F. Wang, et al.
Fitoterapia 134 (2019) 378–381
scandoside methyl ester. The double split peaks of H-1 in 1H NMR
spectrum, with a coupling constant of 5.4 Hz between H-1 and H-9,
suggested that H-1 was oriented at the opposite side to H-9, and the
same to that in E-6-O-p-coumaroyl scandoside methyl ester. Further
investigation of the literature, led us to find that compound 1 showed
the same configuration of C-6, C-5 and C-9 with that of 10-cafeyl
deacetyl daphylioside [23] and 10-O-dehydroferuloyl-10-O-deacetyl
daphylioside [24]. Especially, the similar coupling constant of J5,6
and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). HPLC
separation was performed on an instrument consisting of a Waters 600
controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance
detector (Waters Corporation, Milford, USA), with a YMC-Pack ODS-A
(250 × 10 mm i.d.) column packed with C18 (5 μm) (YMC Co., Ltd.,
JAPAN). TLC was carried out with glass precoated silica gel GF254
plates (Qingdao Marine Chemical Inc.). Spots were visualized under UV
2 4
light or by spraying with 7% H SO in 95% EtOH followed by heating.
(
8.4 Hz in 1 and 8.0 Hz in other two) was also confirmed the Cis or-
Unless otherwise noted, all chemicals were obtained from commercially
ientation of H-5 and H-6 in 1. Based on the above evidence, the
structure of 1 was established as shown in Fig. 1, and was named
camptoside.
available sources and were used without further purification.
3.2. Plant material
Compound 1 was determined as a novel iridoid glycoside with an E-
4
-hydroxystyryl group connected to C-6 position by a carbon‑carbon
The Camptosorus sibiricus Rupr. were collected in April 2017 from
Yuzhou city, Henan Province of People's Republic of China. Plant
identity was verified by professor Qi Guo of the Shandong Academy of
Pharmaceutical Sciences, where a voucher specimen (No. 20160425) is
deposited.
bond. For some amount of E-6-O-p-coumaroyl scandoside methyl ester
was isolated in our recently re-investigated of Camptosorus sibiricus
Rupr. (Aspleniaceae), compound 1 was seemed to be a product through
a rearrangement reaction with de‑carbon-dioxide of E-6-O-p-coumaroyl
scandoside methyl ester, due to this kind of reaction was commonly
occurred in the fragmentation pattern in MS. A similar reaction was
designed and conducted by heating the E-6-O-p-coumaroyl scandoside
methyl ester DMSO solution (2.0 mg/mL, 60 °C) for two hours, how-
ever, there not any amount of 1 was detected in the reaction mixture by
HPLC.
3.3. Extraction and isolation
The air-dried whole herbs of C. sibiricus Rupr. (17.4 kg) were ex-
tracted with 11.0 L of 95% EtOH at room temperature for 3 × 48 h. The
ethanol extract was evaporated under reduced pressure to yield a dark
The structure of the novel compound 1 differs from the one of ir-
idoid esters by formal extrusion of carbon dioxide and coupling be-
tween the resulting alkyl(alkenyl) residues. No example of this reaction
seems to have been reported in biological systems, and an alternative
could be the trapping of a iridoid cation by a nucleophilic C6-C2
acetylenic compound.
Compounds 1–4 were examined for anti-inflammatory activity by
evaluating the inhibition of lipopolysaccharide (LPS) induced NO pro-
duction in RAW 264.7 macrophages. As shown in Table 2, compounds
2
brown residue (528.6 g), which was suspended in H O (1.5 L) and
partitioned with EtOAc (6 × 1 L). The aqueous phase was applied to a
AB-8 macroporous adsorbent resin (1000 g) column. Successive elution
2
of the column with H O, 30% EtOH, 50% EtOH, and 95% EtOH
(5000 mL each) yielded four corresponding fractions after removing
solvents. The fraction eluted by 50% EtOH (97 g) was chromatographed
over silica gel, eluting with increasing amounts of methanol (0–100%)
in dichloromethane, to afford ten fractions (A–E) based on TLC analysis.
Fraction B (22 g) was chromatographed over Sephadex LH-20 eluting
with dichloromethane/methanol (1:1) to yield B-1–B-4. B-3 (4.3 g) was
separated via RP-MPLC eluting with a gradient of MeOH (5–100%) in
1
–3 showed strong potencies in inhibiting NO production and had no
influence on cell viability.
H
2
O to give B-3-1–B-3-6. Separation of B-3-3 (621 mg) by a normal-
phase silica gel column (EtOAc/95%EtOH, 30:1) yielded B-3-3-1–B-3-3-
. B-3-3-2 (126 mg) was separated by RP flash CC (10–90% MeOH in
O) to afford B-3-3-2-1-B-3-3-2-6, and purification of B-3-3-2-4
23 mg) by RP HPLC (38% MeOH in H O) gave 3 (4.5 mg) and 4
3. Experimental
5
H
2
3.1. General experimental procedures
(
(
(
2
11.5 mg). Fractions B-3-3-4 (58 mg) was isolated by preparative TLC
mobile phase: EtOAc/95%EtOH, 15:1), followed by RP HPLC separa-
Optical rotations were measured on P-2000 polarimeter (JASCO,
Tokyo, Japan). UV spectra were measured on a UV-2550PC spectro-
meter (SHIMADZU). IR spectra were recorded on a Nicolet iN 10 Micro
FTIR spectrometer. NMR spectra were obtained at 400 or 600 MHz for
tion (42% MeOH in H
was isolated by preparative TLC (mobile phase: EtOAc/95%EtOH/
CH COOH, 15:1:1), followed by RP HPLC separation (25% MeCN in
O) to yield 1 (22.4 mg).
2
O) to yield 2 (3.3 mg). Fraction B-3-5 (103 mg)
1
13
3
H, and 100 or 150 MHz for C, respectively, on a Bruker Avance
H
2
AVIII-600 or 400 MHz spectrometer with solvent peaks used as refer-
ences. ESIMS and HRESIMS data were measured using an LTQ Orbitrap
XL instrument. Column chromatography (CC) was performed with silica
gel (200–300 mesh, Qingdao Marine Chemical Inc. Qingdao, China)
3
.3.1. Camptoside (1)
20
Yellow gel; [α]
D
+ 50.5 (c 0.18, MeOH); IR (KBr) νmax 3340,
2
8
920, 1691, 1629, 1611, 1514, 1440, 1376, 1288, 1160, 1077, 1047,
−
1
1
13
88, 841, 806, 768 cm
3
; H NMR (CD OD, 600 MHz) and C NMR
Table 2
(
[
CD
3
OD, 150 MHz) spectral data, see Table 1; (+)-ESIMS m/z 524
Inhibitory effects of compounds 1–4 against LPS induced NO production in
RAW 264.7 macrophage cells.
4
]
; (+)-HRESIMS m/z 529.1658 [M + Na]+ (calcd for
+
M + NH
11Na, 529.1680).
25 30
C H O
a
cell viability (%)b
Compounds
IC50 (μM)
3
.3.2. Enzymatic hydrolysis of 1
A solution of compound 1 (10 mg) in H O (3 mL) was treated with β-
1
2
3
11.2
8.3
9.4
87.5 ± 3.7
92.5 ± 2.1
90.1 ± 3.4
83.4 ± 2.5
2
glucosidase from almonds (Fluka) (10 mg) at 37 °C for 30 h. The reac-
tion mixtures were extracted with EtOAc (3 × 3 mL). The H O phases of
the hydrolyzate was concentrated to dryness, and chromatographed on
a silica gel column, eluting with CH CN-H O (8:1), to yield glucose with
2
O). The solvent system CHCl -MeOH-H O
Curcuminc
10.1
2
a
The IC50 value of each compound was defined as the concentration (μM)
that caused 50% inhibition of NO production in LPS-activated RAW 264.7
macrophage cells. Compounds 4 were inactive (IC50 > 50 μM).
3
2
20
[
α]
D
+ 43.1 (c 0.12, H
2
3
b
(8:5:1) was used for TLC identification of glucose (Rf, 0.32).
Cell viability was expressed as a percentage (%) of the LPS-only treatment
group.
c
Positive control. The results are averages of three independent experiments,
3.3.3. Anti-inflammatory bioassay
and the data are expressed as means ± SD.
The experiment was performed as previously described [25], and
380