Phenylpropanoids from the stems of Ephedra sinica

Article abstract of DOI:10.1080/10286020.2015.1070831

Two new compounds of phenylpropanoids, (S)-N-((1R,2S)-1-hydroxy-1-phenylpropan-2-yl)-5-oxopyrrolidine-2-carboxamide (1) and (3R)-3-O-β-d-glucopyranosyl-3-phenylpropanoic acid (2), were isolated from the stems of Ephedra sinica. Their structures were elucidated by in-depth examination of spectroscopic data, mainly including those from the 1D and 2D NMR and HRESIMS techniques, and chemical method. The absolute configurations of the two compounds were also corroborated through CD procedure.

Full text of DOI:10.1080/10286020.2015.1070831

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Phenylpropanoids from the stems of Ephedra
sinica
Dan Zhang, An-Jun Deng, Lin Ma, Zhi-Hong Li, Zhi-Hui Zhang, Jian-Dong Jiang
&
Hai-Lin Qin
To cite this article: Dan Zhang, An-Jun Deng, Lin Ma, Zhi-Hong Li, Zhi-Hui Zhang, Jian-Dong
Jiang & Hai-Lin Qin (2015): Phenylpropanoids from the stems of Ephedra sinica, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2015.1070831
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Date: 04 November 2015, At: 16:41

Journal of Asian Natural Products Research, 2015
http://dx.doi.org/10.1080/10286020.2015.1070831
Phenylpropanoids from the stems of Ephedra sinica
a
Dan Zhang , An-Jun Deng , Lin Ma , Zhi-Hong Li , Zhi-Hui Zhang , Jian-Dong Jiang
a
a
a
a
a,b
a
and Hai-Lin Qin *
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
b
00050, China; Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China
1
(Received 27 March 2015; final version received 6 July 2015)
Two new compounds of phenylpropanoids, (S)-N-((1R,2S)-1-hydroxy-1-phenylpro-
pan-2-yl)-5-oxopyrrolidine-2-carboxamide (1) and (3R)-3-O-b-D-glucopyranosyl-
3-phenylpropanoic acid (2), were isolated from the stems of Ephedra sinica. Their
structures were elucidated by in-depth examination of spectroscopic data, mainly
including those from the 1D and 2D NMR and HRESIMS techniques, and chemical
method. The absolute configurations of the two compounds were also corroborated
through CD procedure.
Keywords: Ephedra sinica; Ephedraceae; amphetamine-type alkaloid; glycoside of
phenylpropanoic acid
1
.
Introduction
investigation on literatures, that there were
only limited amphetamine-type alkaloids
from the Ephedra species [7,8], of which,
Ephedra sinica Stapf. (Ephedraceae) is one
of the original plants of Ephedrae Herba
that was recorded in the Chinese Pharma-
copoeia as “Ma Huang.” Just like the other
two original plants, E. intermedia Schrenk
et C. A. Mey. and E. equisetina Bunge, the
stem of E. sinica has been widely used to
treat rheums, asthma, and cough with
dyspnea, inter alia, in traditional Chinese
medicine (TCM) for thousands of years
(
^)-2-imino-1-phenylpropan-1-ol was
reported by our laboratory for the first
time [9]. On the other hand, there were not
many literatures about the NMR studies on
the isolated amphetamine-type alkaloids
yet. In our ongoing search for new
amphetamine-type alkaloids, including
amphetamine-type compounds with alka-
lescence, from Ephedra species, a new
amide of amphetamine-type was isolated
from the neural n-BuOH-soluble fraction
of the ethanol extract from the stem of
E. sinica. In the meantime, one new
glycoside of phenylpropionic acid was
also isolated from the same neural fraction.
In an exact sense, both new compounds
belong in phenylpropanoids because of
their shared propylbenzene core. This
paper deals mainly with the isolation and
structural elucidation of these new
compounds.
[1,2]. It has been reported that E. sinica
contains amphetamine-type alkaloids and
flavonoids as the most important and
accepted constituents in pharmaceutics
and chemotaxonomy. In general, the
amphetamine-type alkaloids were illus-
trated to bear some interesting bioactiv-
ities. For instance, the most famous l-
ephedrine and its analogs isolated from the
title plant have been applied to treat
hypotension, obesity, asthma, and allergic
rhinitis [3–6]. and so on, clinically.
However, it was also found, through the
*Corresponding author. Email: qinhailin@imm.ac.cn
q 2015 Taylor & Francis

2
D. Zhang et al.
1
showed that no C NMR signals corre-
3
2
.
Results and discussion
1
lated to the H NMR signals at dH 7.83,
The neutral n-BuOH-soluble section of the
ethanol extract from the stems of E. sinica
was subjected to multiple column chro-
matography (CC) and further purified by
preparative HPLC procedure, affording
two new compounds (1 and 2) reported
herein (Figure 1).
7.70, and 5.43. Also, an AA’BB’X-type
coupling system of ZCHZCH ZCH Z
2
2
was determined, in combination with the
1
1
1
H, HCOSY experiment, by a set of H
NMR signals at d 1.48–1.50 and 2.04–
0
0
2
.06 (1 H each, 2 £ m, H -4 ), 1.98 (2 H,
2
0
0
dd, J ¼ 10.5, 6.5 Hz, H -3 ), and 3.92–
Compound 1 was obtained as a white
amorphous powder, with ½aꢀ 2 29.6 (c
2
0
0
13
2
D
0
3.96 (1 H, m, H-5 ). The
C NMR
spectrum of 1 exhibited 14 carbon signals,
which were classified by DEPT exper-
iment into one methyl, two methylenes,
eight methines including two pairs of
aromatic carbons showing the uniform
0
C H N O , was assigned by C NMR
.61, CH OH). Its molecular formula,
3
1
3
1
4 18 2 3
data and protonated ion at m/z 263.1392
þ
[
M þ H] in the HRESIMS measurement
of positive-ion mode, indicative of seven
degrees of unsaturation. The IR absorp-
tions at 3309, 3091, 1662, 1639, 1554, and
resonance frequencies at d 126.3 (d) and
C
127.7 (d), respectively, one quaternary
carbon, and two carbonyls at d 171.2 and
2
1
13
C
1
NMR spectrum, indicated the existence of
452 cm , in conjunction with the
C
1
77.3 (Table 1). Considering the molecu-
1
hydroxyl, amide, and aryl groups. The H
lar formula of seven unsaturation degrees,
and in association with the HSQC
spectrum, the HMBC experiment unam-
biguously established the AA’BB’X-type
coupling system as a 5-oxopyrrolidine-2-
carboxamide moiety by the correlations
NMR spectrum revealed the diagnostic
signals of the alkaloid of 2-amino-1-
hydroxy-1-phenylamphetamine-type, with
a monosubstituted benzene ring and a 2-
amino-1-hydroxy-1-phenylsubstituted
propane being exhibited by the signals at d
7
7
4
0
0
0
00
00
00
00
00
00
from NH-1 to C-2 , C-3 , C-4 , and C-5 ,
00
0
0
0
0
0
from H -3 to C-2 , C-4 , and C-5 , from
2
.28–7.32 (4 H, m, H-2 , 3 , 5 , 6 ), 7.20–
0
0
0
00
00
H -4 to C-2 and C-6 , and from NH-2 to
2
.23 (1 H, m, H-4 ), 5.43 (1 H, br s, OH),
.50 (1 H, d, J ¼ 5.5 Hz, H-1), 3.86–3.92
0
0
C-6 (Figure 2), which also suggested that
the 2D structure of 1 was N-(1-hydroxy-1-
phenylpropan-2-yl)-5-oxopyrrolidine-2-
carboxamide. With this constitution estab-
lished by critical NMR examination, the
next consideration was to elucidate the
absolute configurations of C-1 and C-2 of
the 1-hydroxy-1-phenylpropan-2-yl and
(
1 H, m, H-2), and 0.98 (3 H, d, J ¼ 7.0 Hz,
H-3), which is very consistent with those
of the known alkaloids of this kind [10].
In addition to the hydroxyl group, two
other labile protons at d 7.83 (1 H, d,
J ¼ 8.5 Hz, NH-2) and 7.70 (1 H, br s, NH-
0
0
1
1
) were also exhibited in the H NMR
0
0
spectrum. All three labile protons were
confirmed by HSQC experiment which
C-5 of the 5-oxopyrrolidine-2-carboxa-
mide moieties. The CD spectrum of
OH
3
''
2
4
''
O 1''
O
3
OH
C1 OOH
H
HO
HO
2
'
5''
O
2''
OH
6'
1'
1
'
2
3
N
1
a
1'' N
''
3'
6
2'
3'
H
4'
O
6
'
5'
5'
4'
1
2
Figure 1. Structures of compounds 1 and 2.

Journal of Asian Natural Products Research
3
1 13
Table 1. H (500 MHz) and C (125 MHz) NMR spectroscopic data for 1 and 2 (in DMSO-d6).
No.
1
2
a
dH (mult , J in Hz)
b
d C (dept )
dH (mult, J in Hz)
d C (dept)
1
2
4.50 d (5.5)
3.86–3.92 m
74.9 d
50.0 d
172.4 s
41.2 t
a 2.93 dd (16.0, 7.5)
b 2.66 dd (16.0, 7.5)
5.06 t (7.5)
3
0.98 d (7.0)
5.43 br s
7.83 d (8.5)
14.9 q
76.2 d
OH-1
NH-2
0
1
143.3 s
126.3 d
127.7 d
126.7 d
127.7 d
126.3 d
141.4 s
126.9 d
127.9 d
127.4 d
127.9 d
126.9 d
101.6 d
73.7 d
0
2
7.28–7.32 m
7.28–7.32 m
7.20–7.23 m
7.28–7.32 m
7.28–7.32 m
7.70 br s
7.38 d (7.5)
7.32 t (7.5)
7.26 t (7.5)
7.32 t (7.5)
7.38 d (7.5)
4.34 d (8.0)
2.94–2.97 m
3.01–3.15 m
3.01–3.15 m
0
3
0
4
0
5
0
6
0
0
0
0
0
0
0
0
1
2
3
4
177.3 s
29.1 t
25.1 t
1.98 dd (10.5, 6.5)
a 2.04–2.06 m
b1.48–1.50 m
3.92–3.96 m
76.6 d
70.0 d
0
0
0
0
5
6
55.4 d
171.2 s
3.01–3.15 m
3.60 dd (12.5, 4.5)
76.9 d
61.0 t
3
.36–3.40 m
a
b
Notes: multiplicity; DEPT NMR.
1
2
displayed a positive Cotton effect at
18 nm (log D1 þ 5.03) (Figure 3),
Compound 2 was obtained as a white
amorphous powder, and it has
00
20
D
13
demonstrating the 5 S configuration [11–
3]. The coupling constant of
J1,2 ¼ 5.5 Hz suggested that 1 bore the
,2-erythro configuration, which was
consistent with the determination of 1R,
S configuration through another positive
½aꢀ 2 19.4 (c 0.112, MeOH). The
2
C
NMR data and the deprotonated ion at m/z
1
327.1087 [M 2 H] in the HRESIMS
1
experiment of negative-ion mode indi-
cated the molecular formula of C H O .
The IR spectrum suggested the existence
1
5 20 8
2
2
1
Cotton effect at 258 nm (log D1 þ 3.68) in
of hydroxyl (3452 cm ), carbonyl
2
1
(1705 cm ), and aromatic groups
the CD spectrum [10,14,15]. Accordingly,
1
2
(1636 cm ). Especially, the broad peak
1
was determined as (S)-N-((1R,2S)-1-
2
1
21
hydroxy-1-phenylpropan-2-yl)-5-oxopyr-
rolidine-2-carboxamide, and was given the
trivial name sinicine.
between 2400 cm and 3400 cm in the
IR spectrum indicated the existence of
1
13
carboxy. The H and C NMR spectra, in
OH
O
O
OH
H
HO
HO
COOH
O
OH
N
N
H
O
1
2
Figure 2. Key HMBC (→) correlations of 1 and 2.

4
D. Zhang et al.
Figure 3. CD spectra of 1.
1
the C NMR signal at d 172.4 (s, C-1)
3
conjunction with the DEPT and HSQC
experiments, showed up a monosubsti-
tuted benzene ring by the resonances at d /
C
matched the determination of carboxylic
acid. With the elucidation of the structural
moieties accomplished, the 2D structure of
2 was established as 3-O-b-D-glucopyr-
anosyl-3-phenylpropanoic acid by the
HMBC experiment which exhibited the
H
0
d 7.26 (1 H, t, J ¼ 7.5 Hz, H-4 )/127.4 (d,
C
0
0
C-4 ), 7.32 (2 H, t, J ¼ 7.5 Hz, H-3 ,
0
0 0
5
)/127.9 (d, C-3 , 5 ), 7.38 (2 H, d,
0
0
0
0
J ¼ 7.5 Hz, H-2 , 6 )/126.9 (d, C-2 , 6 ),
0
and d 141.4 (s, C-1 ). An aliphatic ABX
C
correlations from the oxymethine at d
0 0 0
H
coupling system was also determined by a
set of resonances at d /d 2.66 (1 H, dd,
5.06 (H-3) to C-1, C-2, C-1 , C-2 /6 , and
0
0
00
C-1 , from H-1 to C-3, from H-2a and
0
H
C
J ¼ 16.0, 7.5 Hz, H-2a) and 2.93 (1 H, dd,
J ¼ 16.0, 7.5 Hz, H-2b)/41.2 (t, C-2) and
H-2b to C-1, C-3, and C-1 , and the like
(Figure 2). Now that the NMR technique,
as well as acid hydrolysis, had served its
intended purpose, we sought to address the
issue of elucidating the absolute configur-
ation of C-3. Calculation of electronic
circular dichroism (ECD) using time-
dependent density functional theory
(TDDFT) [17], in conjunction with the
experimental ECD data, has been success-
fully applied to determine the absolute
configuration of compounds in recent
years. In the case of 2, the calculated
ECD value for the 3R configuration is log
D1 þ 8.13 at 212.5 nm, a value closely
comparable to the log D1 þ 2.68 at
212.5 nm of experimental ECD value, as
5
.06 (1 H, t, J ¼ 7.5 Hz, H-3)/76.2 (d, C-3)
(Table 1). One hexopyranse moiety was
detected by the resonances for one
anomeric oxymethine at d /d 4.34 (1 H,
H
C
0
0
00
d, J ¼ 8.0 Hz, H-1 )/101.6 (d, C-1 ), one
oxymethylene at d /d 3.60 (1 H, dd,
H
C
00
J ¼ 12.5, 4.5 Hz, H-6 a) and 3.36–3.40
0
0
00
(1 H, m, H-6 b)/61.0 (t, C-6 ), and four
other oxymethines at d 2.94–3.15 (4 H,
H
0
0
00
00
00
m, H-2 , 3 , 4 , 5 ) as multiplets and at d
C
00 00
7
4
2
3.7 (d, C-2 ), 76.6 (d, C-3 ), 70.0 (d, C-
0
0
00
), and 76.9 (d, C-5 ). Acid hydrolysis of
afforded D-glucose, which was affirmed
by derivatization and GC analysis, as
described in section “3.5 [16]. In addition,
00

Journal of Asian Natural Products Research
5
Figure 4. Calculated and experimental ECD spectra of 2((1) calculated in methanol; (2)
experimental in methanol; (3) calculated in methanol for the S configuration).
shown in Figure 4. Thus, the structure of 2
was characterized as (3R)-3-O-b-D-gluco-
pyranosyl-3-phenylpropanoic acid.
Pack ODS-A 20 £ 250 mm, 5 mm, YMC
CO., Kyoto, Japan) was employed. GC
analysis was undertaken on an Agilent
7
890A instrument (Agilent, Santa Clara,
CA, USA). CC was conducted over silica
gel (200–300 mesh, Qingdao Marine
Chemical Inc. Qingdao, China) or Sepha-
dex LH-20 (Pharmacia Fine Chemicals,
Uppsala, Sweden), and thin layer chroma-
tography (TLC) over glass plate pre-
coated silica gel GF254. Spots were
visualized under UV light of 254 nm and
360 nm, simultaneously, and by spraying
3
.
Experimental
3
.1 General experimental procedures
Optical rotations were measured on a
Perkin 2 Elmer 241 digital polarimeter
(
JASCO, Tokyo, Japan) at a temperature
of 208C. UV spectra were obtained on a
JASCO V-650 spectrophotometer
JASCO, Tokyo, Japan), and IR (KBr)
(
spectra on a Nicolet 5700 infrared
spectrometer (Thermo Nicolet, Waltham,
MA, USA). CD spectra were taken using a
JASCO J-815 CD spectrometer (JASCO,
Tokyo, Japan). 1D and 2D NMR spectra
were recorded on a Bruker Avance-III 500
NMR (Bruker Instruments Inc., F a¨ llanden,
Switzerland) spectrometer, using dimethyl
sulfoxide-d (DMSO-d ) as solvents and
with 10% H SO in 95% EtOH, followed
2 4
by heating. All solvents were of analytical
grade. L-Cysteine methyl ester hydrochlo-
ride, N-trimethylsilylimidazole, and auth-
entic D-glucose were purchased from
Sigma-Aldrich Chemical Co. (St Louis,
MO, USA).
6
6
3
.2 Plant material
tetramethylsilane (TMS) as internal stan-
dard. Both ESIMS and HRESIMS exper-
iments in negative or positive modes were
performed using an Agilent 1100 series
LC/MSD Trap SL mass spectrometer
The stems of Ephedra sinica were
obtained from Bozhou crude drug market,
Anhui Province, China, in December
2011, and was identified by Associate
Prof. Lin Ma (a savant in plant systematics
with Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking
Union Medical College) through careful
examination of the morphological features
(Agilent, Santa Clara, CA, USA). Prepara-
tive HPLC was carried out on a Shimadzu
LC-6AD system (Shimadzu, Kyoto,
Japan) equipped with a SPD-10A detector,
and a reversed-phase C18 column (YMC--

6
D. Zhang et al.
and according to the description for
E. sinica in the state Pharmacopoeia of
China [1]. All the experimental material
was carefully examined to ensure the
purity and a voucher specimen (ID-S-
(5 g, CHCl /MeOH ¼ 50:1) was separated
3
by CC over silica gel using an isocratic
elution of CHCl /MeOH ¼ 50:1 into three
3
subfractions (F3-2-1 to F3-2-3) according
to their TLC profiles. F3-2-2 (3 g) was
separated into three subfractions (F3-2-2-1
to F3-2-2-3) by silica gel CC using an
2
555) was deposited in the Herbarium of
Institute of Materia Medica, Chinese
Academy of Medical Sciences, Beijing,
China.
isocratic elution of EtOAc/Me CO ¼ 15:1
2
and TLC detection. F3-2-2-2 (100 mg) was
purified by preparative RP-HPLC [mobile
phase: CH OH/H O (23%, v/v); flow rate:
3
2
2
1
3
.3 Extraction and isolation
6
ml min ; UV detection at 280 nm] to
Dried and cut stems of E. sinica (19 kg)
were extracted for three times with 85%
aqueous (aq.) EtOH (2 h, 1 h, 1 h) under
reflux condition. After evaporation of the
aq. EtOH solvent under vacuum, a dark-
brown residue of 3.0 kg was obtained. The
residue was suspended in 80% EtOH in
water (8 L) and extracted in a separatory
funnel with petroleum ether (8 L each) for
several times until the upper fraction was
very transparent. The 80% aq. ethanol-
soluble section was evaporated under
vacuum condition to afford another dark-
brown residue, which was re-dissolved in
water (7 L), and then extracted in a
separatory funnel with EtOAc for three
times (7 L each). Next, the water layer was
further extracted in a separatory funnel
with n-BuOH (7 L each) for three times.
The combined n-BuOH solution was
extracted in a separatory funnel with the
afford 1 (2 mg). F3-7 (1 g, CHCl3/
MeOH ¼ 20:1) was submitted to Sepha-
dex LH-20 CC with MeOH as eluent to
yield four subfractions (F3-7-1 to F3-7-4)
according to their TLC profiles. 2 (10 mg)
was obtained from F3-7-2 by using
preparative RP-HPLC [mobile phase:
25% MeOH in H O, containing 0.1%
2
2
1
formic acid; flow rate: 6 mL min ; UV
detection at 280 nm].
3.3.1 (S)-N-((1R,2S)-1-Hydroxy-1-
phenylpropan-2-yl)-5-oxopyrrolidine-2-
carboxamide (1)
2
0
D
½aꢀ 2 29.6 (c 0.61, MeOH); CD (MeOH)
D1
(MeOH) l
þ 5.03, D1
þ 3.68; UV
2
18 nm
258 nm
(log 1) 204.8 (4.31), 229.2
max
(3.09), and 274.8 (2.13) nm; IR (KBr)
nmax3309, 3091, 2978, 1662, 1639, 1554,
1452, 1250, 1124, 1084, 1054, 968, 909,
2
1
1
13
solution of 5% NaHCO in water for three
3
760, and 700 cm
spectral data see Table 1; ESIMS(negative-
; H and C NMR
times (3 £ 1.5 L) and then pure H O (2
2
2
ion mode): m/z 261.5 [M 2 H] ; HRE-
£
1 L), respectively, to pH ¼ 7.0. The
neutral n-BuOH solution was evaporated
under vacuum condition to afford the
corresponding brown residue (220 g) of n-
BuOH extract.
SIMS (positive-ion mode): m/z 263.1392
þ
[M þ H]
(calcd for C H N O ,
14 19 2 3
263.1390).
The n-BuOH extract was separated, by
CC over silica gel using a gradient of
CHCl /MeOH (100:1–1:1) as eluent, into
3.3.2 (3R)-3-O-b-D-Glucopyranosyl-3-
phenylpropanoic acid (2)
3
2
0
seven fractions (designated as F1 to F7)
according to their TLC profiles. F3 (21 g,
½aꢀ 2 19.4 (c 0.112, MeOH); CD
D
(MeOH) D1
þ 2.68; UV (MeOH)
2
12.5 nm
CHCl /MeOH ¼ 10:1) was subjected to
lmax (log 1) 206.6 (3.75) and 258.8
(2.46) nm; IR (KBr) nmax 3452, 3400–
2400, 2951, 2921, 2877, 2665, 2598, 1705,
1636, 1452, 1277, 1208, 1086, 1018, 905,
3
silica gel CC and eluted with a gradient of
CHCl /MeOH (50:1 to 20:1), yielding
seven subfractions (F3-1 to F3-7). F3-2
3

Journal of Asian Natural Products Research
7
2
1
1
13
H and C NMR
7
spectral data see Table 1; ESIMS (nega-
54, and 701 cm
;
was dissolved in anhydrous pyridine
(1 ml). L-Cysteine methyl ester hydrochlo-
ride (2 mg) was then added, and the
reaction mixture was incubated at 608C
for 2 h. After the mixture was concentrated
to dryness under reduced pressure, 0.2 ml
of N-trimethylsilylimidazole was added,
and the mixture was further incubated at
2
tive-ion mode): m/z 327.1 [M 2 H] ;
HRESIMS (negative-ion mode): m/z
2
3
3
27.1087 [M 2 H] (calcd for C H O
15 19 8
27.1085).
3.4 Calculation procedure for CD
spectrum of 2
6
08C for 2 h. Then H O (2 ml) was added,
2
and the solution was extracted with hexane
for three times (2 ml each). The hexane
extract was subjected to GC system under
the following conditions to monitor:
capillary column: HP-5 (30 m £ 0.25 mm,
with a 0.25 mm film, Dikma); detection:
FID; detector temperature: 2808C; injec-
tion temperature: 2608C; initial tempera-
ture 168C, which was raised to 2808C at the
rate of 58C/min and the final temperature
was maintained for 10 min; Carrier: N2
gas. The authentic D-glucose was also
treated by the same procedure as that for 2.
From the acidic hydrolysate of 2, D-
glucose was confirmed by comparing the
retention time of the derivative with that of
authentic sugar, with the retention time of
D-glucose being 27.96 min.
The elucidation of the absolute configur-
ation of 2 was carried out via Monte Carlo
searching in the MMFF94 molecular
mechanics force field using the SPARTAN
0
8 [18]. Geometry re-optimizations at
B3LYP/6-31G(d) in vacuo, with the
PCM solvent model for MeOH followed
by TDDFT calculations using various
functionals (B3LYP, BH&HLYP, PBE0)
and the TZVP basis set were performed by
the Gaussian 09 [19] package. Boltzmann
distributions were estimated from the
ZPVE-corrected B3LYP/6-31G(d) ener-
gies. ECD curves were obtained on the
basis of rotator strengths with a half-band
of 0.25 eV using SpecDis v1.61 [20]. The
Microsoft Excel software was used for
visualization of the results.
Acknowledgment
3
.5 Acid hydrolysis of 2 and
This work was financially supported by the
National Natural Science Foundation of China
under grant No. 81361138020 and the National
Science and Technology Project of China under
grant No. 2012ZX09301002001003.
determination of the absolute
configuration of sugar
The acid hydrolysis of 2 and the
determination of the absolute configur-
ation of sugar were performed using
reported method [16]. About 2 mg of 2
was dissolved in 2 M HCl (2 ml) and
heated at 858C for 10 h in oil bath. The
mixture was concentrated under reduced
pressure to dryness. The resulting residue
Disclosure statement
No potential conflict of interest was reported by
the authors.
References
was suspended in H O and extracted in a
2
[
1] Committee of the Chinese Pharmaco-
poeia, The Chinese Pharmacopoeia,
part 1 (China Medical Science Press,
Beijing, 2010), p. 300.
separatory funnel with CHCl for three
3
times. The aqueous layer was evaporated
under vacuum, then diluted with H O, and
2
[
2] P.G. Xiao, Modern Chinese Material
Medica, Vol. 1 (Chemical Industry
Press, Beijing, 2001), p. 297.
then re-evaporated under vacuum, the re-
dilution and re-evaporation procedure
being repeated for several times, to
produce a neutral residue. The residue
[
3] S. Kobayashi, M. Endou, F. Sakuraya,
N. Matsuda, X.H. Zhang, M. Azuma,

8
D. Zhang et al.
N. Echigo, O. Kemmotsu, Y. Hattori, and
S. Gando, Anesth. Anal. 97, 1239 (2003).
[15] L.A. Mitscher, F. Kautz, and J. LaPidus,
Can. J. Chem 47, 1957 (1969).
doi:10.1139/v69-318.
doi:10.1213/01.ANE.0000092917.96558.
3C.
4] F.L. Greenway, Obes. Rev. 2, 199 (2001).
doi:10.1046/j.1467-789x.2001.00038.x.
5] W.S. Thomas, Am. J. Med. Sci 171, 719
[
16] J.Q. Yu, A.J. Deng, L.Q. Wu, Z.H. Zhang,
Y. Liu, W.J. Wang, and H.L. Qin,
Fitoterapia 85, 101 (2013). doi:10.1016/
j.fitote.2013.01.005.
[
[
[
(
0
1926). doi:10.1097/00000441-192605
00-00012.
[
[
17] P.J. Stephens, D.M. McCann, F.J. Devlin,
and A.B. Smith, J. Nat. Prod. 69, 1055
6] S.M. Mucha, M. deTineo, R.M. Naclerio,
and F.M. Baroody, Arch. Otolaryng.
Head. Neck. Surg. 132, 164 (2006).
doi:10.1001/archotol.132.2.164.
(2006). doi:10.1021/np060112p.
18] Spartan 08 (Wavefunction, Inc, Irvine,
CA.
[
[
7] R. Krizevski, E. Bar, O. Shalit, Y. Sitrit,
S. Ben-Shabat, and E. Lewinsohn, Phy-
tochemistry 71, 895 (2010). doi:10.1016/j.
phytochem.2010.03.019.
8] Y. Kitani, S. Zhu, T. Omote, K. Tanaka,
J. Batkhuu, C. Sanchir, H. Fushimi,
M. Mikage, and K. Komatsu, Biol.
Pharm. Bull. 32, 1235 (2009). doi:10.
[19] M.J. Frisch, G.W. Trucks, H.B. Schlegel,
G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
G Scalmani, V. Barone, B. Mennucci,
G.A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H.P. Hratchian, A.F. Izmaylov,
J. Bloino, G. Zheng, J.L. Sonnenberg,
M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima,
Y. Honda, O. Kitao, H. Nakai, T. Vreven,
J.A. Montgomery, J.E. Peralta Jr, F. Ogliaro,
M. Bearpark, J.J. Heyd, E. Brothers,
K.N. Kudin, V.N. Staroverov, R. Kobayashi,
J. Normand, K. Raghavachari, A. Rendell,
J.C. Burant, S.S. Iyengar, J. Tomasi,
M. Cossi, N. Rega, J.M. Millam, M. Klene,
J.E.Knox,J.B.Cross,V.Bakken,C.Adamo,
J. Jaramillo, R. Gomperts, R.E. Stratmann,
O. Yazyev, A.J. Austin, R. Cammi,
C. Pomelli, J.W. Ochterski, R.L. Martin,
K. Morokuma, V.G. Zakrzewski,
G.A. Voth, P. Salvador, J.J. Dannenberg,
S. Dapprich, A.D. Daniels, O. Farkas,
J.B. Foresman, J.V. Ortiz, J. Cioslowski,
and D.J. Fox, Gaussian 09, Revision B.01
1248/bpb.32.1235.
[
9] W. Zhao, A.J. Deng, G.H. Du, J.L. Zhang,
Z.H. Li, and H.L. Qin, J. Asian Nat. Prod.
Res. 11, 168 (2009). doi:10.1080/
1
0286020802573552.
10] F. Lutz, T. Igarashi, T. Kinoshita,
M. Asahina, K. Tsukiyama,
[
T. Kawasaki, and K. Soai, J. Am.
Chem. Soc. 130, 2956 (2008). doi:10.
1021/ja077156k.
[
[
[
11] T. Konno, H. Meguro, and K. Tuzimura,
Tetrahedron Lett. 16, 1305 (1975). doi:10.
1016/S0040-4039(00)72656-6.
12] G. Pattenden, Saturated Heterocyclic
Chemistry (Royal Society of Chemistry,
London, 1978), p. 123.
13] A.P. Volosov, V.A. Zubkov, and
T.M. Birshtein, Tetrahedron 31, 1259
(Gaussian, Inc, Wallingford, CT Fox, 2010).
(
6
1975). doi:10.1016/0040-4020(75)801
7-0.
[
20] T. Bruhn, A. Schauml o¨ ffel, Y. Hemberger,
and G. Bringmann, SpecDis version 1.61.
Inc. edited by U.O. Wuerzburg (Wuerz-
burg, Germany, 2013).
[
14] K. Soai and A. Ookawa, J. Chem. Soc.
Chem. Commun. 5, 412 (1986) doi:10.
1039/c39860000412.