G Model
CCLET 3701 1–5
2
Y. Zhang et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
51
2.3. Extraction and isolation
nm; CD (MeOH) 234 (De ꢁ5.48), 253.5 (De +2.26), 313 (De +0.89)
nm. IR (KBr) max 3399, 2940, 1678, 1613, 1513, 1462, 1426, 1327,
1262, 1220, 1118, 1032, 835, 802, 722 cmꢁ1 1H NMR (600 MHz,
CD3OD) and 13C NMR (150 MHz, CD3OD) data, see Table 1; ESI-MS
m/z 431 [M+Na]+, 407 [MꢁH]ꢁ; HR ESI-MS m/z 431.1676 [M+Na]+
(calcd. for C21H28O8Na, 431.1676).
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
n
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
Air-dried, powdered roots of A. chinense (200 kg) were
macerated for 12 h with 800 L of aqueous 95% EtOH and refluxed
for 6 h (800 L ꢀ 3). After removal of the solvent under vacuum, the
resultant residue (8 kg) was suspended in acidic H2O (100 L) and
acidified to pH 2 with HCl to afford acidic H2O-soluble and acidic
H2O-insoluble fractions. The acidic mixture was then filtered and
partitioned with petroleum ether. The acidic H2O phase was
basified to pH 10 with NaOH and then partitioned with CHCl3 to
yield the CHCl3 extract (90 g). The alkaline H2O phase was then
acidified to pH 7 with HCl and partitioned with n-BuOH to yield the
n-BuOH extract (210 g). The crude CHCl3 extract (90 g) was
fractionated using a basified silica gel column (pH 8–9, 200–
300 mesh, 1.6 kg), eluting with petroleum ether containing
increasing amounts of EtOAc (1:0, 50:1, 20:1, 10:1, 5:1, 1:1),
and then eluted with CH2Cl2:MeOH (10:1–0:100) to afford eight
fractions (A–H). Fraction F (7.2 g) was fractionated via an ODS
;
2-(Hydroxymethyl)phenol 1-O-
-rhamno-pyrano side (2): White amorphous powder; [
+75.1 (c 0.6, MeOH); IR (KBr) nmax 3382, 2923, 1604, 1493, 1455,
b
-D
-glucopyranose-(1 ! 6)-O-
20
a
-L
a]
D
1234, 1071, 761 cmꢁ1 1H NMR (500 MHz, CD3OD) and 13C NMR
;
(125 MHz, CD3OD) data, see Table 1; ESIMS m/z 455 [M+Na]+,
431[MꢁH]ꢁ; HR ESI-MS m/z 455.1525 [M+Na]+ (calcd. for
C19H28O11Na, 455.1524).
2-(Ethoxymethyl)phenol 1-O-b-D-glucopyranoside (3): White
20
amorphous powder; [
a
]
D
+92.3 (c 0.8, MeOH); IR (KBr) nmax
3461, 3268, 2874, 1677, 1604, 1492, 1454, 1391, 1235, 1197, 1104,
1071, 893, 856, 754 cmꢁ1 1H NMR (500 MHz, CD3OD) and 13C
;
NMR (125 MHz, CD3OD) data, see Table 1; ESI-MS m/z 337 [M+Na]+
; HR ESI-MS m/z 337.1276 [M+Na]+ (calcd. for C15H22O7Na,
337.1258).
column (45–70
mm, 400 g) by eluting with a gradient of MeOH
(5–100%) in H2O to yield six major fractions (F1–F5). Fraction F5
(1.2 g) was chromatographed over a Sephadex LH-20 column with
CH2Cl2:MeOH (5:1) and was further purified by reversed-phase
preparative HPLC (MeOH–H2O–TFA 40:60:0.03) to afford com-
pound 1 (8 mg, Fig. 1). Fraction G (7.2 g) was fractionated via an
2.4. Acid hydrolysis of 2
124
Compound 2 (3 mg) was dissolved in 2 mol/L HCl (aq) (2 mL)
and heated at 90 8C for 10 h under constant stirring. After
extraction with EtOAc (3ꢀ 2 mL), the aqueous layer was
evaporated and cryodesiccated. Each residue was dissolved in
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
ODS column (45–70
mm, 400 g) by eluting with a gradient of
MeOH (5–100%) in H2O to yield five major fractions (G1–G5).
Fraction G3 (1.1 g) was chromatographed over a Sephadex LH-20
column with CH2Cl2:MeOH (5:1) and was further purified by
reversed-phase preparative HPLC (MeOH–H2O–TFA 30:70:0.03) to
afford compounds 3 (17 mg, Fig. 1).
The crude n-BuOH extract (210 g) was fractionated using a
macroporous adsorptive resins column (XAD-D101, 3.9 kg),
eluting with D2O and then eluted with ethanol/D2O (95/5) to
afford ethanol/D2O (95/5) fraction (135 g). The ethanol/D2O (95/5)
fraction (135 g) was fractionated using a polyamide resins column
(30–60 mesh, 2.0 kg), and eluted with D2O containing increasing
amounts of ethanol (88:12, 75:25, 60:40, 5:95) to afford D2O
fraction (86 g). The D2O fraction (86 g) was fractionated using a
silica gel column (200–300 mesh, 1.5 kg), and eluted with
petroleum ether containing increasing amounts of acetone
(25:1, 10:1, 5:1, 3:1) and then with CH2Cl2:MeOH (20:1–0:100)
to afford seven fractions (A–G). Fraction D (22.7 g) was fractionat-
dry pyridine (1 mL), and then L-cysteine methyl ester hydrochlo-
ride (4 mg) was added. Each mixture was stirred at 60 8C for 2 h,
and then 0.4 mL of N-trimethylsilylimidazole was added, followed
by heating to dryness at 60 8C for 2 h. Each dried reactant was
partitioned between n-hexane and H2O (4 mL), and the n-hexane
fraction was subjected to gas chromatography (GC) (column: DM-
5, 0.25 mm ꢀ 30 m ꢀ 25
mmol/L; detector: FID; temperature:
280 8C; injector temperature: 260 8C; carrier: N2 gas). The sugars
from each reactant were identified by comparison of their
retention times with those for authentic standards [tR:
24.77 min for
D-glucose, 24.34 min for L-rhamnose].
2.5. Acid hydrolysis of 3
140
Following the same method used for acid hydrolysis of 2,
compound 3 (2 mg) was hydrolyzed to afford the sugar moieties.
The sugars from each reactant were then identified by comparison
of their retention times with those for authentic standards [tR:
141
142
143
144
145
ed via an ODS column (45–70
mm, 400 g) by eluting with a gradient
of MeOH (5–100%) in H2O to yield five major fractions (D1–D5).
Fraction D1 (3.4 g) was chromatographed over a Sephadex LH-20
column with MeOH:H2O (4:1) and was further purified by
reversed-phase preparative HPLC (MeOH–H2O 22:78) to afford
compound 2 (15 mg, Fig. 1).
Twenty-eight known compounds (4–31) were also isolated and
identified from the roots of A. chinense, the detail were deposited in
Supporting information.
19.84 min for D-glucose].
2.6. In vitro anti-Coxsackie virus B3 activity assay
146
The anti-coxsackie virus B3 activity assay was determined
using the same method as previously described [2]. Briefly,
confluent Vero cells grown in 96-well microplates were infected
with 100 median tissue culture infective doses (100TCID50) of Cox
B3 virus. After 1 h of adsorption at 37 8C, the monolayers were
147
148
149
150
151
(7R,8R)-Threo-4,7,9,90-tetrahydroxy-3,5,20-trimethoxy-8-O-40-
neolignan (1): White amorphous powder; [
20
a
]
D
+55.2 (c 0.3,
MeOH); UV (MeOH)
l
max (log
e) 207 (4.73), 233 (4.08), 280 (3.57)
9'
7'
5'
OH
OH
4''
6'
1'
5'
O
5''
4'
O
8'
7
3
HO
HO
HO
1'
O
7
O
1
9
HO
3'
OMe
5
HO
O
2''
2
1
2'
OH
HO
OH
HO
6
3'
5'
O
2
8
O
9
5
1'
O
MeO
HO
5
HO
7
8
3
OH
1
2
OH
3'
4
3
OMe
2
3
1
Fig. 1. Structures of compounds 1–3 from A. chinense.
Please cite this article in press as: Y. Zhang, et al., Phenolic constituents from the roots of Alangium chinense, Chin. Chem. Lett. (2016),