Norditerpenoids from Flickingeria fimbriata and their inhibitory activities on nitric oxide and tumor necrosis factor-α production in mouse macrophages

Article abstract of DOI:10.3390/molecules19055863

Bioassay-guided fractionation of the ethanolic extract of the leaves of Flickingeria flimbriata led to the isolation of two new degraded diterpenoids 1 and 2, a new ent-pimarane type diterpenoid 3, and four known steroids 4-7. The structures of 1-3 were elucidated by spectroscopic analysis, and their absolute configurations were determined by chemical methods, TDDFT quantum chemical calculations of ECD spectra, and CD exiton chirality method. Compounds 1 and 2, named flickinflimilins A and B, possess a rare 15,16-dinor-ent-pimarane skeleton. Compounds 1-7 were screened for the inhibitory activity against lipopolysaccharide (LPS)-induced NO and TNF-a production in RAW264.7 cells. Compounds 1-3 exhibited potent inhibitory activities, with IC50 values of less than 10 μM.

Full text of DOI:10.3390/molecules19055863

Molecules 2014, 19, 5863-5875; doi:10.3390/molecules19055863
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Norditerpenoids from Flickingeria fimbriata and Their
Inhibitory Activities on Nitric Oxide and Tumor Necrosis
Factor-α Production in Mouse Macrophages
Jin-Long Chen, Wen-Jun Zhong, Gui-Hua Tang, Jing Li, Zhi-Min Zhao, De-Po Yang *
and Lin Jiang *
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, Guangdong, China;
E-Mails: jlchen5858@163.com (J.-L.C.); clockzwj@163.com (W.-J.Z.);
tanggh5@mail.sysu.edu.cn (G.-H.T.); lijing45@mail2.sysu.edu.cn (J.L.);
zhaozhimin1978@hotmail.com (Z.-M.Z.)
*
Authors to whom correspondence should be addressed; E-Mails: lssydp@mail.sysu.edu.cn (D.-P.Y.);
Linderson_jiang@163.com (L.J.); Tel./Fax: +86-20-3994-3043 (D.-P.Y.);
Tel./Fax: +86-20-3994-3041 (L.J.).
Received: 10 March 2014; in revised form: 21 April 2014 / Accepted: 28 April 2014 /
Published: 6 May 2014
Abstract: Bioassay-guided fractionation of the ethanolic extract of the leaves of
Flickingeria flimbriata led to the isolation of two new degraded diterpenoids 1 and 2,
a new ent-pimarane type diterpenoid 3, and four known steroids 4–7. The structures
of 1–3 were elucidated by spectroscopic analysis, and their absolute configurations were
determined by chemical methods, TDDFT quantum chemical calculations of ECD spectra,
and CD exiton chirality method. Compounds 1 and 2, named flickinflimilins A and B,
possess a rare 15,16-dinor-ent-pimarane skeleton. Compounds 1–7 were screened for the
inhibitory activity against lipopolysaccharide (LPS)-induced NO and TNF-α production in
RAW264.7 cells. Compounds 1–3 exhibited potent inhibitory activities, with IC values of
5
0
less than 10 µM.
Keywords: norditerpenoid; TDDFT; exiton chirality method; NO; TNF-α

Molecules 2014, 19
. Introduction
Flickingeria fimbriata (Bl.) Hawkes (Orchidaceae), is widely used as a substitute of the precious
5864
1
and scarce Traditional Chinese Medicine Dendrobium candidum, for treatment of pneumonia,
tuberculosis, asthma, and pleurisy [1]. Phytochemical investigations of Flickingeria species have
revealed the occurrence of diterpenes [2–6], phenanthrenes [4], steroids [5], and bibenzyls [6]. These
natural products are reported possessing P-glycoprotein inhibition [2], anti-inflammation [7–9], and
antimutagenic activities [10].
In our continuing search for anti-inflammatory metabolites from medical plants, a fraction of the
EtOAc extract partitioned from the ethanolic extract of leaves of F. fimbriata showed an inhibitory
activity of 42.2% against NO production and 40.1% against TNF-α production, respectively, at a
concentration of 10 µM. Column chromatographic separations led to the isolation of three new
diterpenoids 1–3 together with four known steroids 4–7 (Figure 1). Flickinflimilins A and B
(compounds 1,2) represent an unusual group of 15,16-dinor-ent-pimarane. The isolated compounds 1–7
were screened for their inhibitory activity against lipopolysaccharide (LPS)-induced NO and TNF-α
production in RAW264.7 cells. The results showed that compounds 1–3 exhibited potent inhibitory
activities with the IC50 values less than 10 µM. Herein, the details of the isolation, structural
elucidation, and inhibitory activities of these compounds are described.
Figure 1. The structures of compounds 1–7.
O
O
1
OGlc
2
17
20
C
H
HO
HO
1
9
HO
O
HO
HO
14
2
8
H
A
H
B
O
4
8
H
H
H
1
19
O
2
1
3
H
H
H
H
H
H
H
H
RO
R1
OH
R2
4
5
R = Ac
R = Glc
6
7
R1 = R2 = OH
R = R = OAc
1
2
2
. Results and Discussion
The air-dried powder of the leaves of F. fimbriata was extracted with 95% EtOH at room
temperature to give a crude extract, which was then suspended in H O and successively partitioned
2
with petroleum ether, EtOAc, and n-BuOH, respectively. Various column chromatographic separations
of the petroleum ether, EtOAc extract afforded compounds 1–7.

Molecules 2014, 19
5865
Compound 1 was obtained as a colorless oil with the molecular formula C H O as established by
1
8
28
3
+
1
an m/z of 315.1943 [M + Na] (calcd for C18
H
28
O
3
Na, 315.1936) from HRESIMS. The H-NMR
2.27, 1.00, 0.94, and 0.87 (each 3H, s)], two
spectrum of 1 showed four tertiary methyls [at δ
H
oxygenated methines [at δ 4.05 (ddd, J = 2.8, 4.4, and 11.8 Hz, H-2) and 3.45 (d, J = 2.8 Hz, H-3)],
H
1
3
H
and an olefinic proton [at δ 6.65 (t, J = 1.6 Hz, H-14)]. The C-NMR spectrum in combination with
DEPT experiments showed 18 carbon resonances, including four quaternary carbons (one conjugated
carbonyl and one olefinic), six methines (two oxygenated and one olefinic), four methylenes, and
four quaternary methyls. The double bond and the carboxyl group accounted for two out of the
five degrees of unsaturation, and the leftover double-bond equivalents required a tricyclic nature of 1.
The gross structure was constructed by two-dimensional (2D) NMR analysis. Three fragments (Figure 2),
a (C-1 to C-3), b (C-5 to C-7), and c (C-14, C-8, C-9, and C-11) were established by the correlations
1
1
observed in the H- H COSY spectrum of 1. The connectivity of these fragments, quaternary carbons
and other substitutes was accomplished mainly by analysis of the HMBC spectrum (Figure 2).
Fragment a, C-10 bearing the angular methyl group (C-20), the C-5 methine, and the gem-dimethyl
group could construct ring A by the HMBC correlations from H -20 to C-1, C-5, and C-10 and from
3
3
H -18/19 to C-3, C-4, and C-5. The HMBC correlations of H-9 to C-5, C-7, and C-10, H-7 to C-9, and
H-8 to C-7, C-9 and C-10 supported that ring B was constructed by C-8–C-10 and fragment b, and
fused with ring A via C-5 and C-10. Fragment c and the α,β-unsaturated ketone group provide ring C
by the HMBC correlations from H-14 to C-8, C-9, C-12, and C-13. The planar structure of 1 thus
emerged from the above spectra analysis.
1
1
Figure 2. (A) Selected H- H COSY (▬) and HMBC (→) correlations of 1; (B) Selected
NOESY correlations of 1 ( ).
The relative configuration of 1 was determined by analysis of the NOESY correlations (Figure 2)
and coupling constants. The NOESY spectrum showed cross peaks between the proton pairs
H-2/H -19, H-2/H -20, indicating that these protons were cofacial and axial oriented, which were
3
3
arbitrarily assigned as α orientation. Accordingly, a chair conformation for ring A was assigned. Based
on the small coupling constant (J = 2.6 Hz) between H-2 and H-3, the OH-3 was assigned as
β orientation. The β assignment of coplanar protons H-5 and H-9 were deduced from the NOE
correlations of H-1β/H-5, H-5/H-7β, and H-5/H-9.
The absolute configuration of 1 was established by comparing the experimental ECD spectrum with
the calculated data. The TDDFT calculations were performed using the B3LYP functional and TZVP
basis set. Then conformer of 1 were subjected to TDDFT calculations for solution CD in MeOH and
also calculated for the gas phase CD. The simulated CD spectra (Figure 3) exhibited negative Cotton

Molecules 2014, 19
5866
effects (CEs) around 320 nm and 240 nm and a positive CE around 210 nm, all generally consistent
with the experimental spectrum, and the weighted spectra in both the gas phase and MeOH solution
(Figure 3) provided the excellent fit with the experimental data, giving a firm support to the
determined absolute configuration of 1.
Figure 3. Calculated CD spectra of compound 1 in MeOH solution (red) and in vacuum
(
gray) and comparison between the calculated and experimental CD (black) spectra.
Vertical bars represent rotational strengths R. σ = 0.20 eV.
Compound 2 was obtained as a white amorphous powder, and its molecular formula was
determined to be C H O (seven degrees of unsaturation) from the quasi-molecular ion peak at m/z
1
8
24
3
+
3
24 3
11.1987 [M + Na] (calcd for C18H O Na, 311.1989) in the HRESIMS spectrum. The IR absorption
−1
bands at 3440, 1708, 1600, 1500, and 1476 cm indicated the presence of hydroxyl, carbonyl, and
1
aromatic groups. The H-NMR spectrum of 2 (Table 1) showed the signals of four tertiary methyl
groups [at δ
H
2.34, 1.25, 1.19, and 1.03 (each 3H, s)], two oxygenated methines [at δ
H
4.26 (ddd, J = 2.6,
4
.5, and 11.5 Hz, H-2) and 3.61 (d, J = 2.6 Hz, H-3)], and three aromatic protons [at δ
H
7.80 (d, J = 1.3 Hz,
1
3
H-14), 7.34 (dd, J = 1.3, 8.0 Hz, H-12), and 7.27 (d, J = 8.0 Hz, H-11)]. The C-NMR spectrum in
combination with DEPT experiments showed 18 carbon resonances, including six quaternary carbons
(
one conjugated carbonyl and three olefinic carbons), six methines (two oxygenated and three olefinic
carbons), two methylenes, and four methyls. The double bonds and ketone group accounted for four
out of the seven degrees of unsaturation, the remaining three double-bond equivalents required 2 to be
tricyclic. The aforementioned data resembled those of compound 1. The major structural differences
were due to the location of the conjugated ketone group and C-ring aromatized in 2. The structure of 2
was further demonstrated by HMBC and NOESY spectra (See supplementary materials Figure S27).
Compound 2 represents the first C-ring aromatized dinor-ent-pimarane reported hitherto.
The absolute configuration of compound 2 was postulated on the basis of comparison between the
experimental ECD spectrum and the calculated data. As can be seen in Figure 4, the Boltzmann
weighted CD spectra in both the gas phase and MeCN solution, in particular the solution spectrum, are
in good agreement with the experimental spectrum.
Compound 3, a colorless oil, had a molecular formula of C28
H
44
O
10 as determined by HRESIMS m/z
−
1
13
5
85.2929 [M + HCOO] (calcd for C29
H
45
O12, 585.2923). Closely inspection of the H and C-NMR

Molecules 2014, 19
5867
spectra indicated compound 3 was a diterpenoid glycoside. Comparison of the NMR data (Table 1)
and molecular formula of 3 with those of ephemeranthoside [4] demonstrated that 3 had an additional
acetyl group. [δ 2.10 s (3H); δ 21.3 and 173.0], which was located at C-3 from analysis of the HMBC
H
C
spectrum. The relative configuration of 3 was established by analysis of the NOESY spectrum.
1
13
Table 1. H-NMR (400 MHz) and C-NMR (100 MHz) data for compounds 1–3 (δ in ppm).
a
b
a
1
2
3
No.
δ
H
(J in Hz)
1.54, m
1.44, t (11.8)
.05, ddd
δ
C
, type
δ
H
(J in Hz)
2.31, m
1.99, m
δ
C
, type
δ
H
(J in Hz)
1.56, m
1.53, m
δ
C
, type
1
1
α
β
40.4, CH
2
40.3, CH
2
41.3, CH
2
4
4.26, ddd
4.00, ddd
2
66.3, CH
66.2, CH
65.9, CH
(
2.8, 4.4, 11.8)
3.45, d (2.8)
(2.6, 4.5, 11.5)
3.61, d (2.6)
(2.6, 4.7, 11.7)
4.91, d (2.6)
3
4
5
79.2, CH
38.2, C
47.6, CH
21.7, CH
30.2, CH
78.8, CH
38.8, C
42.8, CH
81.5, CH
39.9, C
49.9, CH
1.37, m
1.58, m
1.37, m
2.13, m
2.07, m
2.29, m
2.64, d (1.4)
2.62, m
1.44, m
1.42, m
1.57, m
6
α
β
α
β
2
36.0, CH
2
22.6, CH
2
6
7
7
2
199.4, C
2.12, m
36.5, CH
2
2.42, d (14.2)
8
9
2.49, t (12.0)
1.59, m
45.8, CH
61.3, CH
37.1, C
131.1, C
153.7, C
39.2, C
142.9, C
52.0, CH
40.5, C
1.92, t (9.1)
1
0
2
.37, dd
1
1
1
1
1α
1β
2α
2β
29.2, CH
2
7.27, d (8.0)
124.2, C
1.65, m
1.24, m
1.10, m
2.31, d
21.3, CH
2
(
3.8, 11.6)
2.15 br, s
7
.34, dd
197.4, C
135.0, C
33.5, CH
48.7, C
2
(
1.3, 8.0)
1
3
145.3, C
148.0, CH
135.8, C
127.7, C
1
1
4α
4β
6.65, t (1.6)
7.80, d (1.3)
5.54, s
125.5, CH
1
5
213.5, C
1
1
1
1
1
2
CH
CH
6a
6b
7
8
9
0
4.49, d (18.4)
4.88, d (18.4)
1.14, s
72.4, CH
2
2.27, s
0.87, s
1.00, s
0.94, s
26.1, CH
21.3, CH
28.7, CH
16.0, CH
3
3
3
3
2.34, s
1.03, s
1.19, s
1.25, s
20.6, CH
21.5, CH
28.4, CH
24.3, CH
3
27.5, CH
28.5, CH
22.4, CH
16.0, CH
21.3, CH
173.0, C
104.2, CH
75.0, CH
78.2, CH
71.5, CH
77.6, CH
3
3
3
3
3
3
3
3
0.89, s
0.98, s
0.77, s
2.10, s
3
CO
CO
3
1
’
’
’
’
’
4.24, d (7.6)
3.16, dd (7.6, 9.2)
3.27, dd (2.7, 5.0)
3.28, d (8.8)
2
3
4
5
3.24, dd (2.7, 5.0)
6
’a
3.87, dd (3.6, 12.0) 72.4, CH
3.64, dd (3.6, 12.0)
2
6
’b
a
b
Measured in CDCl
3
; Measured in Pyridine.

Molecules 2014, 19
5868
Figure 4. Calculated CD spectra of compound 2 in MeCN solution (red) and in vacuum
gray) and comparison between the calculated and experimental CD (black) spectra.
Vertical bars represent rotational strengths R. σ = 0.24 eV.
(
To determine the absolute configuration of compound 3, the chemical transformation from 3 to 3c
was performed (as shown in Scheme 1), then the exciton chirality method was applied on 2,3,16-tri-p-
methoxybenzoate derivative (3c). The negative chirality resulting from the exciton coupling between
the two chromophores of p-methoxybenzoate at 267 nm (∆ε −18.41, π-π* transition) and 243 nm
(
∆ε +2.85, π-π* transition) indicated that the transition dipole moments of the two chromophores were
oriented in a counterclockwise manner (Figure 5) [11]. Thus, the absolute configurations of C-2 and
C-3 of 3c were determined to be 2S and 3R, respectively. The absolute configuration of the β-glucose
was identified to be D-configuration by HPLC analysis. Therefore, compound 3 was established as
2
,16-dihydroxyl-15-keto-2-acetoxy-ent-pimar-8(14)-ene-16-O-β-D-glucopyranoside.
Scheme 1. The chemical transformation from 3 to 3c.
The known compounds sitost-5-en-3β-ol acetate (4) [12], β-sitosterol-3-O-β-D-glucopyranoside (5) [13],
,5,6-trihydroxysitostane (6) [14], stigmastane-3β,5α,6β-triol-3,6-diacetate (7) [15] were identified by
3
comparison of their spectroscopic data with the literature values.

Molecules 2014, 19
5869
The anti-inflammatory activity of all isolates were evaluated. The results (Table 2) showed that
compounds 1–3 exhibited potent inhibitory activity against NO and TNF-α production in RAW264.7
cells and the steroids were inactive with the IC values more than 20 µM.
5
0
Figure 5. CD and UV spectra of compound 3c in MeOH. The arrow denotes the electric
transition dipole of the chromophores.
O
O
O
O
O
O
O
O
O
O
3c
Table 2. IC values of the active compounds 1–3 against NO and TNF-α production in
5
0
RAW264.7 cells.
IC50 (μM)
Compound
NO TNF-α
1
2
3
19.2
6.7
6.2
5.6
8.9
0.9
13.8
1.1
a
Celastrol
a
Positive control for NO and TNF-α production.
3
. Experimental
.1. General
Optical rotations were measured on a Rudolph Autopol I automatic polarimeter. IR spectra were
3
determined on a Bruker Tensor 37 infrared spectrophotometer. NMR spectra were measured on a
Bruker AM-400 spectrometer at 25 °C. ESIMS was measured on a Finnigan LC QDECA instrument,
and HRESIMS was performed on a Waters-Micromass Q-TOF. A Shimadzu LC-20 AT equipped with a
SPD-M20A PDA detector was used for HPLC. A YMC-pack ODS-A column (250 × 10 mm, S-5 µM,
1
2 nm) was used for semipreparative HPLC separation. Silica gel (300–400 mesh, Qingdao Haiyang
Chemical Co., Ltd., Qingdao city, China), C reversed-phase silica gel (12 nm, S-50 µM, YMC Co., Ltd.,
18
Taiwan Chu-Pei city, China), Sephadex LH-20 gel (Amersham Biosciences, Piscataway, NJ, USA) and
MCI gel (CHP20P, 75–150 µM, Mitsubishi Chemical Industries Ltd., Tokyo, Japan.) were used for
column chromatography. All solvents used were of analytical grade (Guangzhou Chemical Reagents
Company, Ltd., Guangzhou, China). All cell lines were obtained from the China Center for Type
Culture Collection of the Chinese Academy of Sciences.

Molecules 2014, 19
.2. Plant Material
Plant of F. fimbriata were collected in July 2010 from Yunnan Province, China, and were identified
5870
3
by one of the authors (Prof. D. P. Yang). A voucher specimen (accession number: LSJSH201010) has
been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University.
3.3. Extraction and Isolation
The air-dried powder of the leaves of F. fimbriata (1 kg) was extracted with 95% EtOH (3 × 10 L)
at room temperature (rt) to give 80 g of crude extract. The extract was suspended in H O (1 L) and
2
successively partitioned with petroleum ether (PE, 3 × 1 L), EtOAc (3 × 1 L) and n-BuOH (3 × 1 L),
respectively. The PE extract (15 g) was subjected to silica gel column chromatography (CC) and eluted
with PE/dichloromethane (0:10→10:0) successively to afford three fractions (I–III). Fraction I (2.5 g)
2 2
was chromatographed over an silica gel column eluting with PE/CH Cl (0:10→10:0) to afford five
fractions (Fr. Ia–Ie), Fr. Ia was then subjected to silica gel column eluting with PE/CH Cl2
2
(
0:10→10:0) and obtained compound 4 (113 mg). Fraction III (3.5 g) was chromatographed over an
silica gel column eluting with PE/Acetone (0:10→10:0) to afford five fractions (Fr. IIIa–IIIe). Fr. IIIa
1.0 g) was separated by silica gel CC (PE/CH Cl , 50:1→10:1) to afford five fractions (Fr. IIIaa–IIIae).
Fr. IIIac was subjected to Rp-C18 CC using a gradient of MeOH/H O (v/v from 7:3 to 10:0) to yield 6
22 mg) and another fraction, which after chromatography on a Sephadex LH-20 column using
(
2
2
2
(
CHCl
3
/MeOH (1:1) as eluent to obtain 7 (23 mg). The EtOAc extract (42 g) was subjected to MCI gel
O gradient (3:7→10:0) to afford three fractions (XI–XIII). Fraction XI (6.5 g)
was subjected to silica gel CC (PE/EtOAc, 2:1→0:1) to give three fractions (XIa–XIc). Fr. XIa (2.1 g)
CC eluted with a MeOH/H
2
was separated by silica gel CC (PE/EtOAc, 2:1), followed by semi-preparative HPLC (CH
3
OH/H
:2, 3 mL/min) to give 1 (6 mg). Fr. XIc (1.3 g) was separated by Rp-C18 silica gel CC (MeOH/H
:5→0:0) to yield 2 (8 mg). Fraction XII (5.5 g) was subjected to silica gel CC (CHCl
0:1→0:1) to give three fractions (XIIa–XIIc). Fr. XIIb (2.5 g) was subjected to silica gel CC
, 1:1→0:1) to give three fractions (XIIb –XIIb ). Fr. XIIb (0.9 g) was subjected to Rp-C18
CC (MeOH/H O, 2:8→10:0) and followed by silica gel CC (EtOAc/acetone, 3:1→0:1) to afford 5 (38 mg).
2
O,
8
5
3
2
O,
3
/MeOH,
(PE/CHCl
3
1
4
1
2
Fr. XIIb (1.2 g) was subjected to Rp-C CC (MeOH/H O, 0:10→10:0) and Sephadex LH-20 (EtOH)
4
18
2
to yield 3 (25 mg).
3
.4. Spectral Data
2
0
Flickinflimilin A (1). Colorless oil; [α] −46.2° (c 0.24, CHCl
3
); UV (MeOH) λmax (log ε) 242 (2.18) nm,
D
−
3
2
03 (2.46) nm; CD (c 3.4 × 10 M, CH
3
CN), λmax (∆ε) 242 (−0.26), 278 (−0.01), and 323 (−0.07); IR
−1
1
13
(
KBr) νmax 3437, 1718, 1591, 1461, 1378, 1129, 1038, 949, and 764 cm ; H and C-NMR data, see
+
+
+
Table 1; positive ESIMS m/z 315.3 [M + Na] , 607.6 [2M + Na] ; HREIMS m/z 315.1943 [M + Na]
calcd for C18 Na, 315.1936).
(
8 3
H O
2
0
Flickinflimilin B (2). White amorphous powder; [α] +16.0° (c 0.50, MeOH); UV (MeOH) λmax (log ε)
D
−
3
2
07 (2.94), 251 (2.45) nm; CD (c 1.7 × 10 M, CH
3
CN), λmax (∆ε) 207 (−6.64), 251 (+2.29), 270 (−0.34),
−1
3
00 (+2.19), and 337 (−3.08); IR (KBr) νmax 3440, 1708, 1600, 1500, 1476, 1129, 767, and 750 cm ;

Molecules 2014, 19
5871
1
13
+
H and C-NMR data, see Table 1; positive ESIMS m/z 289.1 [M + H] ; negative ESIMS m/z 333.2
−
+
24 3
[M + HCOO] ; HRESIMS m/z 311.1987 [M + Na] (calcd for C18H O Na, 311.1989).
2
0
Flickinflimilin C (3). Colorless oil; [α] −55.0° (c 0.22, CHCl
3
); IR (KBr) νmax 3445, 1714, 1646,
D
−
1
1
13
1
5
461, 1379, 1257, 1126, 1085, and 950 cm ; H and C-NMR data, see Table 1; positive ESIMS m/z
+
+
−
−
63.3 [M + Na] , 1103.5 [2M + Na] ; negative ESIMS m/z 585.3 [M + HCOO] , 1125.8 [2M + HCOO] ;
−
HRESIMS m/z 585.2929 [M + HCOO] (calcd for C H O , 585.2923).
2
9
45 12
3
.5. Computational Methods for Electronic Circular Dichroism
Molecular mechanics calculations were carried out with Spartan’14 software package
(
Wavefunction Inc., Irvine, CA, USA, 2013) and quantum chemical computations were run with
Gaussian 09 program package (Gaussian, Inc., Pittsburgh PA, USA, 2011) using default grids and
convergence criteria. MMFF conformational search generated conformers within a 10 kcal/mol energy
window were optimized using DFT method at B3LYP/6-31G (d) level. Frequency calculations were
run at the same level to verify that each optimized conformer was a true minimum and to estimate their
relative thermal free energies (∆G) at 298.15K. The TDDFT calculations were performed using the
hybrid B3LYP functional, and Ahlrichs’ basis sets SVP (split valence plus polarization) and TZVP
(
triple zeta valence plus polarization). The number of excited states per each molecule was 20–30.
Solvent effects were taken into account by using polarizable continuum model (PCM). CD spectra
were generated by the program SpecDis (University of Würzburg, Würzburg, Germany, 2012) using a
Gaussian band shape with 0.28 eV exponential half-width from dipole-length dipolar and rotational
strengths; the difference with dipole-velocity values was negligible (<10%) for most transitions.
3
.6. Determination of Sugar Configuration [16]
Compound 3 (6 mg) was refluxed with 2 M HCl (2 mL, dioxane/H O, 1:1) at 100 °C for 4 h. After
2
removing the dioxane under vacuum, the solution was then diluted with H
EtOAc (3 × 1 mL). The EtOAc layer was evaporated under vacuum, then subjected to CC over silica
gel eluteing with CHCl :MeOH (30:1) to afford 3a. The aqueous layer was evaporated under vacuum,
diluted repeatedly with H O, evaporated under vacuum to obtain neutral residue, and then analyzed by
2
O and then extracted with
3
2
2 2 f
TLC over silica gel (Me CO/n-BuOH/H O, 6:3:1) together with authentic sugar sample (glucose, R = 0.49).
The remaining residue was dissolved in pyridine (200 µL), to which 2 mg of L-cysteine methyl ester
hydrochloride was added. The mixture was stirred at 60 °C for 1 h; then 50 µL of o-tolyl
isothiocyanate was added, and the mixture was stirred at 60 °C for another 1 h. The reaction mixture
was directly analyzed by standard C18 HPLC [a YMC-pack ODS-A column (250 × 10 mm, S-5 µM,
1
2 nm), CH CN/H O, 25:75, 3 mL/min]. The peak (t = 19.0 min) coincided with a derivative of
3 2 R
D-glucose, as compared with authentic D-glucose with t
.7. Chemical Transformation of 3a to 3c [17]
To a stirred solution of 3a (10 mg) in MeOH (2 mL), NaOH (1 mg) was added. The mixture was
R
at 19.1 min.
3
stirred at room temperature for 0.5 h to obtain 3b, and then 3b (7.2 mg) was transferred into a clean
NMR tube and was dried completely under the vacuum of an oil pump. Deuterated pyridine (0.55 mL)

Molecules 2014, 19
5872
and 4-methoxybenzoyl chloride (12 µL) were added into the NMR tube immediately under dry
conditions, and then the NMR tube was shaken carefully to mix the sample and 4-methoxybenzoyl
1
chloride evenly. The reaction NMR tube was permitted to stand at 45 °C and monitored by H-NMR.
1
The reaction was found to be completed after 2 h. H-NMR data of the 4-methoxybenzoate derivative
(
3c) of 3b was obtained from the reaction NMR tube directly. The reaction mixtures were transferred
from the NMR tube and subjected to Sephadex LH-20 CC eluting with CHCl
3
:MeOH (1:1). CD
1
(
CH OH, ∆ε) 212 (−17.20), 243 (+2.85), 267 (−18.41), 297 (+1.12) nm; H-NMR (CDCl , 400 MHz)
3
3
H
δ 5.43, (1H, s, H-14), 5.40, [1H, (ddd, J = 2.1, 4.3, 12.4 Hz, H-2)], 4.15, [1H, (dd, J = 7.6, 14.2 Hz,
H-16a)], 3.67, [1H, (d, J = 2.1 Hz, H-3)], 3.15, [1H, (dd, J = 7.6, 14.2 Hz, H-16b)], 2.29–2.42, (4H, m,
H-6, H-7), 1.96–2.14, (4H, m, H-1, H-12), 1.83, (1H, m, H-9), 1.44, (1H, m, H-5), 1.07, (3H, s, H-17),
1
.01, (3H, s, H-18), 0.92, (3H, s, H-19), 0.88, (3H, s, H-20), [7.97–8.05, (6H, m), 6.91–6.96, (6H, d,
1
3
J = 8.5 Hz ), 3.86–3.87, (9H, s, CH O-p), p-methoxybenzoyl]; C-NMR (CDCl , 100 MHz) δ 207.2
3
3
C
(C-15), 142.1 (C-8), 124.1 (C-14), 76.8 (C-3), 71.3 (C-2), 66.7 (C-16), 50.8 (C-9), 47.7 (C-5), 47.3 (C-13),
3
2
9.5 (C-10), 38.7 (C-4), 36.2 (C-7), 35.6 (C-12), 32.6 (C-1), 28.6 (C-18), 27.2 (C-17), 22.6 (C-19),
1.6 (C-11), 20.2 (C-6), 15.4 (C-20), (165.7, 165.5, 163.6, 163.5, 132.0, 131.6, 113.7, 113.6, 55.4)
−
(
p-methoxybenzoyl); negative ESIMS m/z 737.3 [M − H] .
3
.8. Biological Assays
3
.8.1. Cytotoxic Assay [18]
The RAW264.7 cell line was obtained from ATCC (Manassas, VA, USA), and was cultured in
DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS,
Hyclone) at 37 °C in a humidified atmosphere with 5% CO
2
. Cell viability was assessed by MTT
5
(Sigma, St. Louis, MO, USA). Briefly, 100 µL of adherent cells with an initial density of 1 × 10 cells/mL
were seeded into a 96-well plate and allowed to adhere for 24 h. Cells were exposed to the test
compounds at various concentrations for 48 h. After the incubation, MTT (5 mg/mL) was added to
each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 µL of 20%
SDS–50% DMF after removal of the medium. The optical density of the lysate was measured at 595 nm
in a 96-well microtiter plate reader (Bio-Rad 680, Hercules, CA, USA). The IC50 value of each
compound was calculated by Reed and Muench’s method [19].
3
.8.2. Nitric Oxide Inhibitory Assay [20]
Inhibition of NO production was determined in a LPS-stimulated RAW264.7 macrophage cell line.
5
Cells were seeded in 96-well plates (1 × 10 cells/well) and allowed to adhere for 24 h at 37 °C in a
2
humidified atmosphere containing 5% CO . The medium was then replaced with fresh medium
containing LPS (2 µg/mL) and test compounds at 10 µM, and the cells were incubated for 24 h. NO
production was determined by measuring the accumulation of nitrite in the culture supernatant with
Griess reagent (0.5% sulfanilamide and 0.05% naphthylene-diamide dihydrochloride in 2.5% H PO )
3
4
and then allowed to stand for 5 min at rt. The absorbance at 540 nm was measured using a HTS
000 microplate reader. The nitrite concentration in the medium was determined from the calibration
curve (r = 0.9998) obtained by using different concentrations of sodium nitrite (NaNO ) in the culture
7
2

Molecules 2014, 19
5873
medium as the standard. Blank correction was performed by subtracting the absorbance due to medium
only from the absorbance reading of each well.
3
.8.3. Assay for the Production of Pro-inflammatory Cytokines (TNF-α) [21]
Compounds were dissolved in DMSO, and suspensions of RAW264.7 cells were cultured in
complete RPMI 1640 medium (Hyclone) containing 10% FBS. The cultured cells were incubated with
the tested compounds (10 µM) for 24 h, followed by LPS stimulation (2 µg/mL). Supernatants were
collected to analyze cytokine levels. The mouse TNF-α ELISA Kit was used to determine the cytokine
concentration in the culture supernatants.
4
. Conclusions
Three new diterpenoids including two 15,16-dinor-ent-pimaranes and one diterpenoid glycoside,
and four steroids were isolated from the leaves of F. flimbriata. The absolute configurations of the new
compounds were determined by calculated ECD method, chemical transformation and exiton chirality
method. All the isolates were screened for the anti-inflammation, the new compounds exhibited potent
inhibitory against NO and TNF-α production in RAW264.7 cells.
Supplementary Materials
Supplementary material can be accessed at http://www.mdpi.com/1420-3049/19/5/5863/s1.
Acknowledgments
We gratefully acknowledge financial support for this project from the National Natural Science
Foundation of China (No. 81102782), the China Postdoctoral Science Foundation (No. 2011M501368),
and the Priming Scientific Research Foundation for Junior Teachers of Medicine at Sun Yat-sen University.
Author Contributions
Jin-Long Chen, De-Po Yang and Lin Jiang designed research; Jin-Long Chen, Wen-Jun Zhong,
Jing Li and Zhi-Min Zhao performed research and analyzed the data; Jin-Long Chen and Gui-Hua
Tang wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–7 are available from the authors.
©
2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(