Glycosides from the stem bark of Fraxinus sieboldiana

Article abstract of DOI:10.1021/np0700467

A norditerpene glucopyranoside with a novel carbon skeleton (1), eight new aromatic glycosides (2-9), and 25 known glycosides have been isolated from a H₂O-soluble portion of an ethanolic extract of the stem bark of Fraxinus sieboldiana. Their structures were determined by spectroscopic and chemical methods. Based on analysis of the NMR data of threoand erythro-arylglycerols in different solvents, an application of Δδ5_C8-C7 values to distinguish threo-arylglycerol and erythro-arylglycerol isomers was proposed. In the in vitro assays, compound 5 displayed TNF-α secretion inhibitory activity with an IC₅₀ value of 1.6 μM, compound 6 showed antioxidative activity inhibiting Fe ⁺²-cystine-induced rat liver microsomal lipid peroxidation with an IC₅₀ value of 0.9 μM, and plantasioside (10) showed selective activity against the human colon cancer cell line (HCT-8) with an IC ₅₀ value of 3.4 μM.

Full text of DOI:10.1021/np0700467

J. Nat. Prod. 2007, 70, 817-823
817
Glycosides from the Stem Bark of Fraxinus sieboldiana
Sheng Lin, Sujuan Wang, Mingtao Liu, Maoluo Gan, Shuai Li, Yongchun Yang, Yinghong Wang, Wenyi He, and
Jiangong Shi*
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Key Laboratory of BioactiVe
Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Beijing 100050, People’s Republic of China
ReceiVed January 31, 2007
A norditerpene glucopyranoside with a novel carbon skeleton (1), eight new aromatic glycosides (2-9), and 25 known
glycosides have been isolated from a H O-soluble portion of an ethanolic extract of the stem bark of Fraxinus sieboldiana.
2
Their structures were determined by spectroscopic and chemical methods. Based on analysis of the NMR data of threo-
and erythro-arylglycerols in different solvents, an application of ∆δC8-C7 values to distinguish threo-arylglycerol and
erythro-arylglycerol isomers was proposed. In the in Vitro assays, compound 5 displayed TNF-R secretion inhibitory
+
2
activity with an IC50 value of 1.6 µM, compound 6 showed antioxidative activity inhibiting Fe -cystine-induced rat
liver microsomal lipid peroxidation with an IC50 value of 0.9 µM, and plantasioside (10) showed selective activity
against the human colon cancer cell line (HCT-8) with an IC50 value of 3.4 µM.
Table 1. NMR Data (δ) for Compounds 1 and 1aa
Fraxinus sieboldiana Blume (Oleaceae) is widely distributed in
eastern Asia, especially in southern China. Its dried bark has long
been used as a folk medicine “Qin Pi” in China and Japan, as it is
reported to have diuretic, antifebrile, analgesic, and antirheumatic
1
(MeOH-d4)
1a (MeOH-d4)
no.
H
C
H
C
activities.1 Many coumarins, lignans, secoiridoid glucosides,
,2
3,4
5
6-8
1
2
3
4
5
6
7
8
9
176.6
130.4
152.0
86.0
45.8
124.8
138.7
68.5
14.5
10.5
136.2
131.2
33.1
26.8
87.5
42.3
22.0
22.5
25.6
106.5
75.7
78.3
71.7
77.8
62.8
176.5
130.5
151.9
86.0
9
and phenylethanoids have been reported from species of this genus.
As part of a program to assess the chemical and biological diversity
of traditional Chinese medicines, we undertook investigation of the
stem bark of F. sieboldiana and describe herein isolation and
structural elucidation of a new norditerpene glucopyranoside (1)
and eight new aromatic glycosides (2-9), along with 25 known
glycosides. Some biological assay results are also reported.
7.21 t (2.0)
7.20 s
5.30 dt (9.4, 1.6)
2.96 dd (9.6, 9.4)
5.72 d (9.6)
5.30 d (9.6)
2.97 t (9.6)
5.71 (10.0)
46.0
124.8
138.8
68.4
14.5
10.5
136.5
131.0
33.0
27.7
76.6
3.90 s
1.63 s
1.85 t (2.0)
3.90 s
1.63 s
1.82 s
1
1
2
3
4
5
0
′
Results and Discussion
′
′
′
′
2.06 m
1.99 m, 1.75 m
3.32 dd (10.8, 2.8)
2.06 m
1.67 m
3.31 dd (14.0, 7.2)
The ethanolic extract of the stem bark of F. sieboldiana was
partitioned between H O and EtOAc. The H O phase was subjected
2 2
to separation using various column chromatographic techniques to
afford nine new glycosides (1-9).
6′
7′
42.2
21.7
22.0
25.6
1.75 s
0.91 s
0.95 s
4.26 d (7.6)
3.13 dd (8.4, 7.6)
3.20 t (8.4)
3.24 t (8.4)
3.26 m
1.75 s
0.82 s
0.88 s
8
9
1
2
′
′
′′
′′
Compound 1 was obtained as a colorless gum, and the presence
-
1
-1
of hydroxyl (3389 cm ) and carbonyl (1747 cm ) groups was
evident in its IR spectrum. The positive mode ESIMS of 1 gave a
+
quasi-molecular ion peak at m/z 505 [M + Na] . The molecular
3′′
4′′
5′′
38 9
formula C25H O , with seven degrees of unsaturation, was indicated
1
by HRESIMS. The H NMR spectrum of 1 in MeOH-d
two quaternary methyl singlets at δ 0.91 (H -8′) and 0.95 (H
two olefinic methyl singlets at δ 1.63 (H -9), and 1.75 (H -7′), and
an olefinic methyl triplet at δ 1.85 (J ) 2.0 Hz, H -10). The H
4
showed
6
6
′′a
′′b
3.79 dd (12.0, 2.0)
3.60 dd (12.0, 5.2)
3
3
-9′),
3
3
a
1
NMR data (δ) were measured in MeOH-d for 1 and 1a at 400
1 13
4
3
MHz for H NMR and at 100 MHz for C NMR. Proton coupling
NMR spectrum also displayed signals attributed to two olefinic
methines at δ 7.21 (J ) 2.0 Hz, H-3) and 5.72 (J ) 9.6 Hz, H-6),
two oxygen-bearing methines at δ 5.30 (dt, J ) 9.4 and 1.6 Hz,
H-4) and 3.32 (dd, J ) 10.8 and 2.8 Hz, H-5′), and one oxygen-
bearing methylene at δ 3.90 (s, H-8). It also had a double doublet
attributed to a deshielded methine at δ 2.96 (H-5) and partially
overlapped multiplets due to two methylenes between δ 1.75 and
constants (J) in Hz are given in parentheses. The assignments were
1
1
based on DEPT, H- H COSY, HSQC, HMBC, and phase-sensitive
1
1
H- H COSY experiments.
enzymatic hydrolysis of 1 with â-glucosidase (Table 1 and
Experimental Section). The glucose isolated gave a positive optical
2
D
0
rotation, [R] +46.2 (c 0.11, H O), indicating that it was D-
2
glucose.13
2.20, together with characteristic signals due to a â-glucopyranosyl
unit (Table 1). In addition to protonated carbon signals correspond-
ing to the above protons, the 13C NMR and DEPT spectra of 1
showed six quaternary carbons that were identified as a carboxylic
carbon, four sp olefinic carbons, and a sp carbon (Table 1). These
data suggested that 1 was a glycosidic norditerpene containing a
carboxylic acid group, two rings, and three double bonds. This was
confirmed by spectroscopic data of the aglycone (1a) obtained from
The proton and protonated carbon signals of 1 were assigned
1
1
unambiguously by the HSQC experiment. H- H COSY correla-
tions from H-5′ through H -4′ to H -3′ and HMBC correlations
2
2
2
3
3 3 3
from H -7′ to C-1′, C-2′, and C-3′ and from both H -8′ and H -9′
to C-1′, C-5′, and C-6′, together with their chemical shift values,
indicated the presence of a 5′-oxygen-bearing 2′,6′,6′-trimethylcy-
clohexen-1′-yl moiety in 1. HMBC correlation of the anomeric
proton (H-1′′) to C-5′ indicated that the â-D-glucopyranosyl unit
was located at C-5′. This was supported by comparison of the
*
To whom correspondence should be addressed. Tel: 86-10-83154789.
1
1
Fax: 86-10-63017757. E-mail: shijg@imm.ac.cn.
chemical shifts of C-5′ between 1 and 1a (Table 1). The H- H
1
0.1021/np0700467 CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/27/2007

8
18 Journal of Natural Products, 2007, Vol. 70, No. 5
Lin et al.
Chart 1
COSY correlations from H-6 through H-5 to H-4 and H-3, in
combination with two- and three-bond HMBC correlations from
restricted due to the bulky 2-methylacrylic acid unit. Therefore, 1
was assigned as an atropisomer possessing the relative configuration
H-5 to C-4 and C-6, from H-6 to C-5, C-8, and C-9, from H
C-4, C-6, C-7, and C-9, from H-4 to C-2, C-3, C-5, and C-6, from
H-3 to C-1, C-2, C-4, and C-10, from H -10 to C-1, C-2, and C-3,
and from H -9 to C-6, C-7, and C-8, together with their chemical
shifts, indicated a 5-substituted 4,8-epoxy-2,7-dimethyl-∆ -octa-
dienoic acid moiety in 1. This was supported by long-range
2
-8 to
illustrated in the structure drawing. On the basis of the empirical
rule of â-D-glucosylation-induced shifts,1
0-13
the deshielded chemi-
3
cal shift of the anomeric carbon of 1 (δ 106.5) and chemical shift
differences of C-4′ (∆δ -0.9), C-5′ (∆δ +10.9), and C-6′ (∆δ
3
2,6
13
+0.1) between the C NMR data of 1 and 1a indicated a 5′S
configuration for 1. Accordingly, the structure of 1 was determined
as (-)-(aS)-(4R,5S,2Z)-5-[(5S)-5-O-â-D-glucopyranosyloxy-2,6,6-
homonuclear correlations of H-6 with both H
2
-8 and H
3
-9 and of
1
1
2,6
3
H -10 with both H-3 and H-4 in the H- H COSY spectrum. In
trimethylcyclohexen-1-yl]-4,8-epoxy-2,7-dimethyl-∆ -octadieno-
addition, HMBC correlations from H-5 to C-1′, C-2′, and C-6′, and
from H-6 to C-1′ indicated unambiguously a connection between
C-5 and C-1′. Therefore, the planar structure of 1 was elucidated
as 5-(5-O-â-D-glucopyranosyloxy-2,6,6-trimethylcyclohexen-1-yl)-
ic acid and was named fraxinuacidoside.
Compound 2 was obtained as a yellowish, amorphous powder,
and HRESIMS indicated the molecular formula to be C29H O .
32 17
The IR spectrum of 2 exhibited absorption bands at 3396, 1712,
2,6
-1
4
,8-epoxy-2,7-dimethyl-∆ -octadienoic acid.
1612, 1570, and 1510 cm , indicating the presence of hydroxyl,
1
The stereochemistry including the absolute configuration of 1
carbonyl, and aromatic ring functional groups. The H NMR
was elucidated by a combination analysis of the coupling constants
in the H NMR spectrum and enhancements in the NOE difference
6
spectrum of 2, in DMSO-d , had signals indicating that it was a
1
6,7-disubstituted coumarin, which was confirmed by HMBC
correlations from H-3 to C-2 and C-4a, from H-4 to C-2, C-5, C-4a,
and C-8a, from H-5 to C-4, C-6, C-7, C-4a, and C-8a, and from
experiments, as well as the empirical rule of â-D-glucosylation-
induced shifts of the 13C NMR data.
10-12
The splitting pattern and
1
coupling constants of H-5′ (dd, J5′,4′a ) 10.8 Hz, J5′,4′e ) 2.8 Hz)
indicated that H-5′ had a pseudoaxial orientation. In the NOE
H-8 to C-6, C-7, C-4a, and C-8a. The H NMR spectrum showed
signals attributed to a 1,3,4-trisubstituted aromatic ring at δ 7.15,
7.44, and 7.54 and a methoxy group at δ 3.74. Two doublets
attributed to anomeric protons (δ 4.88 and 5.04), together with
coupling patterns of oxymethylene and oxymethine protons between
δ 3.00 and 4.61, as well as seven exchangeable OH protons,
indicated the presence of two â-glucopyranosyl units in 2. This
was confirmed by acidic hydrolysis of 2, which produced D-glucose
as the sole sugar, identified by TLC comparison and optical rotation.
The 13C NMR and DEPT spectra of 2 had signals corresponding
to the above units (Table 3). An additional carbonyl carbon (δ 165.1,
C-7′′) in the 13C NMR spectrum, together with the chemical shifts
of the protons and carbons assigned to the 1,3,4-trisubstituted
aromatic ring (Tables 2 and 3), suggested that there was a 4-oxygen-
substituted 3-methoxybenzoyl unit in 2.
difference experiment of 1, NOE enhancement of H
3
-9′ by
irradiation of H-1′′ indicated that H -9′ and the â-D-glucopyranosyl
3
unit at C-5′ were oriented on the same side of the cyclohexene
ring system. The splitting patterns and coupling constants of H-5
(dd, J5,6 ) 9.6 Hz and J4,5 ) 9.4 Hz) suggested that H-4 and H-6,
opposite H-5, were oriented on the same side of the dihydropyran
ring. This was confirmed by a NOE enhancement of H-6 by
irradiation of H-4, which, in turn, was enhanced by irradiation of
H-6. A cis-configuration of the double bond between C-2 and C-3
was indicated by a NOE enhancement of H
irradiated. In addition, in the NOE difference experiments, H
and H -9′ were enhanced by irradiation of H-5, but H -7′ was not
enhanced, whereas irradiation of H-4 caused an enhancement of
-7′. These NOE effects indicated that H-5 was close to both H
′ and H -9′, and H-4 to H -7′, suggesting that free rotation of the
bond connecting the two rings, in the solution state of 1, was
3
-10 when H-3 was
3
-8′
3
3
H
3
3
-
This was confirmed by the NOE difference experiment of 2
showing enhancement of H-2′′ by irradiation of the methoxy
protons. In addition, H-5 was enhanced by irradiation of H-1′, while
8
3
3

Glycosides from the Stem Bark of Fraxinus sieboldiana
Journal of Natural Products, 2007, Vol. 70, No. 5 819
Table 2. 1H NMR Data (δ) for Compounds 2-9a
no.
2 (DMSO-d6)b
3 (DMSO-d6)
4 (DMSO-d6)
5 (DMSO-d6)
6 (DMSO-d6)
7 (D2O)
6.68 s
8 (D2O)
6.67 s
9 (D2O)
6.67 s
2
6.83 brs
6.60 brs
3
4
5
6.13 d (9.5)
7.27 d (9.5)
7.22 s
6.37 s
6.37 s
6.70 d (3.0)
6.52 dd (9.0,
6.64 d (8.0)
6.61 d (8.5)
6.47 dd (8.5,
3.0)
6
6.98 d (9.0)
6.61 brd (8.0)
6.68 s
6.67 s
6.67 s
2.0)
7
8
2.73 t (7.0)
3.86 m,
2.67 m
3.81 m,
3.54 m
4.51 d (7.0)
3.76 m
4.54 d (6.0)
3.74 m
4.53 d (6.0)
3.73 m
6.79 s
3.59 m
9
a
3.65 dd (12.0,
3.43dd (12.0,
2.0)
3.34 dd (12.0,
7.0)
4.90 d (7.0)
3.42 dd (8.5,
7.0)
3.41 dd (9.5,
8.5)
3.43 dd
(12.0, 2.0)
3.35 dd
(12.0, 6.4)
4.90 d (7.2)
3.42 dd
(8.4, 7.2)
3.41 dd
2
.0)
3.49 dd (12.0,
.5)
9
b
6
1
2
′
′
4.88 d (7.5)
3.35 m
4.74 d (7.0)
4.73 d (7.5)
4.16 d (8.0)
2.95 dd (8.0,
8.5)
3.12 dd (8.5,
9.5)
4.15 d (7.5)
2.93 dd (8.0,
8.5)
3.12 dd (8.5,
9.5)
4.91 d (7.0)
3.44 dd (8.5,
7.0)
3.42 dd (9.5,
8.5)
3.19 ddd (8.5,
3.19 ddd (8.5,
c
c
7.5, 5.0)
7.5, 5.0)
3
4
′
′
3.36 m
3.30 m
3.16 ddd (8.5,
3.15 ddd (8.5,
c
c
8.5, 5.0)
5.0)
(9.6, 8.4)
3.36 t (9.6)
3.10 ddd (9.5,
3.10 ddd (9.0,
2.97 dd (9.0,
9.5)
2.97 dd (9.0,
9.5)
3.36 t (9.5)
3.36 t (9.5)
c
c
8.5, 5.0)
8.5, 5.0)
5
6
′
′a
3.80 m
4.60 br d
3.00 m
3.58 ddd (11.5,
5.5, 2.0)
3.39 ddd (11.5,
6.0, 5.0)
4.78 d (7.5)
3.20 ddd (8.5,
3.19 m
3.70 ddd (11.5,
5.5, 2.0)
3.43 ddd (11.5,
6.0, 5.0)
4.75 d (7.5)
3.19 ddd (8.5,
7.5, 5.0)
3.25 m
3.84 m
3.26 m
3.81 m
3.22 m
3.68 brd (13.0)
3.23 m
3.69 brd (12.5)
3.22 m
3.69 brd
(12.4)
3.59 dd
(12.4, 5.2)
c
c
(
12.0)
4.32 dd
12.0, 7.0)
6
′b
3.39 dd
3.40 dd (11.0,
7.0)
4.84 d (3.0)
3.73 d (3.0)
3.58 dd (13.0,
5.5)
3.59 dd (12.5,
5.5)
c
c
(
(11.0, 7.0)
4.89 d (3.0)
3.73 d (3.0)
1
2
′′
′′
7.44 d (2.0)
c
c
7.5, 5.0)
3
4
′′
3.24 ddd (8.5,
3.23 ddd (8.5,
8.5, 5.0)
c
c
8.5, 5.0)
′′a
3.09 ddd (9.5,
3.21 ddd (9.5,
3.83 d (9.5)
3.83 d (9.5)
c
c
8.5, 5.0)
8.5, 5.0)
4
5
6
′′b
′′
′′a
3.56 d (9.5)
3.32 s
3.57 d (9.5)
3.32 s
7.15 d (9.0)
7.54 dd
3.35 m
3.70 ddd (11.5,
5.0, 2.0)
3.21 m
3.65 ddd (11.5,
5.0, 2.0)
c
c
(9.0, 2.0)
6
′′b
3.41ddd (11.5,
3.44 ddd (11.5,
6.0, 5.0)
c
c
6.0, 5.0)
OMe
3.74 s
3.71 s
3.73 s
3.73 s
3.76 s
3.75 s
3.75 s
a 1H NMR data (δ) were measured in DMSO-d
for 2-6 and D O for 7-9 at 500 or 400 MHz. Proton coupling constants (J) in Hz are given
6
2
1
1
b
in parentheses. The assignments were based on DEPT, H- H COSY, HMQC, and HMBC experiments. Data of the terminal glucopyranosyl unit
of 2: δ 5.04 (1H, d, J ) 7.5 Hz, H-1′′′), 3.30 (2H, m, H-2′′′, and H-3′′′), 3.18 (1H, m, H-4′′′), 3.35 (1H, m, H-5′′′), 3.64 (1H, dd, J ) 10.0 and
5
.0 Hz, H-6′′′a), and 3.46 (1H, ddd, J ) 10.0, 7.0, and 5.0 Hz, H-6′′′b). c Coupling with adjacent hydroxyl proton was included.
H-5′′ was enhanced by irradiation of H-1′′′. These enhancements
indicated that the two â-D-glucopyranosyl units were located at C-6
of the coumarin moiety and C-4 of the 3-methoxybenzoyl unit,
respectively. In the NMR spectrum, the proton and carbon signals
attributed to the hydroxymethylene of one â-D-glucopyranosyl unit
glucopyranosyls indicated that the magnetic environments of the
two were different. This suggested a 2,5-diglucosyl-1,3-dimethoxy
substitution pattern for 3, which was supported by the NOE
difference experiment showing enhancement of H-1′′ by irradiation
of the two overlapped aromatic protons and further confirmed by
the HMBC experiment showing correlations from H-1′ to C-1 and
H-1′′ to C-4. Therefore, 3 was determined to be 2,5-di-O-â-D-
glucopyranosyloxy-1,3-dimethoxybenzene.
H C
were deshielded by ∆δ 0.96 (H-6′a) and 0.86 (H-6′b), and ∆δ
3
.5 ppm, respectively. This indicated that the benzoyl unit was
located at C-6 of the sugar unit that was connected to the coumarin
moiety. The above deductions were confirmed by gHSQC and
gHMBC experiments of 2. Thus, compound 2 was determined to
be 6-[6-(4-O-â-D-glucopyranosyloxy-3-methoxybenzoyl)]-O-â-D-
glucopyranosyloxy-7-hydroxycoumarin.
Compound 4 exhibited a quasi-molecular ion peak at m/z 487
+
[M + Na] in its ESIMS. The molecular formula C19
28
H O13 was
indicated by HRESIMS. The IR and NMR spectra of 4 resembled
those of 3 except that the NMR signals of the symmetrically
tetrasubstituted benzene moiety and two methoxys of 3 were
replaced by signals attributed to a 1,2,4-trisubstituted benzene
moiety and one methoxy of 4 (Tables 2 and 3). These data indicated
that 4 was a demethoxy derivative of 3, which was further
confirmed by enzymatic hydrolysis and HMBC experiments of 4.
Therefore, 4 was determined to be 1,4-di-O-â-D-glucopyranosyloxy-
2-methoxybenzene.
Compound 3 showed IR absorption bands for hydroxyl (3398
cm- ) and aromatic ring (1601 and 1508 cm ) functional groups.
1
-1
Its positive mode ESIMS gave a quasi-molecular ion peak at m/z
+
5
17 [M + Na] , and the molecular formula C20
H
30
O
14 was indicated
1
by HRESIMS. The H NMR spectrum of 3 in DMSO-d
6
showed
a six-proton methoxy singlet at δ 3.71 and a two aromatic proton
singlet at δ 6.37, in addition to signals attributed to two â-glu-
copyranosyl units. Enzymatic hydrolysis of 3 produced â-D-glucose
as the sole sugar. In addition to a methoxy carbon signal and two
Compound 5 showed quasi-molecular ion peaks at m/z 485 [M
+
+
+ Na] and 501 [M + K] in the ESIMS. Its molecular formula,
20 30
C H O12, was indicated by HRESIMS. The NMR spectra of 5
sets of carbon signals due to â-glucopyranosyl moieties, the 13
C
14
NMR spectrum of 3 showed four signals in the aromatic region
Table 3). These data suggested that 3 was a phenolic diglucoside
were similar to those of the co-occurring osmanthuside H, except
that resonances of the para-disubstituted aromatic moiety of
osmanthuside H were replaced by resonances ascribed to a 1,2,4-
trisubstituted aromatic moiety and an additional methoxy in 5
(
substituted symmetrically by the two â-D-glucopyranosyl and two
methoxy groups. The separation of signals of the two â-D-

8
20 Journal of Natural Products, 2007, Vol. 70, No. 5
Lin et al.
Table 3. 13C NMR Data (δ) for Compounds 2-9a
no.
2 (DMSO-d6)b
3 (DMSO-d6)
4 (DMSO-d6)
5 (DMSO-d6)
6 (DMSO-d6)
7 (D2O)
8 (D2O)
9 (D2O)
1
129.3
153.0
95.1
141.4
149.6
102.5
152.8
129.4
113.0
147.3
144.7
129.2
115.4
143.5
144.9
138.4
105.0
152.5
133.1
138.5
104.5
152.6
133.0
138.5
104.5
152.5
133.0
2
3
4
4
5
6
7
8
8
9
1
2
3
4
5
6
1
2
3
4
5
6
7
160.4
112.0
143.8
110.5
113.7
142.5
151.1
103.2
150.3
153.9
a
a
95.1
153.0
107.3
116.4
115.2
120.9
35.1
116.3
119.5
35.1
152.5
105.0
74.1
152.6
104.5
74.2
152.5
104.5
74.2
69.8
70.0
74.8
75.6
75.6
62.7
103.1
73.8
75.9
69.5
76.5
60.6
62.7
103.1
73.8
75.9
69.5
76.5
60.7
62.7
103.1
73.8
75.9
69.5
76.5
60.6
′
′
′
′
′
′
′′
′′
′′
′′
′′
′′
′′
99.4
73.1
75.8
70.1
74.0
102.9
74.1
76.5
70.0
77.1
60.9
100.9
73.2
76.8
70.0
77.2
60.8
100.9
73.3
76.8
69.9
77.1
60.8
101.3
73.3
76.7
69.7
76.9
60.7
102.7
73.3
76.6
70.2
75.5
67.7
109.2
75.8
78.8
73.2
63.1
102.8
73.3
76.6
70.2
75.4
67.6
109.2
75.8
78.8
73.2
63.1
64.0
122.9
112.7
148.7
150.8
114.3
122.8
165.1
55.7
OMe
56.2
55.6
55.5
56.4
56.4
56.4
a 13
1
1
C NMR data (δ) were measured in DMSO-d
6
for 2-6 and D O for 7-9 at 125 and 100 MHz. The assignments were based on DEPT, H- H
2
b
COSY, HMQC, and HMBC experiments. Data of the terminal glucopyranosyl unit of 2: δ 101.4 (C-1′′′), 73.1 (C-2′′′), 76.8 (C-3′′′), 69.5 (C-4′′′),
7.1 (C-5′′′), 60.5 (C-6′′′).
7
(Tables 2 and 3). In the HMBC spectrum of 5, correlations of C-3
was confirmed by correlations from H-7 and H-8 to C-1 and from
with H-5 and the methoxy protons, and C-2 and C-6 with H -7,
2
H-1′ to C-4 in the HMBC spectrum. Since erythro-arylglycerols
demonstrated that the methoxy group was located at C-3. HMBC
correlations from H-1′ to C-8 and from H-1′′ to C-6′ confirmed
that the connection among the 4-hydroxy-3-methoxyphenylethyl
and the two sugar moieties of 5 was identical to that of osman-
thuside H. Therefore, 5 was determined to be 2-(4-hydroxy-3-
methoxyphenyl)ethanol 1-O-[â-D-apiofuranosyl-(1f6)-â-D-glu-
copyranoside].
with 7R,8S configuration were reported to have negative [R]
D
values,1
6,17
the absolute configuration at C-7 and C-8 of 7a was
assigned as 7R,8S. Thus, the structure of 7 was determined to be
(-)-(7R,8S)-erythro-1-C-syringylglycerol 4-O-â-D-glucopyranoside.
The spectroscopic data of 8 (Tables 2 and 3 and Experimental
Section) were similar to those of 7. Comparison of the NMR data
of 7 and 8 indicated that H-7 of 8 was deshielded by ∆δ 0.03 ppm
and that H-9a and H-9b were shielded by ∆δ 0.22 and 0.15 ppm,
respectively, while C-8 of 8 was deshielded by ∆δ 0.8 ppm. This
suggested that it was a threo-isomer of 7, which was further
confirmed by enzymatic hydrolysis and 2D NMR experiments of
Compound 6 was obtained as a colorless gum, and HRESIMS
+
at m/z 471.1498 [M + Na] indicated the molecular formula to be
19 28
C H O12. The IR and NMR spectra of 6 were very similar to those
of 5 (Tables 2 and 3), except for the absence of methoxy resonances
in the NMR spectra of 6. In addition, as compared to those of 5,
C-3 and C-6 of 6 were shielded by ∆δ 3.8 and 1.4 ppm,
respectively, while C-2 and C-5 were deshielded by ∆δ 2.4 and
2
0
8. The enzymatic hydrolysis of 8 gave 8a with [R] -22.0 (c
D
20
0.15, MeOH) and â-D-glucose with [R]D +41.0 (c 0.38, H O).
2
The NMR data of 8a were consistent with those of threo-1-C-
1.1 ppm, respectively. These changes revealed that 6 was a demethyl
syringylglycerol.15 The negative optical rotation of 8a indicated that
derivative of 5. Thus, 6 was determined to be 2-(3,4-dihydrox-
yphenyl)ethanol 1-O-[â-D-apiofuranosyl-(1f6)-â-D-glucopyrano-
side].
the configuration of the glycerol moiety of 8 and 8a was 7R,8R.16,17
Therefore, 8 was determined to be (-)-(7R,8R)-threo-1-C-sy-
ringylglycerol 4-O-â-D-glucopyranoside.
Compound 7 showed IR absorption bands for OH (3277 cm-1)
Compound 9 showed IR, ESIMS, and NMR spectroscopic data
completely identical to those of 8 (Tables 2 and 3 and Experimental
Section). However, 8 and 9 were separable by reversed-phase HPLC
with retention times of 27.9 and 29.8 min (Supporting Information),
respectively. Enzymatic hydrolysis of 9 yielded 9a and â-D-glucose.
The spectroscopic data of 9a were identical to those of 8a except
that the optical rotation of 9a was opposite that of 8a, indicating
that 9a was (+)-threo-syringylglycerol. Thus, 9 was determined to
be (-)-(7S,8S)-threo-1-C-syringylglycerol 4-O-â-D-glucopyrano-
side.
-1
and aromatic ring (1601 and 1512 cm ) functional groups. ESIMS
+
gave a quasi-molecular ion peak at m/z 429 [M + Na] , and
HRESIMS indicated the molecular formula to be C17
H
26
O
11. The
1
H NMR spectrum of 7 in D
2
O showed a two-proton aromatic
singlet at δ 6.68, a six-proton methoxy singlet at δ 3.76, two
deshielded oxymethine doublets at δ 4.91 and 4.51, and partially
overlapped oxymethylene and/or oxymethine multiplets integrated
for nine protons between δ 3.20 and 3.75 (Table 2). The 13C NMR
and DEPT spectra of 7 displayed characteristic signals for 1-C-
syringylglycerol and â-glucopyranosyl moieties (Table 3). Enzy-
The known compounds were identified by comparison of
2
D
0
1
13
matic hydrolysis of 7 with â-glucosidase yielded 7a with [R]
-
spectroscopic data (UV, IR, ESIMS, H and C NMR) with those
20
3
4
19.8 (c 0.11, MeOH) and â-D-glucose with [R]D +39.8 (c 0.55,
O). The NMR data of 7a (Experimental Section) were in good
reported in the literature as fraxin, esculin, 6,7-di-O-glucopyra-
18
H
2
nosylaesculetin, (+)-syringaresinol O-â-D-glucopyranoside, liri-
15
19
agreement with those of erythro-1-C-syringylglycerol, indicating
that 7 was (-)-erythro-1-C-syringylglycerol â-D-glucopyranoside.
Comparison of the NMR data of 7 and 7a indicated that C-1 and
C-3/C-5 (overlapped) of 7 were significantly deshielded by ∆δ 4.3
and 5.1 ppm, respectively. This suggested that â-D-glucopyranosyl
was located at C-4 of (-)-erythro-1-C-syringylglycerol in 7, which
odendrin, (+)-1-hydroxypinoresinol 4′-O-â-D-glucopyranoside,
2
0
(+)-1-hydroxypinoresinol 4′′-O-â-D-glucopyranoside; 4-(2-hy-
21
droxyethyl)-2-methoxyphenyl â-D-glucopyranoside, 2-(4-hydrox-
22
yphenyl) ethyl â-D-glucopyranoside, 2-(3,4-dihydroxyphenyl)
23
ethyl â-D-glucopyranoside, 2-hydroxy-5-(2-hydroxyethyl) phenyl
â-D-glucopyranoside,24 2-(4-hydroxyphenyl) ethyl â-D-apiofurano-

Glycosides from the Stem Bark of Fraxinus sieboldiana
syl-(1f6)-â-D-glucopyranoside (osmanthuside H), calceolari-
Journal of Natural Products, 2007, Vol. 70, No. 5 821
1
4
Plant Material. Stem bark of F. sieboldiana (20 kg) was collected
at Lu Mountain, Jiangxi Province, China, in August 2004. Plant
identification was verified by Prof. Lin Ma (Institute of Materia
Medica). A voucher specimen (No. ZH02272) was deposited at the
Herbarium of the Department of Medicinal Plants, Institute of Materia
Medica, Beijing 100050, China.
25
26
27
osides A and B, chiritoside C, ferruginoside A, acteoside,
28
plantasioside; 2,6-dimethoxy-p-hydroxyquinone 1-O-â-D-glucopy-
15
ranoside, 2,6-dimethoxy-p-hydroxyquinone 4-O-â-D-glucopyra-
noside,29 4-hydroxy-3-methoxyphenyl â-D-glucopyranoside, 4-hy-
droxy-3-methoxyphenyl â-D-xylopyranosyl-(1f6)-O-â-D-gluco-
pyranoside;30 linarionoside B, (9S)-linarionoside B,31,32 and (3R,9R)-
Extraction and Isolation. The air-dried stem bark of F. sieboldiana
20 kg) was powdered and extracted with 11.0 L of 95% EtOH at room
(
3
-hydroxy-7,8-dihydro-â-ionol 9-O-â-D-apiofuranosyl-(1f6)-â-D-
temperature for 3 × 48 h. The EtOH extract was evaporated under
33
29,33
glucopyranoside. (In the literature,
used.)
different nomenclature was
reduced pressure to yield a residue (428.6 g). The residue was suspended
in H O (1500 mL) and then partitioned with EtOAc (5 × 1000 mL).
2
The aqueous phase was applied to a HDP100 macroporous adsorbent
Threo- and erythro-arylglycerols either in optically pure forms
2
resin (1000 g) column. Successive elution 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 (82.2 g)
or enantiomeric mixtures have been reported from several
plants,1
5,17,20,23,24,34-36
and coupling constants of the deshielded
1
benzylic proton (H-7) signal in the H NMR spectra of their acetates
were used to distinguish threo (J7,8 > 7.0 Hz) and erythro (J7,8
eluted by H
2
O was separated by MPLC over reversed-phase silica gel
O to give
<
eluting with a gradient of increasing MeOH (0-50%) in H
2
0,23,34,36
13
6
.5 Hz) isomers.2
A systematic analysis of the C NMR
four fractions (A-D) on the basis of TLC analysis. Separation of
fraction B (5.52 g) on normal silica gel CC, eluting with a gradient of
increasing MeOH (0-100%) in CHCl , afforded five fractions (B -
data of the reported threo- and erythro-arylglycerols in different
solvents indicated that the chemical shift difference of C-7 and C-8
3
1
(∆δC8-C7) of the threo- and erythro-arylglycerols may be directly
B
5
). Fraction B
3
(1.98 g) was subjected to CC over Sephadex LH-20,
O (70:30) as the eluting solvent, to give three
subfractions (B3-1-B3-3). Subfractions B3-2 (0.32 g) and B3-3 (0.24
g) were separately purified by reversed-phase preparative HPLC, using
applicable to distinguish threo- and erythro-arylglycerols without
substituent(s) at C-7 or/and C-8 of the glycerol moiety. In order to
confirm the validity of the ∆δC8-C7 for distinguishing threo- and
using MeOH-H
2
MeOH-H
and 6 (56.9 mg). Fraction B
Sephadex LH-20 eluting with MeOH and then separated by reversed-
phase preparative HPLC, using MeCN-H O (0.8:99.2), to afford 7
12.1 mg), 8 (7.3 mg), and 9 (11.0 mg).
The fraction eluted by 30% EtOH (73.0 g) was subjected to MPLC
over reversed-phase silica gel (C-18), with a gradient of increasing
2
O (20:80), to afford 3 (85.0 mg), 4 (61.6 mg), 5 (73.0 mg),
erythro-arylglycerols, the 13C NMR data of 7-9 in DMSO-d
,
,
6
4
(0.61 g) was chromatographed over
pyridine-d
MeOH-d , and Me
C8-C7 values of erythro-arylglycerols 7 and 7a in the tested
5
, and D
2
O, as well as 7a-9a in DMSO-d
6
, pyridine-d
5
4
2
CO-d
6
, were obtained. Without exception, the
2
∆δ
(
solvents were smaller than those of threo-arylglycerols 8, 9, 8a,
and 9a (Tables 1 and 2, Supporting Information), which were
consistent with literature reports.
MeOH (0-100%) in H
2
O, to give five fractions (E-I). Fraction G
(
2
1.25 g) was subjected to CC over Sephadex LH-20 (MeOH) to give
Compounds 1-9 were tested for their TNF-R secretion inhibitory
(75.2 mg). Fraction H (2.15 g) was chromatographed over silica gel,
, to afford
(0.42 g) was purified by reversed-
activities of mouse peritoneal macrophages, as well as antioxidant
with a gradient of increasing MeOH (10-50%) in CHCl
3
+2
activities inhibiting Fe -cystine-induced rat liver microsomal lipid
subfractions H . Subfraction H
1
-H
3
3
-
5
peroxidation. At a concentration of 10 M, compounds 2-9
phase preparative HPLC, using MeCN-H
2
O (16:84), to yield 1 (3.1
showed inhibition rates of 30.8%, 25.2%, 16.3%, 44.3%, 28.8%,
mg).
26.6%, 27.5%, and 28.2%, respectively, to TNF-R secretion of
20
Fraxinuacidoside (1): colorless gum; [R]D -17.5 (c 0.52, MeOH);
UV (MeOH) λmax (log ꢀ) 215 (1.9), 279 (sh); IR (KBr) νmax 3389, 2921,
mouse peritoneal macrophages. The inhibition rate of 5 was higher
than the positive control indomethacin, which gave an inhibition
rate of 33.2% at the same concentration. Compound 6 showed
antioxidant activity with an IC50 value of 0.9 µM, which was
stronger than the positive control vitamin E, with an IC50 value of
2853, 1747, 1654, 1573, 1484, 1364, 1077, 1038 cm-1
1
;
H NMR
(MeOH-d
4
, 400 MHz) and 13C NMR (MeOH-d
4
, 100 MHz) data, see
Table 1; ESIMS m/z 505 [M + Na] ; HRESIMS m/z 505.2386 [M +
+
+
Na] (calcd for C25
-[6-(4-O-â-D-Glucopyranosyloxy-3-methoxybenzoyl)]-O-â-D-glu-
copyranosyloxy-7-hydroxycoumarin (2): yellowish, amorphous pow-
38 9
H O Na, 505.2414).
6
4.6 µM, while others gave IC50 values larger than 10 µM. In
addition, in the in Vitro cytotoxic assay against human cancer cell
lines including ovary (A 2780), colon (HCT-8), hepatoma (Bel-
2
D
0
der; [R] -82.2 (c 0.30, DMSO); UV (MeOH) λmax (log ꢀ) 226 (3.9),
2
1
(
56 (3.8), 296 (2.9), 336 (3.2) nm; IR (KBr) νmax 3396, 2908, 1712,
7
402), stomach (BGC-823), and lung (A549), plantasioside (10)
showed selective activity against the human colon cancer cell line
HCT-8) with an IC50 value of 3.4 µM. The other compounds were
-
1 1
612, 1570, 1510, 1419, 1296, 1277, 1219, 1074, 760 cm ; H NMR
DMSO-d
6
, 500 MHz) and 13C NMR (DMSO-d
6
, 125 MHz) data, see
(
-
+
Tables 2 and 3; ESIMS m/z 651 [M - H] , 675 [M + Na] , and 691
inactive to all tested cell lines (IC50 > 5 µM).
+
+
[
M + K] ; HRESIMS m/z 675.1547 [M + Na] (calcd for C29
Na, 675.1537).
2,5-Di-O-â-D-glucopyranosyloxy-1,3-dimethoxybenzene (3): amor-
32 17
H O -
Experimental Section
2
D
0
phous powder; [R] -52.1 (c 0.08, H
2
O); UV (MeOH) λmax (log ꢀ)
General Experimental Procedures. Optical rotations were mea-
sured on a Rudolph Research Autopol III automatic polarimeter. IR
spectra were recorded as KBr disks on a Nicolet Impact 400 FT-IR
spectrophotometer. NMR spectra were obtained at 500 or 400 MHz
2
1
5
3
26 (3.9), 280 (sh) nm; IR (KBr) νmax 3398, 2939, 2893, 1601, 1508,
-
1 1
6
469, 1425, 1242, 1173, 1130, 1078, 816 cm ; H NMR (DMSO-d ,
00 MHz) and 13C NMR (DMSO-d
, 125 MHz) data, see Tables 2 and
6
+
+
; ESIMS m/z 517 [M +Na] ; HRESIMS m/z 517.1547 [M + Na]
1
13
for H and 125 or 100 MHz for C, respectively, on Inova 500 and
(
calcd for C20
30
H O14Na, 517.1533).
4
00 MHz spectrometers in DMSO-d
6
, MeOH-d
4
, pyridine-d
5
, Me
2
CO-
1
,4-Di-O-â-D-glucopyranosyloxy-2-methoxybenzene (4): amor-
d
6
, or D O with solvent peaks (or TMS, in the case of D
2
2
O) being
20
phous powder; [R]D -25.7 (c 0.09, H
2
O); UV (MeOH) λmax (log ꢀ)
used as references. ESIMS data were measured with a Q-Trap LC/
MS/MS (Turbo ionspray source) spectrometer. HRESIMS data were
measured using an AccuToFCS JMS-T100CS spectrometer. Column
chromatography was performed using silica gel (200-300 mesh,
Qingdao Marine Chemical Inc., China) and Sephadex LH-20 (Phar-
macia 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 with an Alltima
2
1
30 (3.8), 280 (sh) nm; IR (KBr) νmax 3402, 2927, 1597, 1506, 1466,
423, 1333, 1238, 1128, 1074 cm ; H NMR (DMSO-d
-1
1
6
, 500 MHz)
13
and C NMR (DMSO-d
6
, 125 MHz) data, see Tables 2 and 3; ESIMS
+
+
m/z 487 [M + Na] ; HRESIMS m/z 487.1446 [M + Na] (calcd for
13Na, 487.1428).
-(4-Hydroxy-3-methoxyphenyl)ethanol 1-O-[â-D-apiofuranosyl-
1f6)-â-D-glucopyranoside] (5): colorless gum; [R]D -61.8 (c 0.05,
MeOH); UV (MeOH) λmax (log ꢀ) 226 (3.8), 280 (3.4) nm; IR (KBr)
νmax 3392, 2935, 2883, 1604, 1518, 1273, 1045, 822 cm-1
H NMR
(DMSO-d , 500 MHz) and C NMR (DMSO-d , 125 MHz) data, see
19 28
C H O
2
20
(
(
250 × 10 mm) preparative column packed with C18 (5 µm). TLC was
carried out on precoated silica gel GF254 plates. Spots were visualized
under UV light (254 or 356 nm) or by spraying with 7% H SO in
5% EtOH followed by heating.
;
1
13
6
6
+
+
2
4
Tables 2 and 3; ESIMS m/z 485 [M + Na] and 501 [M + K] ;
+
9
30
HRESIMS m/z 485.1650 [M + Na] (calcd for C20H O12Na, 485.1635).

8
22 Journal of Natural Products, 2007, Vol. 70, No. 5
-(3,4-Dihydroxyphenyl)ethanol 1-O-[â-D-apiofuranosyl-(1f6)-
Lin et al.
2
Institute of Experimental Animal, Chinese Academy of Medical
Sciences & Peking Union Medical College), 4 days after the injection
(i.p.) of Brewer’s thioglycollate medium, washed twice with D-Hank’s
buffer, and resuspended in RPMI-1640 (Gibco/BRL, Gaithersburg, MD)
2
0
â-D-glucopyranoside] (6): colorless gum; [R] -53.5 (c 0.54, MeOH);
D
UV (MeOH) λmax (log ꢀ) 228 (3.6), 278 (3.1) nm; IR (KBr) νmax 3388,
-
1 1
2
933, 2885, 1606, 1527, 1282, 1049 cm ; H NMR (DMSO-d
6
, 500
1
3
6
MHz) and C NMR (DMSO-d
ESIMS m/z 471 [M + Na] and 487 [M + K] ; HRESIMS m/z
4
6
, 125 MHz) data, see Tables 2 and 3;
at 10 cell/mL. The macrophage cells were plated in 48-well tissue
+
+
5
culture plates at 2 × 10 cells per well and incubated at 37 °C in 5%
+
71.1498 [M + Na] (calcd for C19
28
H O12Na, 471.1478).
2
(v/v) CO for 2 h, the medium was removed, and the cells were then
washed twice with D-Hank’s buffer to remove cells not adhered to the
well wall. After RPMI 1640 containing test compounds at a final
concentration of 10 M, or stimulator lipopolysaccharide (LPS, 1 µg/
mL), and 5% fetal calf serum was supplemented, the adhered mac-
(
-)-(7R,8S)-erythro-1-C-Syringylglycerol 4-O-â-D-glucopyrano-
2
0
side (7): amorphous powder; [R] -32.7 (c 0.28, H
2
O); UV (MeOH)
D
-5
λ
1
5
max (log ꢀ) 215 (3.3), 230 (sh) nm; IR (KBr) νmax 3277, 2943, 2896,
-
1
1
661, 1601, 1512, 1461, 1249, 1126, 1027, 839 cm ; H NMR (D
2
O,
1
3
2
rophage cell line was incubated at 37 °C in 5% (v/v) CO for 24 h.
2
00 MHz) and C NMR (D O, 125 MHz) data, see Tables 2 and 3;
+
+
The supernatant was collected and kept for later use.
ESIMS m/z 429 [M + Na] ; HRESIMS m/z 429.1379 [M + Na] (calcd
for C17 11Na, 429.1373).
-)-(7R,8R)-threo-1-C-Syringylglycerol 4-O-â-D-glucopyranoside
5
L929 cells (200 µL, 10 cells/mL) were inoculated in 96-well plates
and incubated at 37 °C in 5% CO for 24 h, and the supernatant was
2
removed. Then 100 µL of RPMI 1640 containing actinomycin D (0.5
µg/mL) was supplemented, and the supernatant was prepared as
described above or RPMI 1640 was added. After incubation at 37 °C
26
H O
(
2
0
(
8): amorphous powder; [R] -28.2 (c 0.40, H
2
O); UV (MeOH) λmax
D
(log ꢀ) 215 (3.2), 230 (sh) nm; IR (KBr) νmax 3370, 2958, 2921, 1596,
-
1 1
1
504, 1465, 1421, 1335, 1239, 1132, 1066, 834 cm ; H NMR (D
2
O,
in 5% CO for 20 h, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
2
1
3
5
00 MHz) and C NMR (D
2
O, 125 MHz) data, see Tables 2 and 3;
diphenyltetrazolium bromide (MTT) (5 mg/mL) was added to the wells,
and incubation continued for an additional 4 h. The supernatant was
removed, and the cells were decomposed with DMSO (100 µL) for 10
min. Absorbance was measured at 570 nm using a MK 3 Wellscan
+
+
ESIMS m/z 429 [M + Na] and 445 [M + K] .
-)-(7S,8S)-threo-1-C-Syringylglycerol 4-O-â-D-glucopyranoside
(
2
0
(
9): amorphous powder; [R] -21.1 (c 0.47, H
2
O); UV (MeOH) λmax
D
(log ꢀ) 215 (3.3), 230 (sh) nm; IR (KBr) νmax 3377, 2959, 2842, 1645,
(
Labsystem Drogon) plate reader. Each value is the mean of reactions
-
1 1
1
597, 1504, 1465, 1422, 1335, 1240, 1131, 1065, 1005, 835 cm ; H
in three wells for a single compound. The absorbance of the wells with
only RPMI 1640 added was used as blank. The percent TNF-R secretion
inhibition of peritoneal macrophages was calculated as follows, by using
a MS Excel 2000 (Microsoft Corp.) based program developed for this
purpose:
13
2 2
NMR (D O, 400 MHz) and C NMR (D O, 100 MHz) data, see Tables
+
+
2
and 3; ESIMS m/z 429 [M + Na] and 445 [M + K] .
Enzymatic Hydrolysis of 1. Compound 1 (3.0 mg) was hydrolyzed
with 7.0 mg of â-glucosidase (Almonds Lot 1264252, Sigma-Aldrich)
in 1.5 mL of H O at 37 °C for 12 h. After removal of solvent under
reduced pressure, the residue was extracted with MeCN, and the MeCN
extract was chromatographed over silica gel, eluting with CHCl
MeCN (25:1), to give 1a (1.6 mg), and then eluting with CHCl
2
percent inhibition ) [(L - S)/(L - B)] × 100
3
-
3
-
Here L, S, and B are the absorbances for the stimulator LPS, test
samples, and blank, respectively. Indomethacin was used as the
reference compound.
Antioxidative Activity Assay. Antioxidative activity was evaluated
as the inhibitory activity of compounds against lipid peroxidation in
rat liver microsomes according to a modified thiobarbituric acid (TBA)
method.40 In the TBA assay, microsomes were isolated from rat livers
and suspended in 100 mM Tris-HCl buffer (pH 7.4). The microsomal
suspension (1 mg protein/mL), different concentrations of compound
or vehicle, and 0.2 mM cysteine in 0.1 M PBS (pH 7.4) were incubated
2
D
0
MeCN (3:1) to yield a sugar with [R] +46.2 (0.11, H
1
2
O). Compound
a was a colorless gum: [R] -68.2 (c 0.36, MeOH); 1H NMR
D
13
2
0
4 4
(MeOH-d , 400 MHz) and C NMR (MeOH-d , 100 MHz) data, see
-
+
Table 1; ESIMS m/z 319 [M - H] , 343 [M + Na] .
Acidic Hydrolysis of 2. Compound 2 (6.6 mg) was refluxed in 2 N
HCl (5.0 mL) at 80 °C for 3 h. The reaction mixture was extracted
with CH
N NaOH and dried using a stream of N
to CC over silica gel with CHCl -MeCN (3:1) to yield a sugar (3.2
O, 24 h after being dissolved in the
3
Cl (3 × 5 mL), and the aqueous phase was neutralized with
1
2
. The residue was subjected
3
2
D
0
at 37 °C for 15 min, 50 µM FeSO
was then incubated at 37 °C for 15 min again. An equal volume of
0% TCA was added to terminate the reaction, and the mixture was
4
was added, and the reaction mixture
mg), [R] +40.2 (0.45, H
2
solvent).
2
Enzymatic Hydrolyses of 3, 4, and 7-9. A solution of each
compound in H O (3 mL) was individually hydrolyzed with â-glu-
cosidase (10 mg) at 37 °C for 16 h. The reaction mixtures of 3 and 4
were extracted separately with CH Cl extracts
Cl (3 × 3 mL). The CH
were chromatographed over silica gel, eluting with CH Cl-MeCN (100:
), for the hydrolyzates from 3 (10.2 mg) and 4 (10.8 mg), to yield
-hydroxy-2,6-dimethoxyphenol (3.3 mg) and 1,4-dihydroxy-2-meth-
centrifuged at 3000g for 10 min. The supernatant (1 mL) was mixed
with 0.67% (w/v) TBA and kept in a boiling water bath for 10 min.
After cooling, lipid peroxidation was assessed by measuring the
thiobarbituric acid reactive product at 532 nm. Lipid peroxidation
inhibitory activity was calculated as follows: [1 - (T- B)/(C - B) ]
100 (%), in which T, C, and B are absorbance values of the sample
treated, the control without sample, and the zero time control,
respectively. Vitamin E was used as the positive control.
2
3
3
3
1
4
×
oxyphenol (3.5 mg), respectively. The aqueous phases of the hydro-
lyzates of 3 and 4 were dried using a stream of N and then subjected
to CC over silica gel eluted with CHCl -MeCN (3:1) to yield glucose
4.3 mg) from 3, [R] +46.6 (c 0.43, H O), and glucose (5.1 mg)
O). The hydrolyzates of 7-9 were
dried under reduced pressure, and the residues were chromatographed
over silica gel, eluting with CH Cl-MeCN (25:1), to yield 7a (5.1
mg), 8a (3.6 mg), and 9a (4.2 mg), and then eluting with CHCl
MeCN (3:1) to give a sugar, respectively, from the hydrolyzates of 7
2
3
Acknowledgment. Financial support was provided by the New
Century Excellent Talent (NCET) Program of the Chinese Ministry of
Education, the Natural Sciences Foundation of China (NSFC, Grant
No. 20432030), the National “973” Program of China (Grant No.
2
0
(
2
D
2
D
0
from 4, [R] +48.6 (c 0.51, H
2
3
2
004CB13518906), and the Program for Changjiang Scholars and
3
-
Innovative Research Team in University (PCSIRT, Grant No. IRT0514).
We thank Professors G. T. Liu, X. G. Chen, and G. F. Chen for the
bioassays.
1
(9.6 mg), 8 (6.2 mg), and 9 (7.8 mg). For the H NMR (500 or 400
1
3
MHz) and C NMR (125 or 100 MHz) data of 7a, 8a, and 9a in
different solvents, see Tables 2 and 4 in the Supporting Information.
The optical rotations of the sugar obtained from 7, 8, and 9 were
Supporting Information Available: 1D NMR spectra of com-
pounds 1-9 and 1a; SI Tables 1-4 of the NMR data of 7-9 and 7a-
9a in different solvents. This material is available free of charge via
the Internet at http://pubs.acs.org.
2
D
0
20
d
20
[
(
R] +39.8 (c 0.55, H
2
O), [R] +41.0 (c 0.38, H
2
O), and [R]d +38.9
2
c 0.48, H O), respectively. The optical rotations of the sugars were
measured after the samples were dissolved in H
systems CHCl
n-BuOH-AcOH-H O (4:1:5) for PC were used in glucose identifica-
2
tion.
2
O for 24 h. Solvent
-MeOH (2.5:1) for TLC and the upper layer of
3
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