Q.-W. Chen, et al.
Phytochemistry 164 (2019) 206–214
(
2:1, v/v) to yield 1 (26.1 mg, t
0.4 min). HPLC separation of Fraction Ⅰ
3 mL/min) with MeOH/H O (7:3, v/v) resulted in purification of 5
256.8 mg, t : 13.4 min). A portion of Fraction Ⅱ was purified using
/MeOH/H (40:10:1−12:10:1) and
SilGreen C18 column (3 mL/min) with MeOH/H O (2:1, v/v) to obtain 8
17.7 mg, t : 15.3 min). The 60% MeOH fraction was separated on an
ODS silica gel column (46 × 3.6 cm) under reduced pressure with a
stepwise MeOH/H O gradient (1:9–100% MeOH, v/v), to yield 3 sub-
fractions (A−C). Fraction B was separated by silica gel CC using CHCl
MeOH/H O (50:10:1−12:10:1, v/v/v) into 5 subfractions (B.1−B.5).
HPLC separation of Fraction B.2 on a SilGreen C18 column (3 mL/min)
with MeOH/H O (13:7, v/v) yielded pure 6 (273.8 mg, t : 14.1 min).
R
: 16.1 min) and 2 (104.2 mg, t
R
:
13C-NMR (C
5
D
5
N, 125 MHz), see Table 5; HRESIMS (positive-ion mode)
m/z 925.4799 [M + Na] , m/z 903.5007 [M + H]+
+
.
2
h
using a SilGreen C18 column
(
(
2
Osaundersioside F (6): Amorphous solid; [a]20 D− 45.0 (c = 1.2,
MeOH); UV (MeOH) λmax (log ε) 203.4 (4.12) nm; IR νmax 3375, 2927,
R
−
1 1
silica gel CC with CHCl
3
2
O
a
1449, 1377, 1076, 1047, 634 cm
5 5
; H-NMR (C D N, 500 MHz) and
13
2
C-NMR (C
5 5
D N, 125 MHz), see Table 6; HRESIMS (positive-ion mode)
+
(
R
m/z: 763.4253 [M + Na] .
Osaundersioside G (7) Amorphous solid; [a]20 D− 65.0 (c = 1.0,
MeOH); UV (MeOH) λmax (log ε) 203.2 (4.14) nm; IR νmax 3410, 2931,
2
−
1 1
3
/
1710, 1451, 1376, 1043, 636 cm
5 5
; H-NMR (C D N, 500 MHz) and
13
2
C-NMR (C
5 5
D N, 125 MHz), see Table 7; HRESIMS (positive-ion mode)
+
m/z 1083.5087 [M + Na]
.
2
R
Osaundersioside H (8): Amorphous solid; [a]20 D− 90.0 (c = 0.7,
MeOH); UV (MeOH) λmax (log ε) 202.8 (4.08) nm; IR νmax 3402, 2932,
−
1 1
3.4. Determination of the absolute configuration of sugar moieties
1710,1444, 1376, 1072, 1034, 629 cm
and 13C-NMR (C
D N, 125 MHz), see Table 8; HRESIMS (positive-ion
5 5
5 5
; H-NMR (C D N, 500 MHz)
+
Determination of the absolute configurations of sugar moieties was
performed as described previously (Shao et al., 2017). Compounds
1–8) (2 mg) were separately dissolved in 1M hydrochloric acid (2 mL)
and refluxed for 12 h at 80 °C. The mixtures were concentrated under
vacuum, and the residues were suspended in H O and extracted three
times with ethyl acetate. The aqueous layers were evaporated under
vacuum, diluted repeatedly with H O and evaporated under vacuum to
mode) m/z 937.4488 [M + Na] .
(
3.6. Cytotoxicity assay
2
The cytotoxicity of compounds 1–11 against HCT-116 (human colon
cancer cell line), HepG2 (human hepatocyte carcinoma cell line), BGC-
823 (human gastric carcinoma cell line), A549 (human non-small-cell
lung cancer cell line), and MCF-7 (human breast carcinoma cell line)
cells was tested as described previously (Tang et al., 2013; Liu and
2
produce neutral residues. The residues were dissolved in fresh anhy-
drous pyridine (1.0 mL). L-cysteine methyl ester hydrochloride (2 mg)
was added, and the reaction mixtures were heated at 60 °C for 2 h.
Then, the mixtures were concentrated under vacuum and placed in a
drying oven for 2 h at 37 °C. N-trimethylsilylimidazole (0.2 mL) was
−
5
Kong, 2018). Paclitaxel (10 M, final concentration) was used as the
positive control.
added to the mixtures and heated at 60 °C for 2 h H
2
O (2 mL) was added
3.7. Inhibition of LPS-induced NO production in macrophage
to the mixtures to stop the reactions, and each mixture was extracted
three times with n-hexane (2 mL each extraction). The n-hexane ex-
tracts were subjected to GC analysis under the following conditions:
HP-5 (60 m × 0.25 mm, with a 0.25 μm film) column; FID detector;
injector temperature of 300 °C; detector temperature of 300 °C; initial
column temperature 200 °C, which was then raised to 280 °C at 8 °C/
The inhibitory effects of compounds 1–11 on NO production in LPS-
activated mouse peritoneal macrophages were evaluated according to
an established procedure (Wang et al., 2014). Dexamethasone (DEX,
−
5
10 M, final concentration) was used as the positive control.
2
min, and the final temperature maintained for 30 min; N as the carrier
Conflicts of interest
gas. D-glucose and L-rhamnose were confirmed by comparing the re-
tention time of the derivatives of the purified molecules to the standard
derivatized sugars, which had retention times of 29.662 min and
The authors declare no conflict of interest.
Acknowledgments
23.576 min, respectively.
3
.5. Structural characterization
This research was financially supported by the Drug Innovation
Major Project (2018ZX09711001-006), CAMS Innovation Fund for
Medical Sciences (CIFMS) (2016-I2M-3-012) and Beijing Natural
Science Foundation (7172143).
Osaundersioside A (1): Amorphous solid; [a]20 D− 27.8 (c = 0.9,
MeOH); UV (MeOH) λmax (log ε) 203.2 (4.07) nm; IR νmax 3391, 2933,
−
1 1
1
449, 1381, 1076, 1048, 633 cm
;
H-NMR (C
5 5
D N, 600 MHz) and
1
3
C-NMR (C
5
D
5
N, 150 MHz), see Table 1; HRESIMS (positive-ion mode)
References
+
+
m/z 925.4797 [M + Na] , m/z 903.4997 [M + H]
.
Osaundersioside B (2): Amorphous solid; [a]20 D− 60.0 (c = 1.3,
MeOH); UV (MeOH) λmax (log ε) 202.8 (3.94) nm; IR νmax 3403, 2935,
Andriamisaina, N., Mitaine-Offer, A.C., Miyamoto, T., Tanaka, C., Paululat, T., Lirussi, F.,
Bie, Q., Chen, C., Yu, M., Guo, J., Wang, J., Liu, J., Zhou, Y., Zhu, H., Zhang, Y., 2019.
−
1 1
1450, 1382, 1128, 1049, 637 cm
5 5
; H-NMR (C D N, 500 MHz) and
1
3
C-NMR (C
5 5
D N, 125 MHz), see Table 2; HRESIMS (positive-ion mode)
+
+
m/z 909.4841 [M + Na] , m/z 887.5031 [M + H]
.
Challinor, V.L., Stuthe, J.M.U., Parsons, P.G., Lambert, L.K., Lehmann, R.P., Kitching, W.,
Hirano, T., Oka, K., Mimaki, Y., Kuroda, M., Sashida, Y., 1996. Potent growth inhibitory
Iguchi, T., Kuroda, M., Naito, R., Watanabe, T., Matsuo, Y., Yokosuka, A., Mimaki, Y.,
Osaundersioside C (3): Amorphous solid; [a]20 D− 38.2 (c = 1.1,
MeOH); UV (MeOH) λmax (log ε) 202.8 (3.98) nm; IR νmax 3375, 2935,
−
1 1
1
450, 1380, 1124, 1050, 625 cm
5 5
; H-NMR (C D N, 500 MHz) and
1
3
C-NMR (C
5 5
D N, 125 MHz), see Table 3; HRESIMS (positive-ion mode)
m/z 1055.5354 [M + Na] , m/z 1033.5536 [M + H]
+
+
.
Osaundersioside D (4): Amorphous solid; [a]20 D− 46.7 (c = 0.9,
MeOH); UV (MeOH) λmax (log ε) 202.8 (3.93) nm; IR νmax 3367, 3236,
−
1 1
2
5 5
936, 1449, 1380, 1161, 1110, 629 cm ; H-NMR (C D N, 500 MHz)
Kicha, A.A., Kalinovsky, A.I., Ivanchina, N.V., Malyarenko, T.V., Dmitrenok, P.S.,
1
3
and C-NMR (C
mode) m/z 1071.5427 [M + Na]
5 5
D N, 125 MHz), see Table 4; HRESIMS (positive-ion
+
.
Osaundersioside E (5): Amorphous solid; [a]20 D− 92.0 (c = 1.0,
MeOH); UV (MeOH) λmax (log ε) 203.2 (4.05) nm; IR νmax 3376, 2926,
−
1 1
1449, 1378, 1126, 1073, 636 cm
5 5
; H-NMR (C D N, 500 MHz) and
213