Fitoterapia 139 (2019) 104408

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Sesquiterpenes and diterpenes from Euphorbia thymifolia T

a a b b b b,⁎ a,⁎
Jin-Long Liu , Min Yu , Hai-Bing Liao , Ting Liu , Yan-Hui Tan , Dong Liang , Gui-Jie Zhang
a
College of Pharmacy, Guilin Medical University, Guilin 541004, People's Republic of China
b
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University,
Guilin 541004, People's Republic of China

A R T I C LE I N FO A B S T R A C T

Keywords: One new pseudoguaianolide (1), one new megastigmane (6), and one new ent-abietane diterpene (9), together
Euphorbia thymifolia with seven known compounds (2–5, 7, 8, and 10) were isolated from the aerial parts of Euphorbia thymifolia. The
Sesquiterpenes structures of the new compounds and their relative configurations were determined by spectroscopic data
Diterpenes analysis. The absolute configurations of compounds 1, 6, and 9 were determined by single-crystal X-ray crys-
Nitric oxide production
tallographic analysis, modified Mosher's method and calculated ECD experiment, respectively. All compounds
BV-2 microglial cells
were tested for their inhibitory effects against LPS-induced NO production in BV-2 microglial cells, and pseu-
doguaianolides (1–5) showed significant activity with IC50 values of 0.41–15.32 μM.

1. Introduction spectrophotometer and the IR spectra were acquired on a PerkinElmer

Euphorbia thymifolia Linn, an annual herb belonging to the family
Euphorbia, is distributed widely in southern China [1]. The whole plant
has been used for the treatment of dysentery, enteritis, diarrhea, der-
matitis, eczema and mastitis [2]. The extract of the plant has been re-
ported to have anti-inflammatory activity [3]. There are few reports on
the phytochemical research of E. thymifolia [4], while the genus Eu-
phorbia contained diterpenes and triterpenes as principal constituents
[5,6]. In our continuing search for anti-neuroinflammatory agents from
folk medicine [7,8], the phytochemical investigation of E. thymifolia
was undertaken and resulted in the isolation of one new pseudoguaia-
nolide (1), one new megastigmane (6), one new ent-abietane diterpene
(9), and seven known terpenes (2–5, 7, 8, and 10) (Fig. 1). This is the
first report of pseudoguaianolides from the genus Euphorbia. The anti-
neuroinflammatory activity assay showed that pseudoguaianolides
(1–5) could significantly inhibit the production of NO in LPS-stimulated
BV-2 microglial cells (Table 2). Herein, we report the isolation, struc-
tural elucidation, and anti-neuroinflammatory activity of the com-
pounds from E. thymifolia. In addition, the taxonomic consideration of
E. thymifolia is discussed.
2. Experimental
2.1. General experimental procedures
UV absorption spectra were recorded on a PerkinElmer 650
Spectrum Two FT-IR spectrometer. CD spectrum was determined on
JACSO J-1500 spectrometer. NMR spectra were recorded on a 600 or
400 MHz Bruker AVANCE. HR-ESIMS were carried out on a Agilent
6545 Q-TOF LC-MS spectrometer. The X-ray crystallographic data of 1
was measured using a SuperNova diffractometer (equipped with Atlas
detector) with Cu Ka (λ = 1.54184 Å) radiation. Column chromato-
graphy (CC) was performed on silica gel (200–300 mesh, Qingdao
Marine Chemical Ltd., People's Republic of China), RP C18 silica gel
(50 μm, YMC, Japan), MCI gel (CHP20, 75–150 μm, Mitsubishi
Chemical Ltd., Japan), and Sephadex LH-20 gel (Pharmacia Biotech,
Sweden). TLC analyses were carried out on the precoated silica gel
GF254 plates (Qingdao Marine Chemical Ltd.). Semipreparative re-
versed-phase HPLC (RP-HPLC) was performed using a YMC-Pack ODS-A
column (250 × 20 mm, 5 μm) on an LC3000 instrument (Chuang Xin
Tong Heng Science and Technology Ltd., Beijing, People's Republic of
China) equipped with a UV3000 detector (Chuang Xin Tong Heng
Science and Technology).
2.2. Plant material
The aerial part of Euphorbia thymifolia Linn was collected in July
2015 in Guilin, Guangxi Province, People's Republic of China. The plant
material was identified by Professor Shao-Qing Tang of the College of
Life Science, Guangxi Normal University. A voucher specimen (No. ET-
201507) has been deposited at the State Key Laboratory for Chemistry
and Molecular Engineering of Medicinal Resources, Guangxi Normal

⁎
Corresponding author.
E-mail addresses: liangdonggxnu@163.com (D. Liang), zhangguijie001@126.com (G.-J. Zhang).

https://doi.org/10.1016/j.fitote.2019.104408
Received 2 October 2019; Received in revised form 30 October 2019; Accepted 4 November 2019
Available online 04 November 2019
0367-326X/ © 2019 Elsevier B.V. All rights reserved.
J.-L. Liu, et al.
Fig. 1. Structures of compounds 1–10.
University.
2.3. Extraction and isolation
The aerial part of E. thymifolia Linn (10 kg) were extracted with 95%
aqueous EtOH (3 × 100 L, each 3 h) under reflux. The filtrate was
evaporated under reduced pressure, and the residue (2.1 kg) was dis-
persed in H2O, and then extracted with EtOAc. The EtOAc fraction
(726.9 g) was placed on a silica gel column using CH2Cl2/MeOH (100:1
to 6:1) to yield eight fractions (A − H) on the basis of TLC analysis.
Fr.D (215.3 g) was purified by MCI CC (MeOH/H2O, 20:80 to 100:0)
to obtain eight fractions (Fr.D1-Fr.D8). Fr.D4 (2.9 g) was separated by
RP-C18 CC, Sephadex LH-20 CC (MeOH), and further purified by
semipreparative RP-HPLC to yield compounds 7 (20:80 CH3CN-H2O,
8 mL/min, tR = 33.3 min, 17.0 mg) and 10 (20:80 CH3CN-H2O, 8 mL/
min, tR = 25.0 min, 2.0 mg). Fr.D8 (10.9 g) was separated by RP-C18
CC, Sephadex LH-20 CC (MeOH), and further purified by semi-
preparative RP-HPLC to yield compounds 1 (50:50 CH3CN-H2O, 8 mL/
min, tR = 46.6 min, 12.7 mg), 2 (50:50 CH3CN-H2O, 8 mL/min,
tR = 80.3 min, 4.5 mg), 3 (50:50 CH3CN-H2O, 8 mL/min, tR = 70.8 min,
1.1 mg), 4 (43:57 CH3CN-H2O, 8 mL/min, tR = 55.4 min, 17.5 mg), 5
(40:60 CH3CN-H2O, 8 mL/min, tR = 64.1 min, 5.5 mg), and 9 (50:50
CH3CN-H2O, 8 mL/min, tR = 49.6 min, 1.3 mg).
Fr.E (42.6 g) was separated by MCI CC (MeOH/H2O, 30:70 to 100:0)
to obtain nine fractions (Fr.E1-Fr.E9). Fr.E4 (1.5 g) was separated by
RP-C18 CC, Sephadex LH-20 CC (MeOH), and further purified by
semipreparative RP-HPLC to yield compounds 6 (25:75 CH3OH-H2O,
6 mL/min, tR = 59.6 min, 4.8 mg), 7 (15:85 CH3CN-H2O, 8 mL/min,
tR = 32.2 min, 7.4 mg), and 8 (30:70 CH3OH-H2O, 6 mL/min,
tR = 29.8 min, 15.9 mg).
2.3.1. (1S, 2R, 5R, 6S, 7R, 8R, 10R, 11S)-4-oxo-2-methoxy-6-
angeloyloxy-pesudoguai- 8,12-olide (1)
Colorless crystal; [α]D20 + 8 (c 0.2, CH3OH); UV (MeOH) λmax (log
ε) 220 (3.71) nm; IR (KBr) νmax 3431, 2929, 1770, 1742, 1710, 1459,
1378, 1235, 1178, 1082, 1045, 996, 941, 844, 749 cm−1; 1H and 13C
NMR data see Table 1; HR-ESIMS m/z 401.1941 [M + Na]+ (calcd for
C21H30O6Na, 401.1935).
2.3.2. (3R, 4S, 5R, 7S)-3,4,5-trihydroxy-6,7-megastigmadien-9-one (6)
White amorphous powder; [α]D20 − 43 (c 0.2, CH3OH); UV (MeOH)
λmax (log ε) 230 (3.99) nm; IR (KBr) νmax 3422, 2924, 1623, 1384,
1063 cm−1; 1H and 13C NMR data see Table 1; HR-ESIMS m/z 263.1253
2
Fitoterapia 139 (2019) 104408
[M + Na]+ (calcd for C13H20O4Na, 263.1254).
2.3.3. (2R, 3S, 5S, 9S, 10R)-2,3-dihydroxy-ent-abieta-8(14),12(13)-dien-
7-one (9)
White amorphous powder; [α]D20 + 75 (c 0.2, CH3OH); UV (MeOH)
λmax (log ε) 204 (3.59), 296 (3.70) nm; IR (KBr) νmax 3425, 2925, 1664,
1634, 1462, 1384, 1260, 1037, 802, 598 cm−1; 1H and 13C NMR data
see Table 1; HR-ESIMS m/z 319.2273 [M + H]+ (calcd for C20H31O3,
319.2268).
2.4. Single crystal X-ray diffraction analysis
Crystallographic data for 1: C21H30O6, CH3OH, colorless orthor-
hombic crystal, a = 9.2659(3) Å, b = 10.8491(3) Å, c = 19.9917(6) Å,
V = 2009.69(10) Å3, Z = 4, T = 100.01(10) K,
−1
μ(CuKα) = 0.743 mm , Dcalc = 1.251 g/cm3, 11,667 reflections
measured (8.846° ≤ 2Θ ≤ 147.112°), 3964 unique (Rint = 0.0286,
Rsigma = 0.0230) which were used in all calculations. The final R1 was
0.0731 (I ≥ 2σ(I)) and wR2 was 0.1993 (all data).
The crystallographic data for 1 (CCDC 1945824) has been deposited
in the Cambridge Crystallographic Data Centre. Copies of these data can
be obtained free of charge via http://www.ccdc.cam.ac.uk/data_
request/cif.cam.ac.uk.
2.5. ECD calculations
In general, conformational analyses were carried out via random
searching in the Sybyl-X 2.0 using the MMFF94S force field with an
energy cutoff of 2.5 kcal/mol. The results showed eight lowest energy
conformers for both compounds. Subsequently, the conformers were re-
optimized using DFT at the b3lyp/6–311 + g (d, p) level in MeOH using
the polarizable conductor calculation model (CPCM) by the GAUSSIAN
09 program. The energies, oscillator strengths, and rotational strengths
(velocity) of the first 50 electronic excitations were calculated using the
TDDFT methodology at the b3lyp/6–311 + g (d, p) level in MeOH. The
ECD spectra were simulated by the overlapping Gaussian function (half
the bandwidth at 1/e peak height, sigma = 0.30 for all). To get the final
spectra, the simulated spectra of the conformers were averaged ac-
cording to the Boltzmann distribution theory and their relative Gibbs
free energy (ΔG) [7,9].
J.-L. Liu, et al. Fitoterapia 139 (2019) 104408

Table 1
1 13
H NMR and C NMR Data of Compounds 1, 6 and 9.
No. 1 6 9

δH (J in Hz)
a
δC b
δHc (J in Hz) δC d
δHa (J in Hz) δ Cb

1a 2.23, dd (10.8, 4.8) 52.8 35.8 2.16, dd (14.4, 3.0) 42.3

1b 1.36, dd (14.4, 3.0)
2a 3.93, t, (4.8) 77.1 1.96, dd (12.4, 4.2) 45.8 4.15, m 70.8
2b 1.50, d (12.4)
3a 2.65, d (19.2) 41.6 4.06, m 68.5 3.24, br s 78.0
3b 2.16, dd (19.2, 5.4)
4 217.4 3.19, d (8.8) 80.8 37.8
5 53.4 73.5 1.34, dd (12.0, 4.5) 48.2
6a 5.38, br s 73.4 118.3 2.46, dd (13.8, 4.5) 37.7
6b 2.31, overlapped
7 2.81, dd (10.0, 6.6) 48.3 211.1 199.7
8 4.70, t (6.6) 79.6 5.91, s 101.0 133.3
9a 2.41, dd (6.6, 1.8) 39.2 198.1 2.35, m 49.4
9b 1.61, m
10 2.38, m 24.2 2.18, s 26.7 34.2
11 3.03, dq (10.0, 7.2) 40.5 1.14, s 31.6 2.31, overlapped 24.3
12 179.2 1.38, s 28.9 6.09, m 132.6
13 1.51, d (7.2) 10.8 1.44, s 26.4 142.1
14 1.12, d (6.0) 20.1 6.71, s 140.9
15 1.09, s 15.8 2.90, m 26.1
16 166.2 1.05, d (7.2) 22.0
17 127.2 1.02, d (7.2) 21.6
18 6.05, qq (7.2, 1.2) 139.6 1.03, s 29.8
19 1.95, dq (7.2, 1.8) 16.0 1.13, s 16.9
20 1.76, m 20.8 1.08, s 15.8
2-OMe 3.24, s 56.9

a
Recorded at 600 MHz in CDCl3.
b
Recorded at 150 MHz in CDCl3.
c
Recorded at 400 MHz in CDCl3.
d
Recorded at 100 MHz in CDCl3.

2.6. NO production measurement and cell viability assay
The accumulation of nitrite (NO2−) in the culture medium super-
natants was measured using the Griess reaction [7,10]. BV-2 cells were
plated in 96-well microtiter plates and treated with compounds at
various concentrations in the presence of LPS (100 ng/mL) for 24 h. The
absorbance was measured on a plate reader (Bio-Tek, Winooski, VT,
USA) at 540 nm. And cell viability was assessed by the MTT assay as
previously reported [10].
3. Results and discussion
Compound 1 was obtained as colorless crystals. Its molecular for-
mula, C21H30O6, was established by HR-ESIMS (m/z 401.1941 for
C21H30O6Na, calcd m/z 401.1935), indicating seven degrees of un-
saturation. The IR absorptions at 1770, 1742 and 1710 cm−1 indicated
the presence of three carbonyl groups. Analysis of the 1D NMR data
(Table 1) and HSQC spectrum exhibited five methyls, one methoxy, two
methylenes, seven methines (three oxygenated), one sp3 tertiary
carbon, two olefinic carbons, and three carbonyls. The remaining de-
grees of unsaturation were requiring three rings in 1. The 2D NMR
spectra also indicated presence of an angelate group [δC 166.2 (C-16),
127.2 (C-17), 139.7 (C-18), 16.0 (C-19) and 20.8 (C-20)]. The above
information suggested that 1 was likely a pseudoguaianolide-type ses-
quiterpene with an angelate group [11].
Analysis of 1H-1H COSY spectrum revealed the long-range spin-
system of H-3/H-2/H-1/H-10/H-9(H3–14)/H-8/H-7/H-11(H-6)/H3–13
(Fig. 2), together with the HMBC correlations from H-3 (δH 2.65) to C-1
(δC 52.8) and C-5 (δC 53.4), H3–15 (δH 1.09) to C-1, C-5 and C-6 (δC
73.4), H-6 (δH 5.38) to C-8 (δC 79.6) and C-11 (δC 40.5), H-8 (δH 4.70)
to C-6 and C-11, led to establishment of the skeleton of compound 1 as
shown in Fig. 2 [12]. The HMBC correlation from H-2 (δH 3.93) to
–OCH3 (δC 56.9) indicated a methoxy group substitute at the C-2. The
angelate group located at C-6 was supported by the HMBC correlation
from H-6 to C-16 (δC 166.2). The relative configuration of 1 was es-
tablished by NOESY experiments (Fig. 2). The NOESY correlations be-
tween H3-13 and H3-15/H-6, and between H3-15 and H-10 confirmed
that H-6, H-10, H3-13 and H3-15 were on the same side. Minewhile, the
NOESY correlations between H-8 and H-7/H-11, and between H3-14
and H-1/H-2 suggested that H-1, H-2, H-7 and H-8 were on the other
side. Finally, the absolute configuration of 1 was determined by single-
crystal X-ray diffraction (Cu Ka) analysis (Fig. 3). On the basis of the
Flack parameter [−0.04(8)], the absolute configuration of compound 1
was established as (1S, 2R, 5R, 6S, 7R, 8R, 10R, 11S). Thus, the
structure of 1 was assigned as (1S, 2R, 5R, 6S, 7R, 8R, 10R, 11S)-4-oxo-
2-methoxy-6-angeloyloxy-pesudoguai-8,12-olide [13].
Compound 6 was obtained as colorless crystals. The molecular
formula of 6 was established as C13H20O4 based on HR-ESIMS data {m/
z 263.1253 [M + Na]+ (calcd for C13H20O4Na, 263.1254)} and 13C
NMR data (Table 1). The NMR data (Table 1) of 6 showed the presence
of four methyl groups, one methylene, two oxygenated methines, two
sp3 quaternary carbons (one oxygenated), three sp2 carbons (one car-
bonyl group) and one sp. carbon. The above evidence indicated that
compound 6 was a megastigmane and included an allenic unit [14], as
shown in Fig. 1. The NMR data of 6 showed a similarity to those of
grasshopper ketone [15], except for the presence of an additional hy-
droxyl group at C-4. This result was supported by the HMBC correla-
tions from H-2 (δH 1.96)/H3-13 (δH 1.44) to C-4 (δC 80.8). The NOESY
correlation of H-4 (δH 3.19)/H3-13 implied that they are cofacial. While
the coupling constant value (J = 8.8 Hz in CDCl3) between H-3 and H-4
suggested their trans relative configuration. The absolute configuration
of secondary hydroxy group at C-3 was then deduced using the mod-
ified Mosher's method [16]. Comprehensive analysis of the 1H NMR
resonances of the R- and S-MTPA ester derivatives of 6 revealed a
systematic distribution of ΔδHS-R values that established a 3R config-
uration (Fig. 4). The absolute configurations of the C-4 and C-5 were

3
J.-L. Liu, et al.
Fig. 2. Key 1H-1H COSY, HMBC, and NOESY correlations for compound 1.
Fig. 3. X-ray crystal structure of compound 1.
Fig. 4. ΔδHS−R values for the Mosher's ester derivatives of 6.
then assigned as 4S and 5R by defined the relative configurations of H-
3/H-4 trans and H-4/H3-13 cis. The configuration of 7-position was
assigned as 7S by comparing the 1H NMR spectrum (Supplementary
data Fig. S15) of 6 with those of cotrosides A and B [16,17], a pair of
epimers as to the allene axis (7R and 7S). In the 1H NMR spectrum of 6
(In pyridine-d5, Supplementary data Fig. S15), the chemical shifts for H-
8 (δH 6.03) and H3-10 (δH 2.23) were the same as those of cotroside B.
Thus, compound 6 was determined to be (3R, 4S, 5R, 7S)-3,4,5-trihy-
droxy-6,7-megastigmadien-9-one.
Compound 9 was obtained as white amorphous powder, whose
molecular formula was determined to be C20H30O3 based on the HR-
ESIMS at m/z 319.2273 [M + H]+ (calcd 319.2268) and the NMR
spectroscopic data (Table 1). The 1H NMR spectrum of 9 showed the
presence of five methyl groups and two olefinic methine signals. The
13
C NMR data (Table 1) of 9 had 20 carbon signals, including one
carboxyl group [δC 199.7 (C-7)]. On the basis of these spectroscopic
data, compound 9 belonged to the abietane diterpene class [18].
4
Fitoterapia 139 (2019) 104408
Comparison of the NMR spectroscopic data of 9 with those of a sessilifol
D [18] led to the primary differences of a hydroxyl group at C-2 and an
olefinic bond at C-12/C-13 in 9. The above conclusion was supported
by the HMBC correlations from H-1 (δH 2.16) to C-2 (δC 70.8), from H-
14 (δH 6.71) to C-12 (δC 132.6), and from H3–16 (δH 1.05)/H3–17 (δH
1.02) to C-13 (δC 142.1). The relative configuration of 9 was assigned
by the NOESY spectrum. The correlations between H-3β (δH 3.24) and
H-2β (δH 4.15)/H-5 (δH 1.34)/H3–18 (δH 1.03), and between H-5 and H-
9 (δH 2.35) indicated that H-2, H-3, H-5, H-9 and H3–18 were β-or-
iented. H3–20 was in an α-orientation due to the NOESY correlations of
H-1α (δH 2.16)/H3–20. The absolute configuration of 9 was established
by comparing the experimental and calculated ECD spectra predicted
by time-dependent density functional theory (TDDFT) at the B3LYP/
6–311 + G (d, p) level. The experimental electronic circular dichroism
(ECD) spectrum of 9 (Fig. 5) showed a positive Cotton effect (CE) at 292
(Δε = +6.2) nm, fitting well with the calculated ECD spectrum for (2R,
3S, 5S, 9S, 10R)-9. Therefore, the structure of 9 was assigned as (2R, 3S,
5S, 9S, 10R)-2,3-dihydroxy-ent-abieta-8(14),12(13)-dien-7 -one.
The known compounds were identified by comparison of their
spectroscopic data with those reported data. They were minimolide B
(2) [13], 4-oxo-2-ethoxy-6-tigloyloxy-pesudoguai-8,12-olide (3)
[13,19], 6-O-angeloylplenolin (4) [11], 6-O-tigloyl-11,13-dihy-
drohelenalin (5) [11,20], dihydrophasei acid (7) [21], corchoionoside
C (8) [22], and phorbol-13-actate (10) [23]. Otherwise, the methoxy or
ethoxy group in pseudoguaianolides (1, 2, and 3) may be artifact
formed during extraction and isolation through precursor 4 or 5.
All the isolated compounds were evaluated for their inhibitory ef-
fects on NO production in LPS-stimulated BV-2 microglial cells using
the Griess reaction [7,10]. As shown in Table 2, the pseudoguaianolides
(1–5) greatly inhibited NO production, with IC50 values ranging from
0.41 to 15.32 μM. Minocycline was used as positive control
(IC50 = 23.53 μM). Meanwhile, the cytotoxicities of these compounds
on BV-2 cells were assessed by the MTT method to avoid possible effects
of reduced viability on NO release. None of them showed cytotoxicity at
the tested concentrations.
The pseudoguaianolides reported in this paper were isolated from
the genus Euphorbia for the first time. However, the pseudoguaianolides
were mainly contained in several Compositae plants, including Inula
hupehensis [12], Centipeda minima [13], Gaillardia pulchella [24], Arnica
montana [25], and Parthenium hysterophorus [26]. The genus Euphorbia
is among of the most difficult genera in angiosperm classification due to
its variety, complex habitat and great variability [27]. Therefore, it is
worthwhile to further study whether there is a taxonomic relationship
between E. thymifolia and Compositae plants.
Declaration of Competing Interest
The authors have declared that there is no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21967007, 21562017, and 81960634), the
J.-L. Liu, et al. Fitoterapia 139 (2019) 104408

Fig. 5. Experimental and calculated ECD spectra of compound 9.

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