New podolactones from the seeds of Podocarpus nagi and their anti-inflammatory effect

Article abstract of DOI:10.1007/s11418-018-1219-5

Podolactones are a class of structural diverse diterpenoid lactones, mainly isolated from the Podocarpus species. Several bioactivities have been disclosed for podolactones, including cytotoxicity and anti-atherosclerosis. In this study, the seeds of P. nagi were isolated by comprehensive chromatographic methods to obtain three new podolatones, named nagilactone B 1-O-β-d-glucoside (1), nagilactone N3 3-O-β-d-glucoside (2), and 2-epinagilactone B (3), as well as a known compound, nagilactone B (4). Their structures were determined by analyses of NMR and HRESIMS data. Compounds 1 and 2 significantly inhibited nitric oxide (NO) production on LPS-stimulated RAW264.7 macrophages, with IC₅₀ values of 0.18 ± 0.04 and 0.53 ± 0.03?μM, respectively. Indomethacin (IC₅₀ 4.21 ± 0.32?μM) was used as a positive control. Compound 1 suppressed the expression of inducible NO synthase (iNOS) in a concentration-dependent manner, mediating through inhibiting nuclear factor-κB (NF-κB) activity. This is the first report regarding the anti-inflammatory effect of podolactones, which could be potential anti-inflammatory agents.

Full text of DOI:10.1007/s11418-018-1219-5

Journal of Natural Medicines
https://doi.org/10.1007/s11418-018-1219-5
ORIGINAL PAPER
New podolactones from the seeds of Podocarpus nagi and their
anti‑infammatory efect
Zhe‑Ling Feng1,2 · Tian Zhang1 · Jing‑Xin Liu1 · Xiu‑Ping Chen1 · Li‑She Gan3 · Yang Ye2 · Li‑Gen Lin1,2
Received: 3 April 2018 / Accepted: 5 May 2018
© The Japanese Society of Pharmacognosy and Springer Japan KK, part of Springer Nature 2018
Abstract
Podolactones are a class of structural diverse diterpenoid lactones, mainly isolated from the Podocarpus species. Several
bioactivities have been disclosed for podolactones, including cytotoxicity and anti-atherosclerosis. In this study, the seeds
of P. nagi were isolated by comprehensive chromatographic methods to obtain three new podolatones, named nagilactone
B 1-O-β-d-glucoside (1), nagilactone N3 3-O-β-d-glucoside (2), and 2-epinagilactone B (3), as well as a known compound,
nagilactone B (4). Their structures were determined by analyses of NMR and HRESIMS data. Compounds 1 and 2 signif-
cantly inhibited nitric oxide (NO) production on LPS-stimulated RAW264.7 macrophages, with IC50 values of 0.18 0.04
and 0.53 0.03 μM, respectively. Indomethacin (IC50 4.21 0.32 μM) was used as a positive control. Compound 1 suppressed
the expression of inducible NO synthase (iNOS) in a concentration-dependent manner, mediating through inhibiting nuclear
factor-κB (NF-κB) activity. This is the frst report regarding the anti-infammatory efect of podolactones, which could be
potential anti-infammatory agents.
Keywords Podocarpus nagi · Podolactones · Anti-infammation · NO production · NF-κB
Introduction
are of great interest due to their unusual structures and broad
biological activities. Podocarpus nagi (Thunb.) Zoll. Et Mor.
exZoll (Podocarpaceae) is widely distributed in the Southern
part of the People’s Republic of China and Southeast Asia,
where it is traditionally used to treat fevers, asthma, coughs,
cholera, and venereal diseases [2]. Tens of podolacones and
several abietane-type and ent-pimarane-type diterpenoids
have been identifed from the diferent parts of P. nagi; many
of these have exhibited cytotoxic [3, 4], anti-infammatory
[3], anti-atherosclerotic [5] and insecticidal [6] properties.
In systematically searching for anti-infammatory natural
products, we have identifed several classes of molecules
with potency activity, including xanthones from Garcinia
mangostana [7, 8] and abietane-type diterpenoids from P.
nagi [9]. In a continuing study, three new podolactones,
named nagilactone B 1-O-β-d-glucoside (1), nagilactone
N3 3-O-β-d-glucoside (2), and 2-epinagilactone B (3), were
isolated from the seeds of P. nagi, as well as a known com-
pound, nagilactone B (4). This study describes the isolation
of the new compounds, elucidates their structure, and inves-
tigates their anti-infammatory efect on lipopolysaccharide
(LPS)-stimulated RAW264.7 macrophages and underlying
mechanisms.
Podolactones are a group of nor- or dinor-diterpenoids con-
taining a γ-lactone between carbons 6–19 and a δ-lactone
between carbons 12–14, which were mainly isolated from
plants of the Podocarpus genus [1]. These natural products
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11418-018-1219-5) contains
supplementary material, which is available to authorized users.
Zhe-Ling Feng and Tian Zhang have contributed equally to this
study.
* Li-Gen Lin
ligenl@umac.mo
1
State Key Laboratory of Quality Research in Chinese
Medicine, Institute of Chinese Medical Sciences, University
of Macau, Avenida da Universidade, Taipa, Macau 999078,
China
2
State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences,
555 Zu-Chong-Zhi Road, Zhangjiang Hi-tech Park,
Shanghai 201203, China
3
College of Pharmaceutical Sciences, Zhejiang University,
866 Yuhangtang Road, Hangzhou 310058, China
Vol.:(0123456789)
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Journal of Natural Medicines
Table 1 1H-NMR (600 MHz, δ in ppm, J in Hz) data for compounds
1‒3 in DMSO-d6
Results and discussion
No.
1
2
3
The n-butanol-soluble fraction of the 95% ethanol extract
of P. nagi was purifed by column chromatography over
silica gel, MCI gel and preparative HPLC to aford three
new podolactones (1‒3, Fig. 1). The absolute confgura-
tion of the sugar components was determined as described
previously [10]. The sugars were converted into thiazo-
lidine derivatives and then into the arylthiocarbamates
using l-cysteine methyl ester and O-tolylisothiocyanate.
By comparing the retention time with the standard sug-
ars using the HPLC–UV method, the sugar moieties in
compounds 1 and 2 were identifed to be d-glucose (Sup-
plementary Fig. S21).
1α
2α
2β
3α
3β
5
4.25, d (5.8)
3.87, m
3.57, d (4.2)
3.70, m
3.51, t (5.1)
3.62, dd (9.7, 5.1)
1.48, m
2.25, dd (14.6, 5.1)
2.00, d (6.5)
1.48, dd (13.0, 4.0) 4.34, d (6.3)
2.09, t (12.8)
2.05, d (6.2)
5.05, m
5.17, dd (8.7, 4.3) 5.21, dd (8.8, 4.2) 5.18, m
7.23, s
2.13, d (6.6)
4.99, dd (8.8, 6.6) 5.04, m
6
7α
11
15
6.28, s
3.28, m
1.17, d (6.8)
1.20, d (6.8)
1.43, s
6.51, s
3.25, m
1.15, d (6.8)
1.18, d (6.8)
1.27, s
3.21, m
16/17 1.15, d (6.8)
1.18, d (6.8)
Compound 1 was isolated as a white amorphous pow-
der, and its HRESIMS indicated a molecular formula
of C25H34O12 from the deprotoned molecular ion at m/z
525.1975 ([M − H]−, calcd for C25H33O12, 525.1972).
Its HRESIMS showed a typical fragment ion at m/z
363.1447 [M − 162]−, indicating the presence of a glu-
cose unit. The IR spectrum exhibited the absorptions for
hydroxy (3327 cm−1), γ-lactone carbonyl (1763 cm−1) and
δ-lactone carbonyl (1708 cm−1) groups. The 1H- and 13C-
NMR spectra of 1 (Tables 1 and 2), with the aid of the
HSQC data, displayed signals for an anomeric carbon (δH
4.77, d, J = 8.0 Hz/δC 103.4), indicating the existence of a
glucose unit [10]. Additionally, the NMR spectra showed
signals ascribable to an olefnic proton (δH 7.23, s/δC
106.8, CH-11), four oxygen-bearing methines (δH 3.87,
m/δC 65.7, CH-2; δH 4.25, d, J = 5.8/δC 78.4, CH-1; δH
5.05, m/δC 73.9, CH-6; δH 5.17, dd, J = 8.7, 4.3/δC 58.3,
CH-7), two singlet methyls (δH 1.27/δC 23.2, CH3-18;
δH 1.35/δC 18.7, CH3-20), and two doublet methyls (δH
1.15, J = 6.8 Hz/δC 20.0; δH 1.18, J = 6.8 Hz/δC 20.2,
CH3-16/17), which quite resembled those of nagilactone
B [11, 12]. The major diference was the down-feld shift
of C-1 (δC from 71.4 to 78.4), indicating the glucose unit
might be linked to C-1. The HMBC correlations from H-1
18
20
1′
2′
3′
4′
5′
6′
1.27, s
1.35, s
4.77, d (8.0)
3.10, m
3.23, m
3.11, m
3.24, m
1.22, s
1.21, s
4.50, d (7.7)
3.02, dd (9.2, 7.7)
3.20, m
3.06, m
3.28, m
3.70, m
3.47, m
3.74, m;
3.58, dt (12.0, 6.0)
1-OH
5.30, d (4.7)
5.04, m
5.84, d (4.4)
2-OH 4.86, d (9.2)
7-OH 5.82, d (4.4)
2′-OH 5.88, d (3.9)
3′-OH 5.06, d (5.9)
4′-OH 4.97, d (5.5)
6′-OH 4.32, t (6.6)
5.89, d (4.5)
4.20, d (1.8)
to C-1′, and from H-1′ to C-1, fully assigned the glucose
moiety at C-1. The relative confguration of 1 was inferred
by ROESY spectrum. The NOE correlations of H-1/H-2,
H-1/H-5, H-2/H-5, H-2/CH3-18, H-5/H-6, H-5/CH3-18,
and H-6/H-7 indicated the α-orientation of H-1, H-2, H-5,
Fig. 1 Chemical structures of compounds 1‒3
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Journal of Natural Medicines
Table 2 13C-NMR (150 MHz, δ in ppm) data for compounds 1‒3 in
DMSO-d6
absolute confgurations of their core skeleton [4]. Accord-
ingly, the structure of nagilactone B 1-O-β-d-glucoside (1)
was established as shown (Fig. 1).
No.
1
2
3
Compound 2 had the molecular formula C25H34O13, as
established by its HRESEMS ([M − H]−, m/z 541.1926,
calcd for C25H33O13, 541.1921), The IR absorptions at 3431
and 1729 cm−1 indicated the existences of hydroxy and car-
1
2
3
4
78.4 CH
65.7 CH
33.8 CH2
42.1 C
57.1 CH
50.3 CH
76.0 CH
48.9 C
71.7 CH
76.8 CH
36.1 CH2
42.8 C
1
bonyl groups. The H-NMR spectrum of 2 (Table 1) dis-
5
6
7
8
45.4 CH
73.9 CH
58.3 CH
111.5 C
164.9 C
41.1 C
106.8 CH
162.8 C
168.6 C
28.3 CH
20.0 CHa3
20.2 CHa3
23.2 CH3
180.7 C
18.7 CH3
103.4 CH
73.8 CH
69.7 CH
76.5 CH
77.2 CH
61.0 CH2
49.8 CH
73.3 CH
58.9 CH
111.7 C
164.6 C
37.1 C
106.6 CH
161.6 C
169.9 C
28.9 CH
20.5 CHa3
20.8 CHa3
26.8 CH3
178.2 C
18.8 CH3
105.9 CH
74.5 CH
70.5 CH
76.8 CH
77.7 CH
61.8 CH2
48.6 CH
74.1 CH
59.4 CH
111.8 C
165.8 C
41.0 C
106.7 CH
162.1 C
169.3 C
28.8 CH
20.5 CHa3
20.7 CHa3
25.5 CH3
181.1 C
15.7 CH3
played signals ascribable to an olefnic proton (δH 6.28, s),
fve oxygen-bearing methines (δH 5.21, dd, J=8.8, 4.2 Hz;
δH 4.99, m; δH 4.34, d, J =6.2 Hz; δH 3.70, m; δH 3.57, d,
J = 4.2 Hz), two singlet methyls (δH 1.43; 1.22), two dou-
blet methyls (δH 1.20, d, J = 6.8 Hz; 1.17, d, J = 6.8 Hz),
and a glucose unit. The 1H- and 13C-NMR data of 2 quite
resembled those of compound N3 in the patent [13]. The
HMBC correlations from H-3 (δH 4.34) to C-1′ (δC 105.9),
as well as from H-1′ (δH 4.50) to C-3 (δC 76.0), supported
the connection of the glucose moiety at C-3. In the ROESY
spectrum, the correlations of H-1/H-3, H-2/H-3, H-3/CH3-
18, H-5/CH3-18, H-6/CH3-18, and H-6/H-7 suggested the
α-orientation of H-1, H-2, H-3, H-5, H-6, H-7 and CH3-18
(Fig. 2). Additionally, compound 2 showed Cotton efects
(215.5 nm, Δε +9.6; 239.5 nm, Δε −0.7) similar to those
of compound 1, indicating the same absolute confguration
of their core skeleton. Thus, the structure of nagilactone N3
3-O-β-d-glucoside was identifed (Fig. 1).
9
10
11
12
14
15
16
17
18
19
20
1′
2′
3′
4′
5′
6′
Compounds 3 and 4 were isolated as a 6:5 mixture.
Both compounds were assigned the molecular formula of
C19H24O7 by its HRESIMS m/z 363.1456 [M−H]− (calcd
for C19H23O7, 363.1444), with 8 degrees of unsaturation.
The 1H-NMR spectra of 3 and 4 (Table 1) displayed two sets
of almost superimposable resonances, each ascribable to two
singlet methyls, two doublet methyls, a pair of geminal pro-
tons, a septet proton, four oxygenated methines, an olefnic
proton, and two active protons. The septet proton and two
doublet methyls indicated the presence of a typical isopropyl
group in the structures of 3 and 4. In the 13C-NMR spectra of
compounds 3 and 4 (Table 2), two sets of resonances were
also observed. After careful analysis, the 1H- and 13C-NMR
aExchangeable
H-6, H-7 and CH3-18 (Fig. 2). The confguration of the
anomeric proton of the glucose was assigned as β con-
fguration based on the coupling constant (J1,1� = 8.0 Hz).
Compound 1 exhibited the positive Cotton effect at
214.0 nm (Δε + 5.8) and the negative Cotton efect at
240.0 nm (Δε − 0.7), which were the similar as those of
1-deoxy-2β-hydroxynagilactone A, suggesting the same
Fig. 2 Key ROESY correlations (H ↔ H) for compounds 1‒3
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Journal of Natural Medicines
data of 4 inferred it as nagilactone B [11, 14]. Thus, com-
pound 3 was deduced to be a stereoisomer of 4. The relative
confguration of 3 was inferred from the ROESY experi-
ment. The ROESY correlations of H-5/H-6, H-6/H-7 and
H-6/H3-18 indicated that H-5, H-6, H-7, and H3-18 were on
the same side and α-oriented. Furthermore, the ROESY cor-
relations between H-1 and H-5, and between H-2 and H3-20,
indicated the hydroxy groups at C-1 and C-2 as being β- and
α-oriented, respectively (Fig. 2). Therefore, the structure of
3 was established as shown, and named 2-epinagilactone B.
All the isolates were tested for their anti-infammatory
efects on LPS-stimulated RAW264.7 macrophages. First,
MTT assay was carried out to evaluate the non-cytotoxic
concentrations of these two compounds on RAW264.7 cells.
As shown in Fig. 3a and Supplementary Fig. S22, the maxi-
mum safe dosages for compounds 1, 2, and a mixture of 3
and 4, were 1.25, 2.50 and 5.00 μM, respectively. Nitric
oxide (NO), a key mediator and regulator of infammatory
responses, is mainly produced in macrophages by induc-
ible nitric oxide synthase (iNOS). The anti-infammatory
efect the podolactones was evaluated by LPS-stimulated
NO production in RAW264.7 cells. The results showed com-
pounds 1 and 2 signifcantly inhibited NO production in a
concentration-dependent manner (Fig. 3b), with IC50 val-
ues of 0.18 0.04 and 0.53 0.03 μM, respectively. While,
the mixture of 3 and 4 did not show inhibitory efects on
Fig. 3 Cytotoxic and NO production inhibitory efects of compounds
1 and 2. a The cell viability of RAW264.7 cells treated with difer-
ent concentrations of compound 1 or 2. b Inhibitory rate of NO
production for compounds 1 and 2. c Compound 1 and indometha-
cin suppressed iNOS expression in a concentration-dependent man-
ner. GAPDH was used as an internal loading control. *p<0.05;
**p<0.01; ***p<0.001, compared with controls
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Journal of Natural Medicines
LPS-induced NO production up to 10 μM (Supplementary
Fig. S22). Indomethacin (IC50 4.21 0.32 μM) was used as
a positive control. Further studies indicated compound 1 and
indomethacin suppressed iNOS expression in a concentra-
tion-dependent manner (Fig. 3c).
The nuclear factor-κB (NF-κB) signaling pathway is
a key regulator of infammatory response. NF-κB dimers
are located in cytosol under normal conditions, combin-
ing with its inhibitor protein IκBα. In response to infam-
matory stimuli, such as LPS, NF-κB is activated through
the phosphorylation and subsequent degradation of IκBα
by the IKK (IκB kinase) complex. The activated NF-κB
dimer dissociates from IκBα and subsequently translocates
to the nucleus, where it induces the expression of various
infammatory genes, including iNOS [15]. As expected, LPS
increased the phosphorylation of IKKα/β, IκBα, and p65
remarkably, which was reversed by compound 1 treatment
(Fig. 4a). The immunofuorescent staining results showed
compound 1 suppressed LPS-induced nuclear translocation
of p65 (Fig. 4b). These results demonstrated that compound
1 suppressed the activity of NF-κB signaling pathway to
exert its anti-infammatory efect. Taken together, compound
1 was found to suppress LPS-induced activation of IKKα/β,
IκBα and p65, and prevent the translocation of NF-κB/p65
from cytoplasm to nucleus, resulting in reduction of iNOS
expression. These data suggested podolactones might be a
new class of inhibitors of the NF-κB signaling pathway.
Experimental
General experimental procedures
Optical rotation data were obtained using an Autopol VI
polarimeter. UV data were recorded with a Varian CARY
50 spectrophotometer. IR spectra were recorded on a Perki-
nElmer spectrum-100 FTIR spectrometer using KBr disks.
NMR spectra were recorded on a Bruker Avance-600
1
Fig. 4 Compound 1 inhibited NF-κB activation in LPS-stimulated
RAW264.7 cells. a The protein levels of phospho-IKKα/β, IKKα,
IKKβ, phospho-IκBα, IκBα, phospho-p65 and p65 were detected
by Western blot analyses. GAPDH was used as an internal loading
control. b The nuclear translocation of p65 was detected by immu-
nostaining. Nuclei were stained with DAPI
(Bruker, Switzerland, 600 MHz for H-NMR, 150 MHz
for 13C-NMR) NMR spectrometer, with the chemical shift
values presented as δ values having TMS as an internal
standard. The coupling constants (J) were given in Hz.
CD spectra were measured on a Jasco J-180 spectropho-
tometer (Japan). HRESIMS spectra were recorded on an
LTQ-Orbitrap XL spectrometer. All solvents were ana-
lytical grade (TianJing Chemical Plant, Tianjing, China).
Silica gel used for column chromatography (CC) and pre-
coated silica gel GF254 plates used for TLC were pro-
duced by Qingdao Haiyang Chemical Co., Ltd. TLC spots
were viewed at 254 nm and visualized by spraying with
1% vanillin in H2SO4. MCI gel (CHP20P, 75‒150 μm,
Mitsubishi Chemical Industries Ltd.) and ODS C18 gel
(50 µm, YMC) were used for CC. Preparative HPLC was
performed on a Shimadzu LC-20AP instrument with a
SPD-M20A PDA detector. Chromatographic separation
was carried out on a C18 column (19 × 250 mm, 5 μm,
Waters, SunFire™), using a gradient solvent system com-
prised of H2O and CH3CN at a fow rate of 10 mL/min.
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Journal of Natural Medicines
2‑Epinagilactone B (3)
Plant material
White amorphous powder; UV (CH3OH) λmax (log ε) 301.0
(0.11) nm; IR νmax (KBr) 2977 (strong, broad), 1632, 1547,
1104, 892 cm−1; 1H- and 13C-NMR data (see Tables 1 and
2); HRESIMS m/z 363.1456 [M−H]− (calcd. for C19H23O7,
363.1444).
The seeds of P. nagi were collected from Lincang county,
Yunnan province, China, and identifed by Professor Jingui
Shen from Shanghai Institute of Materia Medica, Chinese
Academy of Sciences. A voucher was deposited at the her-
barium of Institute of Chinese Medical Sciences, University
of Macau (LL-20150901).
Acid hydrolysis and determination of sugar
confguration in compounds 1 and 2
Extraction, isolation and characterization
of compounds
Each compound (1 mg) was hydrolyzed with 2 M HCl
(0.5 mL) for 1.5 h. After removing the solvent by nitrogen
blowing, the residue was dissolved in pyridine (500 μL) and
reacted with l-cysteine methyl ester HCl (5 mg) at 60 °C
for 1.5 h. Then O-tolyl isothiocyanate (25 μL) was added
to the mixture and reacted at 60 °C for an additional 1.5 h.
The reaction mixture was analyzed by HPLC (SunFire C18
5 μm, 4.6×150 mm; 20% CH3CN‒0.1% formic acid water
to 35% CH3CN‒0.1% formic acid water, 0‒25 min, 1 mL/
min). The sugar was identifed as d-glucose in compounds 1
and 2 (tR, 15.98–16.04 min) [authentic samples, d-glucose
(tR, 16.35 min) and l-glucose (tR, 15.01 min)].
The air-dried seeds of P. nagi (14.97 kg) were ground into
powder and extracted with 95% ethanol at room temperature
(40 L×3 times, each 2 days). After evaporation of the col-
lected percolate, the crude extract (1.37 kg) was suspended
in 4 L H2O and extracted with petroleum ether (2 L × 3),
EtOAc (3 L×3) and n-butanol (2 L×3), successively. The
n-butanol fraction was subjected to CC over MCI gel and
eluted with H2O/CH3OH (19:1–1:19, v/v), to yield fve frac-
tions (B1–B5). Fraction B2 was subjected to CC over ODS
gel eluted with H2O/CH3OH (19:1–1:19, v/v) to yield six
subfractions (B2A–B2E). The subfraction B2B was further
separated over silica gel and eluted with CHCl3/CH3OH
(60:1–5:1, v/v), to yield six subfractions (B2B1–B2B6).
The subfraction B2B5 was further separated by prepara-
tive HPLC, eluting with H2O/CH3CN (13:7, v/v), to obtain
compounds 3 and 4 (22.9 mg). Fractions B3 and B4 were
combined and subjected to CC over silica gel, eluted with
petroleum ether–acetone (14:1–1:1, v/v) to yield nine
subfractions (B3A–B3I). Fraction B3G was purifed with
Sephadex LH-20, eluting with CHCl3/CH3OH (1:1, v/v)
to yield three fractions (B3G1–B3G2). B3G2 was further
separated by preparative HPLC, eluting with H2O/CH3CN
(4:1, v/v), to obtain compounds 1 (1.6 mg) and 2 (3.4 mg).
Reagents
The compounds were dissolved in dimethyl sulfoxide
(DMSO, Sigma-Aldrich, St. Louis, MO, USA) as a stock
solution and stored at −20 °C. Dulbecco’s modifed Eagle’s
medium (DMEM), 0.25% trypsin–EDTA, fetal bovine serum
(FBS), Penicillin–Streptomycin (10,000 units/mL of peni-
cillin and 10,000 μg/mL of streptomycin) and phosphate-
bufered saline (PBS) were purchased from Gibco (Carlsbad,
CA, USA). Indomethacin, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) and LPS were
purchased from Sigma-Aldrich.
Nagilactone B 1‑O‑β‑d‑glucoside (1)
Cell culture
White amorphous powder; [α]2D0 +7.2 (c 0.02, CH3OH); UV
(CH3OH) λmax (log ε) 292.0 (0.19) nm; ECD (CH3OH, nm)
λmax (Δε) 214.0 (+5.8), 240.0 (−0.7), 289.0 (+1.0); IR νmax
(KBr) 3416 (strong, broad), 1758, 1323, 1107, 894 cm−1;
1H- and 13C-NMR data (see Tables 1 and 2); HRESIMS m/z
525.1975 [M−H]− (calcd. for C25H33O12, 525.1972).
The murine macrophage RAW264.7 cells were obtained
from American Type Culture Collection (Rockville, MD,
USA) and maintained in DMEM supplemented with 10%
FBS. Cells were grown in a standard humidifed incubator
with 5% CO2 at 37 °C.
MTT assay
Nagilactone N3 3‑O‑β‑d‑glucoside (2)
White amorphous powder; [α]2D0 -2.0 (c 0.02, CH3OH); UV
(CH3OH) λmax (log ε) 298.0 (0.30) nm; ECD (CH3OH, nm)
λmax (Δε) 215.5 (+9.6), 239.5 (−0.7), 288.0 (+1.6); IR νmax
(KBr) 3431 (strong, broad), 1729, 1469, 1372, 963 cm−1;
1H- and 13C-NMR data (see Tables 1 and 2); HRESIMS m/z
541.1926 [M−H]− (calcd. for C25H33O13, 541.1921).
Viability of the cells after treatment with the compounds was
determined by MTT assay as described previously [16, 17].
Exponentially growing RAW264.7 cells were seeded into
96-well plates. Upon approximately 70–80%, the cells were
treated with series concentrations of diferent compounds for
24 h. DMSO was used as the vehicle control. Then, 1 mg/mL
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Journal of Natural Medicines
MTT solution was added to each well and the 96-well plates
were further incubated for 4 h at 37 °C. 100 μL DMSO
was added to each well to dissolve the formazan crystals.
Absorbance at 570 nm was measured by a microplate reader
(Perkin Elmer, 1420 Multilabel Counter Victor 3, Wellesley,
MA, USA).
NF-κB p65 (1:100 dilution) at 4 °C overnight. The slides
were washed with PBS and incubated with Texas Red-conju-
gated anti-rabbit IgG secondary antibody at 1:1000 dilution
(Thermo Scientifc) at room temperature for 1 h. Finally, the
slides were washed and stained with 4′,6-diamidino-2-phe-
nylindole (DAPI) for nucleus. The translocation of p65 into
nucleus was determined by confocal microscopy (Olympus,
Tokyo, Japan).
NO production assay
RAW264.7 cells were seeded into 24-well plates
(1×105 cells/well). After 24 h, the cells were treated with
diferent concentrations of compounds or vehicle (DMSO)
for 1 h. Then, the cells were stimulated with 1 μg/mL LPS
and further incubated for 18 h. Indomethacin and DMSO
were used as the positive and negative controls, respectively.
To measure the NO content, the culture supernatant was
collected and assayed using the Griess reagent, as described
previously [7, 8].
Statistical analysis
Data were expressed as mean values and standard deviation.
All the experiments were repeated under the same conditions
at least three times (n = 6). Statistical signifcances were
analyzed by one-way analysis of variance using SPSS 17
software (Statistical Package for the Social Sciences, SPSS
Inc., Chicago, IL, USA). p<0.05 was considered as the sig-
nifcant diference.
Western blot analysis
Conclusion
RAW264.7 cells were treated with or without diferent con-
centrations of compound 1 for 1 h, and then treated with or
without LPS (1 µg/mL) for another 18 h. Cells were washed
twice with cold PBS and then lysed with cold RIPA bufer
containing freshly added phosphatase inhibitor cocktails and
protease inhibitor on ice for 30 min. Whole cell lysates were
centrifuged at 13,500 rpm for 20 min and the supernatants
were transferred into new tubes. 15 μg protein extract was
separated by 8% SDS-PAGE. Extracted protein was trans-
ferred onto PVDF membrane (Bio-Rad), blocked with 5%
nonfat milk in TBST bufer for 1 h at room temperature and
incubated with the specifc primary antibodies overnight at
4 °C. After washing with TBST, the membrane was incu-
bated with horseradish peroxidase conjugated secondary
antibody for 2 h at room temperature. The immune-blotting
signals were detected using SuperSignal West Femto Maxi-
mum Sensitivity Substrate (Thermo Scientifc) under visu-
alization in ChemiDoc™ MP Imaging System (Bio-Rad).
Primary antibodies, including iNOS, p-IKKα/β, IKKα,
IKKβ, p-IκBα, IκBα, p-p65, p65, and GAPDH, as well as
the secondary antibodies, were purchased from Cell Signal-
ing Technology (Danvers, MA, USA).
In conclusion, three new podolactones were isolated from
the seeds of P. nagi, including nagilactone B 1-O-β-d-
glucoside (1), nagilactone N3 3-O-β-d-glucoside (2), and
2-epinagilactone B (3). Compounds 1 and 2 suppressed
LPS-induced NO production on RAW264.7 macrophages,
through inhibiting NF-κB activity. To the best of our knowl-
edge, it is the frst report of the anti-infammatory property
of podolactones. The outcome of the current study supplied
a new class of anti-infammatory agents.
Acknowledgements Financial support by Science and Technology
Development Fund, Macao S.A.R (FDCT 102/2017/A), the Research
Fund of University of Macau (MYRG2017-00109-ICMS), and the
State Key Laboratory of Drug Research (SIMM1803KF-16) are grate-
fully acknowledged.
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
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