Anti-inflammatory Ingenane Diterpenoids from the Roots of Euphorbia kansui

Article abstract of DOI:10.1055/a-0646-4306

Bioassay-guided fractionation of the ethanolic extract of the roots of Euphorbia kansui led to the isolation of two new ingenane diterpenoids, euphorkans A ( 1 ) and B ( 2 ), together with 16 known analogues ( 3 – 18 ). Their structures were determined by combined spectral and chemical methods. All the isolates were evaluated for their inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells. Compounds 1 – 6 and 10 – 13 exhibited pronounced inhibitory activity with IC ₅₀ values in the range of 2.78 – 10.6 μM, and were more potent than the positive control, quercetin (IC ₅₀ = 15.8 μM). Compounds 1 and 5 were selected for further assays toward the key inflammation mediators TNF- α and IL-6, and showed a significant inhibition in a dose-dependent manner. The preliminary mechanistic study revealed that 1 and 5 inhibited NF- κ B activity, which may exert a role in their anti-inflammatory activity.

Full text of DOI:10.1055/a-0646-4306

Original Papers
Anti-inflammatory Ingenane Diterpenoids from the Roots
of Euphorbia kansui
Authors
Jun-Sheng Zhang, Han-Zhuang Weng, Jia-Luo Huang, Gui-Hua Tang, Sheng Yin
Affiliation
Supporting information available online at
School of Pharmaceutical Sciences, Sun Yat-sen University,
Guangzhou, Peopleʼs Republic of China
http://www.thieme-connect.de/products
ABSTRACT
Key words
Bioassay-guided fractionation of the ethanolic extract of the
roots of Euphorbia kansui led to the isolation of two new in-
genane diterpenoids, euphorkans A (1) and B (2), together
with 16 known analogues (3–18). Their structures were deter-
mined by combined spectral and chemical methods. All the
isolates were evaluated for their inhibitory effects on lipopoly-
saccharide-induced nitric oxide production in RAW264.7
macrophage cells. Compounds 1–6 and 10–13 exhibited pro-
nounced inhibitory activity with IC50 values in the range of
2.78–10.6 µM, and were more potent than the positive con-
trol, quercetin (IC50 = 15.8 µM). Compounds 1 and 5 were se-
lected for further assays toward the key inflammation media-
tors TNF-α and IL-6, and showed a significant inhibition in a
dose-dependent manner. The preliminary mechanistic study
revealed that 1 and 5 inhibited NF-κB activity, which may ex-
ert a role in their anti-inflammatory activity.
Euphorbia kansui, Euphorbiaceae, ingenane diterpenoids,
NO production, anti‑inflammatory, NF‑κB
received January 21, 2018
revised
May 29, 2018
accepted June 14, 2018
Bibliography
DOI https://doi.org/10.1055/a-0646-4306
Published online | Planta Med © Georg Thieme Verlag KG
Stuttgart · New York | ISSN 0032‑0943
Correspondence
Prof. Sheng Yin
School of Pharmaceutical Sciences, Sun Yat-sen University
No. 132 Waihuan East Road, Guangzhou 510006, People’s
Republic of China
Phone: + 862039943090, Fax: + 862039943090
yinsh2@mail.sysu.edu.cn
tory mediators involved in the NF-κB signaling pathway might be
potential anti-inflammatory candidates.
Introduction
Inflammation is a defensive response of the organism to infection,
physical injury, chemical insult, or radiation [1,2]. Inflammation
has been investigated intensively over the last two decades. How-
ever, owing to its involvement in devastating pathological condi-
tions and the serious side effects of existing anti-inflammatory
drugs, the discovery and development of novel anti-inflammatory
agents remain a crucial challenge [3, 4]. The initiation, promotion,
and progression of various inflammatory diseases are mediated by
a number of proinflammatory mediators such as nitric oxide (NO),
TNF-α, interleukin 1 (IL-1), and interleukin 6 (IL-6) [5]. During the
inflammation process, nuclear factor-κB (NF-κB) is regarded as
one of the most important transcriptional factors in regulating
the expression of genes that encode proinflammatory mediators,
inducible enzymes (iNOS and COX-2), adhesion molecules (e.g.,
ICAM, VCAM, and E-selectin), and growth factors [6, 7]. Therefore,
compounds that downregulate the expression of these inflamma-
Euphorbia kansui L. (Euphorbiaceae) is a perennial herb widely
distributed in the northwestern provinces of China. Its roots have
been extensively used as a traditional medicine for the treatment
of inflammation-related diseases, such as asthma, edema, and as-
cites [8]. In the past decades, a number of diterpenoids, triter-
penes, and phenolic derivatives have been isolated from this
plant, some of which exhibited anti-inflammatory, antileukemia,
antitumor, antiallergic, antiviral, and antinematodal activities [9–
12].
In our continuing efforts toward discovering structurally intri-
guing anti-inflammatory substances from Euphorbiaceae plants
[13, 14], two new ingenane diterpenoids and sixteen analogues
were isolated from the roots of E. kansui. These compounds were
screened for anti-inflammatory activity by using the lipopolysac-
charide (LPS)-induced NO production model, and the action
mechanism of the active compounds was further explored. Here-
in, details of the isolation, structural elucidation, and anti-inflam-
Zhang JS et al. Anti-inflammatory Ingenane Diterpenoids … Planta Med

Original Papers
matory activities as well as preliminary structure-activity relation-
ships (SARs) of these compounds are described.
Results and Discussion
Roots of E. kansui (4 kg) were extracted with 95% EtOH at room
temperature to give a residue, which was suspended in H2O and
successively partitioned with petroleum ether (PE), EtOAc, and n-
BuOH. Each fraction was tested for inhibitory effects on NO gen-
eration in LPS-induced RAW264.7 cells. The PE-soluble fraction
showed promising activity (> 80% inhibition at 50 µg/mL) and
was selected for further chemical investigation, which afforded
1–18 (▶ Fig. 1).
Compound 1, a colorless oil, had a molecular formula of
C
36H54O9, as established by HRESIMS, corresponding to ten de-
grees of unsaturation. The IR absorption bands revealed the pres-
ence of OH (3471 cm−1) and carbonyl (1729 cm−1) groups. The 1H
and 13C NMR data and HSQC spectra exhibited signals for one ke-
tone (δC 205.2), three carbonyl groups (δC 174.0, 171.7, and
171.0), two trisubstituted double bonds [δH 6.09 (1H, d,
J = 4.3 Hz) and 6.03 (1H, s); δC 136.3, 136.1, 131.6, and 128.5],
and seven methyl groups [δH 2.16 (3H, s), 2.05 (3H, s), 1.79 (3H,
s), 1.19 (3H, s), 1.06 (3H, s), 0.98 (3H, d, J = 7.2 Hz), and 0.88 (3H,
t, J = 6.7 Hz); δC 22.5, 21.1, 21.0, 18.2, 16.7, 15.5, and 14.1]. As six
of the ten degrees of unsaturation were accounted for by one ke-
tone, three carbonyls, and two double bonds, the remaining de-
grees of unsaturation required that 1 be tetracyclic. The above-
mentioned data were quite similar to that of the known ingenane
diterpenoid 5,20-O-diacetyl-3-O-(2,3-dimethylbutanoyl)-13-O-
dodecanoylingenol [15], except for the absence of an acetyl
group and an upfield-shifted signal [δH 3.87 (1H, d, J = 6.5 Hz)], in-
dicating that 1 was a deacetylated derivative of the previously de-
scribed compound. HMBC correlations from the upfield-shifted
signal (δH 3.87) to C-3, C-7, C-10, and C-20 indicated that the pro-
ton corresponding to this signal was at C-5. The methylene signals
of the 13-O-dodecanoyl chain were overlapped (δH 1.23–1.30).
The number of methylenes in this substituent was thus assigned
from the MS data. The planar structure of 1 was further confirmed
by detailed analysis of its 2D NMR data (▶ Fig. 2). The relative
configuration of 1 was assigned to be the same as that of 5,20-O-
diacetyl-3-O-(2,3-dimethylbutanoyl)-13-O-dodecanoylingenol by
comparing their 1D NMR data and analyzing the NOESY data. In
particular, NOE interactions of H-3/H-5 indicated that H-3 and H-
5 were cis-oriented and were arbitrarily assigned in the α-orienta-
tion. The structure of 1 was further confirmed by the chemical
correlation with the co-isolated known analogue 3-O-(2,3-dime-
thylbutyryl)-13-O-n-dodecanoyl-13-hydroxyingenol (4) [16]. The
alkaline hydrolysis of 1 and 4 generated the same product, 19
(▶ Fig. 3), which was verified by comparison of their NMR and MS
data. Thus, the structure of 1 was elucidated as depicted and was
given the trivial name euphorkan A.
▶ Fig. 1 Structures of compounds 1–19.
▶ Fig. 2 Selected 1H-1H COSY (blue) and HMBC (→) correlations of 1.
▶ Fig. 3 Chemical correlations of compounds 1, 2, 4, and 19.
Compound 2 had a molecular formula of C42H64O10, as deter-
mined by HRESIMS and 13C NMR data. The 1D NMR spectra of 2
were very similar to those of 20-O-(2,3-dimethylbutanoyl)-13-O-
dodecanoylingenol [17], except for the presence of two addition-
al acetyl groups [δH 2.11 (s) and 2.26 (s); δC 20.8, 21.1, 170.7, and
172.5], indicating that 2 was a diacetylated derivative of the
latter. The two acetyl groups were located at 3-OH and 5-OH on
the basis of the HMBC correlations of H-3 (δH 4.97)/(δC 172.5) and
H-5 (δH 5.39)/(δC 170.7), respectively, which was further sup-
ported by the strongly downfield-shifted H-3 and H-5 signals in 2
with respect to those in 20-O-(2,3-dimethylbutanoyl)-13-O-
Zhang JS et al. Anti-inflammatory Ingenane Diterpenoids… Planta Med

An excess of proinflammatory mediators such as TNF-α, IL-1,
IL-6, NO, and PGE2 produced by macrophages can trigger patho-
logical conditions and lead to multiple chronic inflammatory dis-
eases. Particularly, TNF-α and IL-6 are regarded as potent bio-
markers to assess the inflammatory process [26]. To further ex-
plore the anti-inflammatory potential of 1 and 5, their inhibitory
effects on the LPS-induced expression of TNF-α and IL-6 in
RAW264.7 cells were investigated. As shown in ▶ Fig. 4A,B, the
enhanced levels of TNF-α and IL-6 via the stimulation of LPS were
dramatically decreased (p < 0.01) by pretreatment with 1 and 5. In
particular, 1 and 5 inhibited TNF-α in a dose-dependent manner.
These results suggest that 1 and 5 were able to reduce the inflam-
matory response in RAW264.7 cells. As NF-κB plays a key role in
inflammation via transcription of proinflammatory genes [27],
whether 1 and 5 could suppress the activation of NF-κB was also
investigated. A luciferase assay was performed on a RAW264.7
cell line stably transfected with NF-κB-luciferase reporter plasmid.
As shown in ▶ Fig. 5, compound 5 exhibited about a 2-fold more
potent inhibition on NF-κB activation (IC50 = 11.0 µM) than the
positive control pyrrolidine dithiocarbamate (PDTC), a well-known
▶ Table 1 IC50 values of the active compounds on LPS-induced NO
production in RAW264.7 cellsa.
Compound
IC50 (µM)
1
4.90 (C. I. 4.67–5.13)
10.4 (C. I. 10.8–11.0)
5.69 (C. I. 5.44–5.94)
5.80 (C. I. 5.23–6.33)
2.78 (C. I. 2.72–2.84)
10.6 (C. I. 10.04–11.02)
2.86 (C. I. 2.40–3.30)
9.05 (C. I. 8.40–9.70)
9.45 (C. I. 8.54–10.33)
4.60 (C. I. 4.14–5.03)
8.86 (C. I. 7.40–9.10)
15.8 (C. I. 14.4–16.2)
2
3
4
5
6
10
11
12
13
19
Quercetinb
aCompounds with IC50 > 10 µM were not listed. bPositive control
NF-κB inhibitor (IC50 = 20.9 µM) [25], while compound 1 (IC50
=
17.9 µM) was comparable to PDTC. This study demonstrated that
the effects of 1 and 5 on the production of NO, TNF-α, and IL-6 in
LPS-induced RAW264.7 macrophages were associated with the
inhibition of NF-κB transcriptional activity.
The current study supports the use of E. kansui in folk medicine
for the treatment of inflammation-related diseases. While the
anti-inflammatory activity of ingenanes has been previously re-
ported [28, 29], this is the first study on the anti-inflammatory
mechanism of this group of compounds. Based on the present re-
sults, ingenane diterpenes deserve further investigation as poten-
tial therapeutic candidates for inflammation-related conditions.
dodecanoylingenol (H-3, δH 4.43 and H-5, δH 4.74). The structure
of 2 was further confirmed by the chemical correlation of 2 to 19
via alkaline hydrolysis (▶ Fig. 3). Compound 2 was named euphor-
kan B.
The known compounds 3-O-(2,3-dimethylbutanoyl)-13-O-do-
decanoyl-20-O-acetylingenol (3) [18], 3-O-(2.3-dimethylbutyryl)-
13-O-n-dodecanoyl-13-hydroxyingenol (4) [16], 3-O-(2′E,4′E-
decadienoyl) ingenol (5) [19], 3-O-(2′E,4′Z-decadienoyl) ingenol
(6) [19], 3-O-(2E,4E-decadienoyl)-20-deoxyingenol (7) [18], 3-O-
(2E,4Z-decadienoyl)-20-deoxyingenol (8) [18], 3-O-(2′E,4′E-deca-
dienoyl)-20-O-acetylingenol (9) [19], 3-O-(2′E,4′Z-decadienoyl)-
20-O-acetylingenol (10) [20], 20-O-(2′E,4′E-decadienoyl) ingenol
(11) [19], 20-O-(2′E,4′Z-decadienoyl) ingenol (12) [19], 20-O-ace-
tyl-[5-O-(2′E,4′Z)-decadienoyl]-ingenol (13) [20], kansuiphorin C
(14) [21], 5-O-benzoyl-20-deoxyingenol (15) [22], 3-O-benzoyl-
20-deoxyingenol (16) [22], 20-deoxyingenol (17) [23], ingenol
(18) [24], and 13-O-docecanoylingenol (19) [15] were identified
by comparison of their spectroscopic data with those in the liter-
ature.
Compounds 1–19 were evaluated for their inhibitory effects
on NO production in LPS-activated RAW264.7 macrophages.
Quercetin, a well-known natural NO inhibitor, was used as the pos-
itive control (IC50 = 15.8 µM) [25]. As shown in ▶ Table 1, com-
pounds 1–6, 10–13, and 19 exhibited pronounced inhibitory ac-
tivity with IC50 values ranging from 2.78 to 10.6 µM, while the
others with IC50 values greater than 25 µM were considered to be
inactive. To exclude that the inhibitory activities of the active
compounds on RAW264.7 cells were due to cytotoxicity, cell via-
bility was measured using the MTT method. No obvious cytotox-
icity (> 90% cell survival) was observed at the concentration of
50 µM (Data not shown). Preliminary SARs revealed that the oxi-
dation of C-20 and C-13 may be important to the activity, as all
active compounds, except compound 9, contain a hydroxyl group
or an ester residue at these positions.
Materials and Methods
General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polarime-
ter. UV spectra were recorded on a Shimadzu UV-2450 spectro-
photometer. IR spectra were determined on a Bruker Tensor 37
infrared spectrophotometer with KBr disks. NMR spectra were
measured on a Bruker AM-400 spectrometer at 25°C. ESIMS and
HRESIMS were carried out on a Finnigan LCQ Deca instrument.
Absorbance was recorded at 490 nm and 540 nm using a micro-
plate reader (Molecular Devices) with Soft Max Pro 5 software
(Molecular Devices). Semipreparative HPLC was performed on an
A Shimadzu LC-20AT equipped with an SPD-M20A PDA detector
and a YMC-pack ODS‑A column (250 × 10 mm, S-5 µm, 120 Å).
Silica gel (300–400 mesh, Qingdao Haiyang Chemical Co. Ltd.),
reversed-phase C18 (RP‑C18) silica gel (S-50 µm, 120 Å, YMC Co.
Ltd.), and MCI gel (CHP20P, 75–150 µm, Mitsubishi Chemical In-
dustries Ltd.) were used for column chromatography (CC). All sol-
vents were of analytical grade.
LPSs (from Escherichia coli serotype 055:B5), PDTC (98%), and
quercetin (98%) were purchased from Sigma-Aldrich. MTT,
DMSO, and geneticin (G418) were obtained from Millipore. DMEM
and FBS were purchased from Gibco. A Griess reagent kit was pro-
Zhang JS et al. Anti-inflammatory Ingenane Diterpenoids … Planta Med

Original Papers
▶ Fig. 4 A Effects of 1 on the production of TNF-α in LPS-induced RAW264.7 macrophages. B Effects of 5 on the production of TNF-α in LPS-in-
duced RAW264.7 macrophages. RAW264.7 macrophages were treated with increasing concentrations of 1 (1, 5, and 10 µM) and 5 (1, 3, and 9 µM)
for 30 min followed by LPS stimulation for another 24 h. The expression of TNF-α and IL-6 was measured by an ELISA assay. (**p < 0.01 versus the
group treated with LPS only)
vided by Beyotime. Luciferase assay system reagent was from
Promega. Mouse TNF-α and IL-6 kits were purchased from Neo-
bioscience.
Plant material
The roots of E. kansui were purchased in January 2016 from
Bozhou Medicine Market, Anhui Province, P.R. China, and were
identified by Dr. Gui-Hua Tang of Sun Yat-sen University. A vouch-
er specimen (accession number: GS201607) has been deposited
at the School of Pharmaceutical Sciences, Sun Yat-sen University.
Extraction, bioassay-guided fractionation,
and isolation
▶ Fig. 5 Inhibitory effect of 1 and 5 on LPS-induced NF-κB-depen-
dent promoter activity in RAW264.7 macrophage stably trans-
fected with NF-κB-luciferase reporter plasmid. Pyrrolidine dithio-
carbamate (PDTC) was used as a positive control. Cells were pre-
treated with the indicated concentration (1.56–50 µM) for 1 h prior
to treatment with LPS (1 µg/mL) for 6 h. Then, the activity was
measured using the Luciferase system assay.
The air-dried powder of the roots of E. kansui (4 kg) was extracted
with 95% EtOH (3 × 3 L) at room temperature to give 120 g of
crude extract. The extract was suspended in H2O (3 L) and parti-
tioned successively with PE (3 × 3 L), EtOAc (3 × 3 L), and n-BuOH
(3 × 3 L). Each of these fractions was tested for its inhibitory activ-
ity against NO production induced by LPS in RAW264.7 macro-
phage cells. The PE extract (90 g, 80.2% inhibition at 50 µg/mL)
was subjected to MCI gel CC eluted with a MeOH/H2O gradient
(3:7 → 10:0) to afford four fractions (I–IV). Fr. I (8.8 g) was sub-
jected to silica gel CC (PE/EtOAc, 5:1 → 0:1) to give five fractions
(Ia-Ie). Fr. Ic (1.1 g) was separated by silica gel CC (PE/acetone,
10:1) followed by semipreparative HPLC (MeCN/H2O, 70/30,
3 mL/min) to give 7 (15 mg, tR 8.0 min) and 8 (13 mg, tR
9.8 min). Fr. Id (128 mg) was purified on silica gel CC (CH2Cl2/
MeOH, 100:1) to give 13 (6.7 mg). Fr. Ie (550 mg) was separated
by RP‑C18 silica gel CC (MeOH/H2O, 6:4 → 10:0) to yield 6
(14.2 mg) and 12 (48 mg). Fr. II (16.5 g) was subjected to silica
gel CC (CH2Cl2/MeOH, 0:1 → 100:1) to give five fractions (IIa-
IIe). Fr. IIb (6.1 g) was separated by RP‑C18 silica gel CC (MeOH/
H2O, 5:5 → 10:0) followed by silica gel CC (PE/acetone, 10:1 →
4:1) to give 11 (4.8 mg) and 15 (9.6 mg). Fr. III (2.5 g) was sub-
jected to silica gel CC (CH2Cl2/MeOH, 1:0 → 100:1) to give two
fractions (IIIa and IIIb). Fr. IIIb (0.8 g) was subjected to a Sephadex
LH-20 column using MeOH as the eluent followed by semiprepar-
ative HPLC (MeOH/H2O, 80/20, 3 mL/min) to give 3 (3.1 mg, tR
12.6 min), 14 (4.3 mg, tR 15.3 min), and 16 (6.3 mg, tR 15.6 min).
Fr. IV (7 g) was applied to silica gel CC (PE/acetone, 400:1 → 3:1)
to give four fractions (IVa-IVd). Fr. IVb (862 mg) was separated by
RP‑C18 silica gel CC (MeOH/H2O, 5:5 → 10:0) followed by semi-
preparative HPLC (MeOH/H2O, 85/15, 3 mL/min) to give 9
(9.5 mg, tR 11.1 min) and 10 (8.3 mg, tR 12.2 min). Fr. IVc
(121 mg) was further purified by a Sephadex LH-20 column using
MeOH as the eluent to give 5 (8 mg) and 1 (4.8 mg). Fr. IVd (2.3 g)
was separated by RP‑C18 silica gel CC (MeOH/H2O, 7:3 → 10:0) to
give three subfractions (Fr. IVd1-IVd3). Fr. IVd2 was further sepa-
rated by semipreparative HPLC (MeOH/H2O, 85/15, 3 mL/min) to
give 18 (5 mg, tR 8.6 min) and 17 (8.1 mg, tR 9.3 min). Fr. IVd3 was
applied to silica gel CC (PE/acetone, 8:1) to give 4 (5.2 mg) and 2
(11 mg).
Euphorkan A (1): colorless oil; [α]2D5 + 33.7 (c 0.2, CH2Cl2); UV
(MeOH) λmax (log ε) 204 (4.17) nm; IR (KBr) νmax 3471, 1729,
1377, 1231, 1022 cm−1; HRESIMS m/z 653.3658 [M + Na]+ (calcd.
1
for C36H54O9Na+, 653.3660); H NMR (CDCl3, 400 MHz): δH 6.09
(1H, d, J = 4.3 Hz, H-7), 6.03 (1H, s, H-1), 5.47 (1H, s, H-3), 4.75
Zhang JS et al. Anti-inflammatory Ingenane Diterpenoids… Planta Med

(1H, d, J = 12.6 Hz, H-20a), 4.48 (1H, d, J = 12.6 Hz, H-20b), 4.05
(1H, m, H-8), 3.87 (1H, d, J = 6.5 Hz, H-5), 2.72 (2H, m, H2-12),
2.60 (1H, m, H-11), 1.79 (3H, s, H3-19), 1.19 (3H, s, H3-16), 1.06
(3H, s, H3-17), 1.05 (1H, m, H-14), 0.98 (3H, d, J = 7.2 Hz, H3-18),
3-OAc: 2.05 (3H, s), 13-OCO(CH2)10CH3: 0.88 (3H, t, J = 6.7 Hz),
1.23–1.30 (H-3′−H-10′, overlapped); 20-OAc: 2.16 (3H, s); 13C
NMR (CDCl3, 100 MHz): δC 205.2 (C, C-9), 136.3 (C, C-2), 136.1
(C, C-6), 131.6 (CH, C-1), 128.5 (CH, C-7), 84.5 (C, C-4), 82.9
(CH, C-1), 74.7 (C, C-5), 71.9 (C, C-10), 68.9 (C, C-13), 66.6
(CH2, C-20), 42.8 (CH, C-8), 37.6 (CH, C-11), 35.2 (CH2, C-12),
30.4 (C, C-15), 28.3 (CH, C-14), 22.5 (CH3, C-16), 18.2 (CH3, C-
18), 16.7 (CH3, C-17), 15.5 (CH3, C-19), 3-OAc: 171.7, 21.1, 20-
OAc: 171.0, 21.0; 13-OCO(CH2)10CH3: 174.0 (C, C-1′), 34.4
(CH2, C-2′), 31.9–29.2 (CH2 × 8, C‑3′−C-10′ overlapped), 22.7
(CH2, C-11′), 14.1 (CH3, C-12′).
of G418 (500 µg/mL). DMSO was used to dissolve all compounds.
The final solvent concentration in the assay was 1%.
Analysis of nitric oxide production
RAW264.7 microphages were seeded into 96-well plates at a den-
sity of 5 × 104 cells/well for 24 h and then preincubated with dif-
ferent concentrations of compounds for 1 h before stimulation
with or without LPS (1 µg/mL) for 24 h. The NO concentration in
culture medium was determined by a Griess reagent kit. The ab-
sorbance (A) at 540 nm was measured using a multifunction mi-
croplate reader (Molecular Devices, Flex Station 3). Sodium nitrite
was used as a standard to calculate the nitrite concentration. Inhi-
bition (%) = [1 – (ALPS + sample – Auntreated)/(ALPS – Auntreated)] × 100.
The experiments were performed in triplicate, and the IC50 values
were calculated by nonlinear regression in GraphPad Prism 5.
Quercetin was used as a positive control.
Euphorkan B (2): colorless oil; [α]2D5 + 41.4 (c 0.3, CH2Cl2); UV
(MeOH) λmax (log ε) 208 (3.47) nm; IR (KBr) νmax 2925, 1732,
1369, 1232, 1023 cm−1; HRESIMS m/z 727.4425 [M – H]− (calcd
for C42H63O10−, 727.4427); 1H NMR (CDCl3, 400 MHz): δH 6.22
(1H, brs, H-7), 6.07 (1H, brs, H-1), 5.39 (1H, s, H-5), 4.97 (1H, s,
H-3), 4.53 (1H, d, J = 15.2 Hz, H-20a), 4.25 (1H, d, J = 15.2 Hz, H-
20b), 4.20 (1H, m, H-8), 2.70 (1H, m, H-12a), 2.60 (1H, m, H-11),
2.27 (1H, m, H-12b), 1.76 (3H, s, H3-19), 1.26 (1H, m, H-14), 1.18
(3H, s, H3-16), 1.06 (3H, s, H3-17), 0.99 (3H, d, J = 7.0 Hz, H3-18),
3-OAc: 2.11 (3H, s), 5-OAc: 2.26 (3H, s), 13-OCO(CH2)10CH3: 0.88
(3H, t, J = 6.8 Hz), 1.21–1.32 (H-3′−H-10′, overlapped); 20-
OCOCH(CH3)CH(CH3)2: 2.19 (1H, m, H-2″), 1.86 (1H, m, H-4″),
1.06 (3H, d, J = 7.0 Hz), 0.88 (3H, d, J = 6.9 Hz), 0.86 (3H, d,
J = 6.9 Hz); 13C NMR (CDCl3, 100 MHz): δC 204.4 (C, C-9), 135.8
(C, C-2), 133.9 (C, C-6), 131.6 (CH, C-1), 130.4 (CH, C-7), 85.6
(C, C-4), 82.2 (CH, C-3), 75.0 (C, C-5), 71.8 (C, C-10), 69.1 (C, C-
13), 65.0 (CH2, C-20), 42.9 (CH, C-8), 37.8 (CH, C-11), 34.9 (CH2,
C-12), 30.7 (C, C-15), 28.1 (CH, C-14), 22.7 (CH3, C-16), 18.0
(CH3, C-18), 16.8 (CH3, C-17), 15.3 (CH3, C-19), 3-OAc: 172.5,
21.1, 5-OAc: 170.7, 20.8, 13-OCO(CH2)10CH3: 174.0 (C, C-1′),
34.4 (CH2, C-2′), 31.9–29.2 (CH2 × 8, C-3′−C-10′ overlapped),
22.7 (CH2, C-11′), 14.1 (CH3, C-12′); 20-OCOCH(CH3)CH(CH3)2:
176.0 (C, C-1″), 45.9 (C, C-2″), 30.9 (C, C-4″), 19.0 (C, C-5″),
14.1 (C, C-3″), 13.4 (C, C-6″).
Cytotoxicity assay
The cytotoxicity of the isolated compounds toward RAW264.7
cells was determined by the MTT assay. RAW264.7 cells were
seeded in 96-well plates (5 × 103/well) for 24 h. Next, the test
samples dissolved in DMSO were added into the wells and diluted
with 100 µL DMEM to the final concentration of 50 µM with 1%
DMSO serving as the solvent control. Wells containing only
100 µL DMEM were used as a blank control. Then, 24 h later,
20 µL solution MTT (5 mg/mL) were added to each well. After in-
cubation for 4 h, the medium was removed and 100 µL DMSO
were added to each well. The absorbance (A) was recorded at
490 nm using a microplate reader. The inhibition of cell growth
was calculated according to the following formula: % Inhibition =
[1 – (Asample – Ablank)/(Asolvent – Ablank)] × 100.
Analysis of TNF-α and interleukin 6 production
RAW264.7 macrophages were pretreated with 1 and 5 for 1 h
prior to stimulation with LPS (0.1 µg/mL) for 6 h. The concentra-
tions of TNF-α and IL-6 in the culture medium were determined
using commercial ELISA kits according to the instructions of each
manufacturer.
Luciferase report assay
Alkaline hydrolysis of 1, 2, and 4
The luciferase assay was performed to analyze NF-κB-dependent
reporter gene transcription as previously described [25].
RAW264.7 cell lines stably transfected with p-NF-κB‑Luc reporter
plasmid (4 × 104 cells/well) were plated into 96-well plates over-
night. After pretreating with compounds 1, 5, and PDTC (positive
control), respectively, at indicated concentrations for 1 h, cells
were stimulated with LPS (1 µg/mL) for 6 h. Then, each well was
slightly washed with ice-cold PBS twice followed by lysis in lysis
buffer. Luciferase activity was detected by the Promega luciferase
assay system.
Compounds 1 (0.9 mg), 2 (1.1 mg), and 4 (8 mg) were stirred with
1 mL of 0.1 M NaOH in MeOH for 2 h at room temperature, re-
spectively. Then each mixture was subjected to Sephadex LH-20
using MeOH as the eluent to afford the pure hydrolysis product
19, which was identified by NMR and MS data.
Cell culture
The RAW264.7 murine macrophage cell lines purchased from Cell
Bank of Shanghai Institute of Biochemistry and Cell Biology (Chi-
nese Academy of Sciences, Shanghai, China) were grown in DMEM
supplemented 10% FBS. Cells were cultured under standard con-
ditions (5% CO2 in the air in a humidified environment at 37°C).
RAW264.7 cells stably transfected with NF-κB-luciferase reporter
plasmid were provided by Prof. Depo Yang, School of Pharmaceu-
tical Sciences, Sun Yat-sen University (Guangzhou, China) and
were cultured under the same conditions except for the presence
Supporting information
1D and 2D NMR spectra of 1 and 2, and 1H and 13C NMR spectra of
compounds 3–19 are available as Supporting Information.
Zhang JS et al. Anti-inflammatory Ingenane Diterpenoids … Planta Med

Original Papers
[15] Shi J, Li Z, Nitoda T, Izumi M, Kanzaki H, Baba N, Kauazu K, Nakajima S.
Antinematodal activities of ingenane diterpenes from Euphorbia kansui
and their derivatives against the pine wood nematode (Bursaphelenchus
xylophilus). Z Naturforsch C 2008; 63: 59–65
Acknowledgements
The authors thank the National Natural Science Foundation of China
(No. 81573302), the Science and Technology Planning Project of
Guangdong Province (No. 2013B021100009), and the National High
Technology Research and Development Program of China (863 Project,
No. 2015AA020928) for providing financial support for this work.
[16] Matsumoto T, Cyong JC, Yamada H. Stimulatory effects of ingenols from
Euphorbia kansui on the expression of macrophage Fc receptor. Planta
Med 1992; 58: 255–258
[17] Ott HH, Hecker E. Highly irritant ingenane type diterpene esters from
Euphorbia cyparissias L. Experientia 1981; 37: 88–91
Conflict of Interest
[18] Wang LY, Wang NL, Yao XS, Miyata S, Kitanaka S. Diterpenes from the
roots of Euphorbia kansui and their in vitro effects on the cell division of
xenopus. J Nat Prod 2002; 65: 1246–1251
The authors declare no conflict of interest.
[19] Wang LY, Wang NL, Yao XS, Miyata S, Kitanaka S. Diterpenes from the
roots of Euphorbia kansui and their in vitro effects on the cell division of
xenopus (part 2). Chem Pharm Bull 2003; 51: 935–941
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