing Leng,a Houli Zhang,b Xiaochi Ma,*b and Chao Wang*b
a The first affiliated hospital of Dalian Medical University, No.222 Zhongshan Road,
Dalian 116011, People’s Republic of China
b College of Pharmacy, Dalian Medical University, Dalian 116044, People’s Republic
of China
ABSTRACT
Euphoroids A
known analogues
The structures of these compounds were determined by extensive spectroscopic data
analysis, including UV, HRESIMS, 1D-, and 2D-NMR data The inhibitory
effects of compounds 13 on human cancer cells were determined using the
MTT assay. The results revealed that new compounds and showed moderate

C (
1
3
), three new ent-abietane-type diterpenoids, together with ten
(413) were obtained from the roots of Euphorbia ebracteolata.
.
1

2
3
cytotoxic activities against human cancer cell lines. Especially, compound
displayed selective cytotoxic effect agains cancer cell lines.
3
Keywords: Euphorbia ebracteolata; diterpenoids; abietane; cytotoxic activities
1. Introduction
*Corresponding authors.
E-mail addresses: lidw87@163.com (D.W. Li), maxc1978@163.com (X. Ma),
wach_edu@sina.com (C. Wang)
†These authors contributed equally to this work.

Euphorbia ebracteolata Hayata (Euphorbiaceae) is a biennial herbaceous
plant that is widely distributed and cultivated in China. Its dried roots, named
“Lang Du” in Chinese, are used in traditional Chinese medicine for the
treatments of edema, cough, indigestion, asthma, and chronic bronchitis [1].
Chemical studies of the roots of E. ebracteolata revealed that these roots
contain diterpenoids, triterpenoids, sesquiterpenoids, flavonols, and
acetophenones, etc. of which diterpenoids are the main active constituents
[2-6]. As part of a program to assess the chemical and biological diversity of
traditional Chinese medicines, the ethanol extract of the roots of E.
ebracteolata has been investigated. Our previous studies on the ethanol extracts
led to the isolation of 27 new compounds including monoterpenoids, rosane,
ent-kaurane, ent-atisane, tigliane, ingenane, heterodimeric diterpenoids, and
phenolic glycosides. Some of them exhibited the DPPH scavenging effects,
anti-tuberculosis effects, and inhibitory effects against α-glucosidase and
human carboxylesterase 2 [7-10]. In this study, three new ent-abietane-type
diterpenoids Euphoroids AC (13), together with ten known analogues (413)
were isolated from the ethanol extracts of the roots of E. ebracteolata by
several chromatographic technologies (Fig. 1). Their structures were
determined by spectroscopic analyses. The cytotoxicities against six human
cancer cell lines of 113 were evaluated. The structure-activity relationship of
ent-abietane-type diterpenoids and their cytotoxic activities were firstly also
reported. We present herein the isolation and structural characterization of
Euphoroids AC (13), as well as their cytotoxic activities.
2. Materials and methods
2.1. General methods
UV spectra were recorded on a JASCO P2000 automatic digital polarimeter. 1D-
1
and 2D-NMR spectra were obtained at 600 for H and 150 MHz for 13C, respectively,
on a Burker 600 MHz spectrometer with solvent peaks as references. HRESIMS data
were obtained with an Agilent 1290 Infinity liquid chromatography system and an
Agilent 6540 UHD Accurate-Mass Q-TOF mass spectrometer. Analytical HPLC data

were collected on an UltiMate 3000 instrument (Thermo Scientific Dionex) equipped
with a diode array detector (DAD). Preparative HPLC separation was performed on
an Agel instrument with a UV detector and a YMC C18 column (250  20mm, 5 mm).
Column chromatography (CC) was performed with silica gel (200−300 mesh,
Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China). TLC was
conducted on precoated silica gel GF254 plates (5 × 10 cm, 2.5 × 7.5 cm, Qingdao
Marine Chemical Inc.). Chromatographic grade methanol were purchased from
Sigma-Aldrich. All other solvents were of chemical grade (Kermel Chemical Co. Ltd,
Tianjin, China). Spots were visualized under UV light (254 or 365 nm) or by spraying
with 10% sulfuric acid in EtOH, followed by heating at 105 °C.
2.2. Plant material
The roots of E. ebracteolata were purchased from Bozhou city of Anhui province
in China. The plant material was identified by Prof. Qing-Shan Yang (Anhui
University of Chinese Medicine). A voucher specimen (No. P-231) was deposited in
the College of Pharmacy, Dalian Medical University.
2.3. Extraction and isolation
The pulverized plant material (15 kg) was decocted with 80% EtOH (150 L;
3 × 1.5 h). The extracts were combined and evaporated under reduced pressure
to yield the aqueous residue. The aqueous residue was diluted with H2O and
sequentially partitioned with petroleum ether, EtOAc, and n-BuOH. EtOAc
extract (504 g) was chromatographed over silica gel, eluting with petroleum
ether/acetone (50:1−2:1) mixtures, to yield 55 fractions. Fractions 3 (35 g) and
5 (77 g) were further separated into 14 fractions (A1–A14) and 20 fractions
(B1–B20) by MPLC (ODS) using MeOH:H2O (50:60–100:0) mixtures as the
eluent, respectively. Subfraction A4 (350 mg) was purified by preparative
HPLC (RP C18 column, 8 ml/min, detected at 210 and 250 nm) with a 60:40
MeOH:H2O mixture as the eluent to yield compounds
4 (17.9 mg; tR 33.1 min)
and (6.6 mg; tR 39.0 min). Compounds (7.1 mg; tR 42.1 min) and 10 (4.0
9
8
mg; tR 53.0 min) were isolated from subfraction A5 (200 mg) by preparative
HPLC with a 65:35 MeOH:H2O mixture as the eluent. Subfraction B6 (450 mg)

was separated by preparative HPLC (MeOH/H2O, 66:34) and afforded the
production of compounds (4.5 mg; tR 19.5 min), (3.0 mg; tR 23.0 min),
(4.5 mg; tR 75.0 min), 11 (2.2 mg; tR 21.3 min), 12 (3.6 mg; tR 15.2 min), and
13 (5.4 mg; tR 17.0 min). Compounds (45 mg; tR 31.0 min), (2.2 mg; tR 36.0
min), and (18.3 mg; tR 38.0 min) were isolated from subfraction B7 (400 mg)
1
2
3
5
6
7
by preparative HPLC (MeOH/H2O, 68:32).
2.3.1. Euphoroid A (1)
White amorphous powder; [α]20 + 11.0 (c 0.05 CH3OH); UV (CH3OH) λmax 232.1
D
nm; 1H NMR (CDCl3, 600 MHz) data, see Table 1; 13C NMR (CDCl3, 150 MHz) data,
see Table 1; (+)-HR-ESIMS m/z 333.2069 [M + H]+ (calcd for C20H29O4, 333.2060).
2.3.2. Euphoroid B (2)
White amorphous powder; [α]20 – 8.2 (c 0.05 CH3OH); UV (CH3OH) λmax 290.9
D
nm; 1H NMR (CDCl3, 600 MHz) data, see Table 1; 13C NMR (CDCl3, 150 MHz) data,
see Table 1; (+)-HR-ESIMS m/z 315.1995 [M + H]+ (calcd for C20H27O3, 315.1955).
2.3.3. Euphoroid C (3)
White amorphous powder; [α]20 – 13.0 (c 0.05 CH3OH); UV (CH3OH) λmax 202.8,
D
220.5 nm; 1H NMR (CDCl3, 600 MHz) data, see Table 1; 13C NMR (CDCl3, 150 MHz)
data, see Table 1; (+)-HR-ESIMS m/z 635.4278 [M + Na]+ (calcd for C38H60O6Na,
635.4278).
2.4. Hydrolysis of 3
Compound 3 (2 mg) was dissolved in 95% MeOH (1.0 mL), and K2CO3 (10 mg)
was added. The solution was stirred at 40 °C for 12 h, acidified with HCl (2 N), and
then evaporated under reduced pressure. The residue was dissolved in MeOH and
subjected to HPLC analysis.
2.5. Cells and culture conditions
A549 (human lung cancer cell line), MCF-7 (human breast cancer cell line), Mewo
(human melanoma cell line), SH-SY5Y (human neuroblastoma cell line), Lovo
(human colon adenocarcinoma cell line), and HepG2 (human liver cancer cell line)
were obtained from American Type Culture Collection (ATCC, Rockville,
MD). Cells were maintained in RRMI1640 supplemented with 10% fetal

bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin.
Cultures were incubated at 37
℃ in a humidified 5% CO2 atmosphere.
2.6. Cells proliferation assay
A549, MCF-7, Mewo, SH-SY5Y, Lovo, and HepG2 were seeded in 96-well
microtiter plates at 1200 cells/well. After 24 h, the compounds 113 were added to
the cells. After 96 h of drug treatment, cell viability was determined by measuring the
metabolic conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) into purple formazan crystals by active cells. MTT assay results were read
using a MK 3 Wellscan (Labsystem Drogon) plate reader at 570 nm. Compounds
113 were tested at five concentrations and were dissolved in 100% DMSO to give a
final DMSO concentration of 0.1% in each well. Each concentration of the
compounds was tested in three parallel wells. IC50 values were calculated using
Microsoft Excel software [11].
3. Results and discussion
Compound
1 was obtained as a white amorphous powder. The molecular
formula was determined as C20H28O4 by HRESIMS at m/z 333.2069 [M + H]+
(calcd for C20H29O4, 333.2060), combined with the NMR data (Table 1). The
1H NMR spectrum of
1 exhibited resonances attributable to an olefinic group at
δH 6.11 (m, H-7); three oxymethine groups at δH 4.92 (s, H-14), 5.03 (m, H-12),
and 3.32 (dd, J = 10.8, 8.4 Hz, H-11); a vinylic methyl group at δH 1.88 (d, J =
1.8 Hz, H-17); and three tertiary methyl groups at δH 0.92 (s, H-18), 0.90 (s,
H-19), and 0.92 (s, H-20). The 13C NMR and HSQC spectra displayed 20
carbon signals (Table 1) corresponding to four methyls, four methylenes, six
methines (including one olefinic carbon at δC 133.4 and three oxygenated
carbons at δC 84.3, 77.9, and 70.3), and six quaternary carbons (one carbonyl at
1
δC 174.5 and three olefinic carbons at δC 135.9, 156.1, and 122.3). The H–1H
COSY cross peaks between H-1/H-2/H-3, H-5/H-6/H-7, and H-9/H-11/H-12
and HMBC correlations from H-17 to C-13, C-15, and C-16; from H-12 to
C-11, C-13, and C-15; from H-11 to C-8, C-9, C-10, C-12, and C-13; from
H-14 to C-7, C-9, C-12, and C-15 (Fig. 2); together with chemical shifts of

these proton and carbon resonances, revealed
11β,14β-dihydroxy-ent-abieta-7(8),13(15)-dien-16,12β-olide
A)[12]. However, comparison of the NMR data of with fischeriolide A
indicated that the C-13 and C-15 resonance in were shielded by ΔδC −3.4 and
−4.6 ppm, respectively, whereas the C-7, C-8, C-9, C-11, and C-12 resonances
were deshielded by ΔδC +2.5 ~ + 12.2 ppm. Meanwhile, compound has the
reverse rotation as the reported fischeriolide A. These suggested that the
configuration of should be different from that of fischeriolide A, which was
1
has the same planar structure as
(fischeriolide
1
1
1
1
further supported by a NOESY experiment. The NOE correlations of H-9/H-12,
H-9/H-14, and H-11/H-14 and CH3-20 indicated α-orientations for H-9, H-11,
H-12 and H-14 (Fig. 2). Therefore, the structure of
Euphoroid A.
1 was determined to be
Compound 2, a white amorphous powder, has a molecular formula of C20H26O3 as
determined by HRESIMS and NMR data (Table 1), which showed 18 less mass units
than 1. The 1D NMR spectra (Table 1) displayed similar pattern to 1, while the major
difference was the hydroxy group at C-11 in 1 was replaced by one double bond
which was placed at C-11-C-12 in 2, consistent with the HMBC correlations (Fig. 3)
of H-11 with C-8, C-9, and C-12. The NOESY correlations (Fig. 3) of H-5/H-9, and
H-9/H-14 indicated that these protons were co-facial and assigned a β-orientation.
Thus, compound 2 was elucidated to be Euphoroid B.
Compound 3 was obtained as a white powder. Its molecular formula was
determined to be C38H60O6 based on the HRESIMS ion at m/z 635.4278 [M + Na]+
1
(calcd for C38H60O6Na, 635.4278) and NMR data (Table 1). The H NMR spectrum
suggested the presence of partially overlapped aliphatic methylenes and/or methines.
The NMR data for the tetracyclic substructure of 3 were almost indistinguishable
from those of yuxiandajisu E [13], and ester moiety linked to C-14 of 3 were similar
to those of linoleic acid [14], with the only difference being the position of
carbonyl group (δ 172.3), which were also proved by the 2D NMR data (Fig. 4). The
two parts were linked with an ester bond between C-14 and C-2′ by the key HMBC
cross peak of H-14 to C-1′. The standard linoleic acid and hydrolysate of 3 were also

subjected to HPLC analysis under the same condition. The same retention time
confirmed the existence of linoleic acid in the structure of 3. The relative
configuration was determined by the NOESY spectrum (Fig, 4) in combination with
yuexiandajisu E. The correlations of H11/ CH3-20, H-12/ CH3-20, H-5/ H-9, and
H-12/ H-14 indicated an α-orientation for H-11, H-12, and H-14 and a β-orientation
for H-5 and H-9. Thus, the structure of compound 3 was named Euphoroid C.
The ten known compounds were identified as jolkinolide E (4) [15],
ent-11α-hydroxyabieta-8(14),13(15)-dien-16,12-olide (5) [16], jolkinolide F (6) [17],
helioscopinolide E (7) [18], jolkinolide B (8) [19], jolkinolide A (9) [20],
euphopilolide (10) [21], fischeriolide A (11) [12], yuexiandajisu D (12) [22],
yuexiandajisu E (13) [22] from their spectroscopic data upon comparisons with values
reported in the literatures.
In the bioassay experiment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method, the cytotoxic effects of isolated
diterpenoids against six human cancer cell lines A549, MCF-7, Mewo, SH-SY5Y,
Lovo, and HepG2, have been evaluated (Table 2). Compounds 2, 3, 8, and 11
displayed moderate cytotoxicities on cancer cell lines. Especially, the bioactive
compounds 2 and 3 were determined to be new diterpenoids. Additionally, new
diterpenoid 3 inhibit the proliferation of four cancer cell lines A549, MCF-7, Lovo,
and HepG2 with IC50 values < 30 M. But, no cytotoxicity was observed for 3 against
Mewo and SH-SY5Y (IC50 > 100 M). Thus, diterpenoid 3 displayed selective
cytotoxicity against human cancer cell lines.
4. Conclusions
In summary, the three new and ten known ent-abietane-type diterpenoids were
isolated from the roots of E. ebracteolata. Additionally,
2, 3, 8, 9, and 11 showed
cytotoxic effects against human cancer cell lines, especially for compound
3
showed selective cytotoxic activities against six human cell lines. The result,
combined with our previous studies [7-10], shows that diverse compounds contribute
towards pharmacological efficacy that supports and enriches the traditional uses of E.
ebracteolata roots.

Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
The authors thank National Natural Science Foundation of China (No.
81622047, 81673579, 81503201), Distinguished professors of Liaoning and
Hunan Provinces, and State Key Laboratory of Fine Chemicals (KF1603,
KF1705) for financial supports.
Appendix A. Supplementary data
13
1H NMR, C NMR, 2D NMR, and HRESIMS of new compounds associated with
this article can be found online. Supplementary data associated with this article can be
found in the online version.
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Fig. 1. Structures of ent-abietane diterpenoids 113 from the roots of E. ebracteolata.

1
Fig. 2. (a) Main H-1H COSY (blue thick lines) and HMBC correlations (red arrows,
from 1H to 13C) of 1; (b) The key NOESY correlations of 1.

1
Fig. 3. (a) Main H-1H COSY (blue thick lines) and HMBC correlations (red arrows,
from 1H.

1
Figure 4. (a) Main H-1H COSY (blue thick lines) and HMBC correlations (red
arrows, from 1H to 13C) of 3; (b) The key NOESY correlations of 3.

Table 1
1
The H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data of
compounds
1
,
2
, and
3
(δ in ppm, J in Hz).
1
2
3a
position
1
H
C
H
C
H
C
2.57 m
1.20 m
1.51 m
1.42 m
1.42 m
1.20 m
1.96 m
1.18 m
1.59 m
1.55 m
1.48 m
1.23 m
1.95 m
1.64 m
1.62 m
1.54 m
1.45 m
1.22 m
42.0
19.0
38.8
41.6
2
3
18.7
18.9
42.0
33.4
50.3
23.5
42.1
33.1
49.1
24.0
41.7
33.4
55.1
20.4
4
5
6
1.41 m
1.37 dd (12.0,4.8)
1.07 m
2.07 m
1.92 m
2.20 m
1.92 m
1.62 m
1.42 m
2.04 m
1.72 m
7
6.11 m
133.4
135.9
50.3
36.9
77.9
84.3
156.1
70.3
122.3
174.5
8.8
6.02 t (2.4)
133.1
133.8
47.8
40.2
74.2
62.2
37.9
68.6
78.9
151.8
73.6
129.9
174.2
8.94
34.2
22.1
17.9
8
9
2.45 m
3.05 brs
2.09 s
10
11
12
13
14
15
16
17
18
19
20
36.3
3.32 dd (10.8,8.4)
5.03 m
5.77 d (2.4)
108.2
148.4
146.6
67.7
4.58 brs
5.11 m
4.92 s
5.02 s
5.50 s
122.9
171.3
8.7
1.88 d (1.8)
0.92 s
2.02 s
0.90 s
0.90 s
0.70 s
2.01 d (1.8)
0.92 s
22.8
33.9
14.4
21.8
0.90 s
33.1
0.87 s
0.92 s
14.6
1.71 s
aData for the linoleoyl unit in 3: H 5.30−5.37 (4H, m), 2.76 (2H, t, J = 6.6 Hz), 2.34 (2H, m), 2.03 (4H, m), 1.62 (2H, m), 1.27−1.30
(14H, m), 0.88 (3H, t, J = 7.2 Hz); C 172.3 (C-1'), 34.5 (C-2'), 24.9 (C-3'), 29.2 (C-4'), 29.2 (C-5'), 29.3 (C-6'), 29.7 (C-7'), 27.3 (C-8'),

130.1 (C-9'), 128.3 (C-10'), 25.8 (C-11'), 128.0 (C-12'), 130.4 (C-13'), 27.3 (C-14'), 29.5 (C-15'), 31.7 (C-16'), 22.7 (C-17'), 14.2 (C-18').
Table 2
Cytotoxicities of compounds 113.
Cytotoxicity IC50 (M)
No.
A549
>100
22.87
28.7
MCF-7 Mewo SH-SY5Y
Lovo
>100
95.68
27.0
HepG2
>100
95.63
28
>100
85.7
>100
80.50
>100
>100
>100
>100
>100
62.7
>100
58.63
>100
>100
>100
>100
>100
85.8
1
2
28.57
>100
>100
>100
>100
73.6
3
>100
>100
>100
>100
94.7
>100
>100
>100
>100
63.2
>100
>100
>100
>100
95
4
5
6
7
8
18.56
>100
69.87
>100
>100
2.5
>100
>100
>100
>100
>100
3.0
>100
>100
88.45
>100
>100
2.5
>100
>100
57.56
>100
>100
4.4
>100
>100
85.0
>100
>100
>100
>100
>100
5.1
9
10
11
>100
>100
2.8
12
13
cisplatina
aCisplatin was used as the positive control. The data points represented the mean value of triplicate experiments.

Graphical abstract

Highlights
1. Three NEW and ten known ent-abietane diterpenoids were obtained from
Euphorbia ebracteolata.
2. New diterpenoids
2
and
3 showed moderate cytotoxic activities against
cancer cell lines.
3. New diterpenoid
lines.
3 displayed selective cytotoxic effects against cancer cell