Z.-Q. Wang, et al.
Fitoterapia 144 (2020) 104603
voucher specimen (No. 20180713) was deposited in the Institute of
Traditional Chinese Medicine & Natural Products, College of Pharmacy,
Jinan University, Guangzhou, P. R. China.
ε): 203 (4.01), 286 (3.40), 337 (3.54) nm; IR (KBr) νmax 3286, 1694,
−1
1616, 1516, 1447, 1378, 1272, 1172, 1109, 1051, 813 cm ; ECD
1
(CH
NMR (CD
Table 1; HRESIMS m/z 769.1740 [M + Na] (calcd. for C38
69.1739).
3
OH, Δε) λmax 217 (+28.56), 240 (−4.98), 331 (+10.03) nm; H
13
3
OD, 600 MHz) and C NMR (CD+
3
OD, 150 MHz) data, see
16Na,
2.3. Extraction and isolation
34
H O
7
The air-dried and powdered aerial parts (10 kg) of M. chinensis were
extracted with 95% EtOH (v/v) for four times (4 × 20 L) at room
temperature to obtain a crude extract (2 kg), which was then suspended
in water and successively partitioned with petroleum ether and n-
BuOH. The n-BuOH-soluble fraction (261.2 g) was separated by silica
gel column using chloroform/methanol mixture (100:0 to 0:100, v/v) as
eluent to obtain four major fractions (Fr. A–Fr. D). Fr. B (85 g) was
subjected to an ODS column and eluted with a gradient mixture of
2.4. Sugar identification
Samples of compounds 3 and 4 (each 3 mg) were dissolved in
2 mol/L HCl (2 mL) and refluxed at 90 °C for 2 h, respectively. Each
solution was evaporated to yield the corresponding residue of each
sample. Next, the residues were separately re-dissolved in pyridine
(2 mL) and added in L-cysteine methyl ester hydrochloride (3 mg),
respectively. The solutions were kept at 60 °C for 1 h, after that, O-tolyl
isothiocyanate (20 μL) was added into each mixture and then heated at
60 °C for another 1 h. Subsequently, HPLC was employed to analyze the
reaction mixtures, which was performed on a Cosmosil 5 C18-MS-II
column (250 mm × 4.6 mm, 5 μm) using CH CN/0.05% HCOOH-H O
3 2
(25:75, v/v) as the mobile phase. The sugar moieties of 3 and 4 were
both determined to be D-glucose by comparison of the retention times of
the monosaccharide derivatives (t
CH
Fr. Bc (10.8 g) was chromatographed on a Sephadex LH-20 column
CH OH/H O, 50:50, v/v) to yield three subfractions (Fr. Bc1–Fr. Bc3).
Thus, compounds 1 (30 mg) and 5 (10 mg) were isolated from Fr. Bc1
3 g) by using semi-preparative HPLC (CH CN-H O, 20:80, v/v).
Meanwhile, Fr. Bd (20 g) was separated by a Sephadex LH-20 column
CH OH) and followed by repeated preparative HPLC to obtain com-
pounds 6 (58 mg), 8 (60 mg), and 9 (65 mg), respectively. Fr. C (50.5 g)
was separated by ODS column using a gradient mixture of CH CN–H
30:70 to 100:0, v/v) to afford Fr. Ca-Fr. Cd. After that, Fr. Cb (8.2 g)
was chromatographed on Sephadex LH-20 column (CH OH/H O,
0:50, v/v) to afford subfractions Fr. Cb1 to Fr. Cb4. Then, Fr. Cb2
203 mg) was separated by semi-preparative HPLC using CH CN-H
3 2
CN-H O (20:80 to 100:0, v/v) to afford Fr. Ba–Fr. Bf. Subsequently,
(
3
2
(
3
2
(
3
R
= 16.48 min) to the derivatives of
3
2
O
authentic D-glucose (t
R
= 16.75 min) and L-glucose (t = 15.43 min)
R
(
[23].
3
2
5
2.5. Quantum chemical ECD calculations
(
(
(
3
2
O
15:85, v/v) as eluent to afford compounds 2 (36 mg), 3 (12 mg), and 4
10 mg), respectively. Finally, Fr. Cd (154 mg) was also separated by
The systematic random conformational analyses of the compounds
1, 2, and 5 were performed in the SYBYL-X 2.1 program by using
MMFF94s molecular force field, which afforded 27, 155, and 26 con-
formers for compounds 1, 2, and 5, respectively, with an energy cutoff
semi-preparative HPLC (CH
51 mg).
3 2
OH-H O, 25:75, v/v) to afford compound 7
(
−
1
of 10 kcal mol
to the global minima. All the obtained conformers
2
.3.1. Mesonolate A (1)
Yellow oil; [α]
25+61.2 (c 0.35, CH
01 (3.96), 288 (3.30), 332 (3.39) nm; IR (KBr) νmax 3423, 1697, 1613,
were optimized under B3LYP/6–31+G(d) level in gas phase by using
Gaussion09 software [24]. The number of stable conformers (26 for
compound 1, 26 for compound 2, and 13 for compound 5, respectively)
were subsequently subjected to TDDFT ECD calculations at B3LYP/
6–31+G(d) (1 and 2) or CAM-B3LYP/6–31+G(d) (5) levels, respec-
tively, with the consideration of the first 80 (1 and 3) or 50 (2) ex-
citations. The overall ECD curves of compounds 1, 2, and 5 were all
weighted by Boltzmann distribution. Finally, the calculated ECD
spectra of compounds 1, 2, and 5 were compared with the experimental
ones, respectively, by using SpecDis 1.70 software [25].
D
3 3
OH); UV (CH OH) λmax (log ε):
2
1
2
6
−
1
513, 1450, 1369, 1277, 1169, 1054, 816 cm ; ECD (CH
3
OH, Δε) λmax
15 (+3.09), 236 (−7.45), 337 (+8.61) nm; H NMR (CD OD,
OD, 150 MHz) data, see Table 1; HRESIMS
12Na: 575.1160).
1
3
1
3
00 MHz) and C NMR (CD
3
+
24
m/z 575.1161 [M + Na] (calcd. for C28H O
2.3.2. Mesonolate B (2)
2
5
Yellow oil; [α]
D
3 3
+10.3 (c 0.35, CH OH); UV (CH OH) λmax (log
ε): 202 (3.88), 292 (3.38), 323 (3.29) nm; IR (KBr) νmax 3192, 1699,
1
−
1
607, 1524, 1447, 1366, 1280, 1174, 1117, 1054, 804 cm ; ECD
2.6. In vitro anti-RSV assay
1
(
CH
3
OH, Δε) λmax 225 (−4.90), 243 (+0.53), 323 (−1.54) nm;
H
1
3
NMR (CD
3
OD, 500 MHz) and C NMR (CD
3
OD, 125 MHz) data, see
Human larynx epidermoid carcinoma cell line (HEp-2, ATCC CCL-
23) and respiratory syncytial virus (RSV A2, ATCC VR-1540) were
obtained from Medicinal Virology Institute of Wuhan University, China.
HEp-2 cells were grown in the growth medium comprising Dulbecco's
modified Eagle medium (DMEM, Gibco, USA) supplemented with 10%
fetal bovine serum (FBS, Biological Industries) and 1% penicillin-
streptomycin. Anti-RSV activities of the compounds (1–9) were de-
termined by the cytopathic effect (CPE) reduction assay. HEp-2 cells
were seeded in 96-well culture plates (1.5 × 104 cells per well) and
+
24
Table 1; HRESIMS m/z 575.1161 [M + Na] (calcd. for C28H O12Na,
5
75.1160).
2.3.3. Mesonolate C (3)
2
5
Yellow oil; [α]
D
3 3
−17.6 (c 0.35, CH OH); UV (CH OH) λmax (log
ε): 199 (3.81), 288 (3.37), 320 (3.41) nm; IR (KBr) νmax 3292, 1602,
1
6
−1
1
522, 1453, 1388, 1269, 1177, 974, 807 cm
;
H NMR (CD
OD, 75 MHz) data, see Table 1; HRESIMS
16Na, 707.1583).
3
OD,
1
3
00 MHz) and C NMR (CD
3
+
m/z 707.1583 [M + Na] (calcd. for C33
H
32
O
incubated overnight at 37 °C, 5% CO
2
. The cells were then infected with
100TCID50 of RSV and treated with various concentrations of com-
2
.3.4. Mesonolate D (4)
pounds (1–9) or ribavirin. The RSV-induced CPE was observed and
scored at 60 h post-infection. The concentration required to inhibit 50%
CPE (IC50) was calculated [26]. MTT assay was used to measure the
cytotoxicity of the compounds (1–9) on HEp-2 cells as described pre-
viously [27].
2
5
Yellow oil; [α]
D
3 3
−27.6 (c 0.35, CH OH); UV (CH OH) λmax (log
ε): 197 (3.71), 292 (2.93), 324 (3.04) nm; IR (KBr) νmax 3449, 1688,
1
−
1 1
613, 1519, 1453, 1387, 1278, 1177, 1114, 1045, 813 cm ; H NMR
OD, 500 MHz) and C NMR (CD OD, 125 MHz) data, see Table 1;
3 3
1
3
(
CD
+
HRESIMS m/z 735.1900 [M + Na]
36
(calcd. for C35H O16Na,
735.1896).
3. Results and discussion
2
.3.5. Mesonolate E (5)
Compound 1 was obtained as yellow oil. The HRESIMS of 1 dis-
played a sodiated molecular ion peak at m/z 575.1161 [M + Na]
2
5
+
Yellow oil; [α]
D
+81.1 (c 0.35, CH
3
3
OH); UV (CH OH) λmax (log
3