Complete assignments of 1H and 13C NMR spectroscopic data for three new stigmastane glycosides from Vernonia cumingiana

Article abstract of DOI:10.1002/mrc.2371

Three new steroidal saponins, Vernoniosides S1 (1), Vernoniosides S2 (2) and Vernoniosides S3 (3) were isolated from the stem of Vernonia cumingian. Their chemical structures were elucidated on the basis of MS, NMR spectroscopic and chemical analysis. Com

Full text of DOI:10.1002/mrc.2371

Spectral Assignments and Reference Data
Received: 12 May 2008
Revised: 14 October 2008
Accepted: 15 October 2008
Published online in Wiley Interscience: 8 December 2008
(www.interscience.com) DOI 10.1002/mrc.2371
Complete assignments of 1H and 13C NMR
spectroscopic data for three new stigmastane
glycosides from Vernonia cumingiana
Maorong Suo∗ and Junshan Yang∗
Three new steroidal saponins, Vernoniosides S1 (1), Vernoniosides S2 (2) and Vernoniosides S3 (3) were isolated from the stem
of Vernonia cumingian. Their chemical structures were elucidated on the basis of MS, NMR spectroscopic and chemical analysis.
Complete assignment of 1H and 13C NMR spectroscopic data were achieved by 1D and 2D NMR experiments (HMQC, HMBC,
c
ROESY). Copyright ꢀ 2008 John Wiley & Sons, Ltd.
1
Keywords: NMR; H NMR; 13C NMR; 1D NMR; 2D NMR; Vernonia cumingian; steroidal saponins; Vernoniosides S1; Vernoniosides S2;
Vernoniosides S3
Introduction
methyl proton signals at δ 1.09 (d, J = 7.0, H-26), 1.22 (d, J = 7.0,
H-27) and 1.41(d, J = 6.5, H-29), and two characteristic multiplet
at δ 3.93 (H-3) and 4.30 (H-28), which was shifted downfield by
glycoslation.[5] Besides that, two olefine protons at δ 5.35 (br s) and
5.41 (br d J = 6.0) were displayed in the 1H NMR spectrum of 1.
An unsaturated functionality evident from the 13C NMR spectrum
were two trisubstituted double bond (120.9 and 136.3, 144.2
and 118.5 ppm), situated between C-7 and C-8, C-9 and C-11, as
indicated by analysis of the HMQC and HMBC correlations. These
data suggested that 1 is based on a 3, 28-dihydroxy-stigmastane
skeleton, possessing a ꢀ7,9(11) diene system.[6] Key correlations
peaks were observed between H-26, 27, 29 and C-24 (δ 76.5) by the
analysis of DEPT, HMQC and HMBC spectra (Fig. 1) of 1, indicating
thattherewasahydroxyllinkedatC-24.TheNMRdataforthesugar
moiety (Table 1) linked at C-3 and C-28 of the aglycon revealed
the presence of two β-glucopyranosyl units. Their β-linkages were
shown by the coupling constant values (J = 8.0, 7.5 Hz) of two
anomeric proton signals at δ 5.00, 4.92 and by their chemical shifts
in the 13C NMR spectra. NMR, HMQC, DEPT and HMBC experiments
allowed the assignments of all proton and carbon signals and the
identification of two substituted β-glucopyranosyl residues. The
configuration of the sugar units was assigned after acid hydrolysis,
the hydrolysate was trimethylsilated, and gas chromatography
(GC) retention times of sugar were compared with authentic
samples. In this way, the sugar units of 1 were determined to be
D-glucose.
Vernonia cumingiana Benth.(Compositae) was mainly distributed
inGuangxi,YunnanandGuangdongprovincesinChina,asaknown
Chinese folk medicine, it was extensively used for the treatment
of malaria, larynx aches, cold, tooth aches and rheumatism.[1]
Members of Vernonia family were well known for the production
of sesquiterpene lactone and stigmastane type glycosides.[2–5] In
the course of searching for new and bioactive constituents from
traditional Chinese medicines, three new stigmastane glycosides,
named Vernonioside S1 (1), Vernonioside S2 (2), Vernonioside
S3 (3), were isolated from the stems of V. cumingiana. This
paper describes the isolation and structure elucidation of these
stigmastane glycosides from V. cumingiana. This is the first report
that sugar unit is linked to C-28 of aglycone of stigmastane
glycosides in the Vernonia family.
Results and Discussion
Compound 1 was obtained as colorless powder, and the
Liebermann-Burchard and Molisch reaction of 1 were positive,
indicating 1 to be steroidal or triterpenoids glucosides. The
molecularformula-of1wasdeducedasC41H66O15 byHR–FAB–MS
(m/z 821.4282 [M + Na]+), and the degree of unsaturation
was 9. The UV spectrum of 1 showed absorption bands at
235.4 and 242.4 nm, suggesting the presence of a conjugated
diene chromophore. The infrared (IR) spectrum displayed strong
absorptions of hydroxyls (3600−3100 cm−1) and carbonyl group
(1699 cm−1).
In the ROESY spectrum of 1, the key correlation peaks between
H-3 (δ 3.93) and H-5 (δ 1.32), H-17 (δ 2.25) and H-14 (δ 2.08),
H-20 (δ 2.48) and H-18 (δ 0.81), H-28 (δ 4.30) and H−2ꢁꢁ (δ 4.04)
were observed. Thus, compound 1 was established as 3β,24,
The FAB–MS (positive ion) showed the loss of two hexoses
(162 mass units) from the positive ion [M + Na]+ at m/z 821.
The 13C NMR spectrum of 1 displayed 29 carbon signals for the
aglycon moiety ascribable to an stigmastane nucleus including
two oxymethines (77.0 ppm, CH, C-3), (81.3 ppm, CH, C-28), and
∗
Correspondence to: Maorong Suo and Junshan Yang, Institute of Medicinal
Plant Development, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing, 100094, China. E-mail: suomaorong@163.com;
junshanyang@sina.com
1
a carboxyl(ic) group (178.7 ppm, C-21). The H NMR spectra for
the aglycon part of 1 were typical of a sterol structure displaying
two angular methyl singlets at δ 0.81 and 0.82, three secondary
InstituteofMedicinalPlantDevelopment,ChineseAcademyofMedicalSciences
and Peking Union Medical College, Beijing, 100094, China
c
Magn. Reson. Chem. 2009, 47, 179–183
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Copyright ꢀ 2008 John Wiley & Sons, Ltd.

M. Suo and J. Yang
Figure 1. Structure and the key HMBC correlations of compound 1.
Figure 2. Structure and the key HMBC correlations of compound 2.
28- trihydroxy-stigmastane-7(8),9(11)-dien-21-oicacid-3,28-dioxy-
β-D-glucopyranoside, named Vernonioside S1.
Compound2wasobtainedasacolorlesspowder. ItsHRFAB–MS
atm/z 803. 4163[M+Na]+ correspondedtothemolecularformula
of C41H64O14, suggesting 10 degrees of unsaturation. Positive
results from both Liebermann-Burchard and Molisch reactions
indicated it to be stigmastane glycoside. The UV spectrum of
2 exhibited absorption bands at 242.8 and 235.6 nm, and its
IR spectrum showed absorptions of hydroxyl (3421 cm−1) and
carbonyl groups (1718 cm−1). Two D-glucose units were identified
by GC analysis after acid hydrolysis as 1.
The1HNMRspectrumof2exhibitedtwoangularmethylsinglets
at δ 0.74 (s, H-18) and 0.83 (s, H-19), three secondary methyl proton
signals at 0.91 (d, H-26), 1.01 (d, H-27), 1.38 (d, H-29), and two
olefine protons δ 5.32 (br s) and 5.48 (br s). The analysis of the NMR
data of 2 revealed the same aglycon as in 1 and two sugar chains
comprised of two glucosides (Table 1). Comparison of the 13C NMR
spectrum of 2 with that of 1 showed the evident downfield shift
of the signal ascribable to C-24 (δ 88.6 in 2 vs δ 76.5 in 1), and
Table 1. 1H NMR and 13C NMR spectra data for compound 1–4a
1
2
3
Position
1
δ(C)
δ(H)
δ(C)
δ(H)
δ(C)
δ(H)
35.0
1.17 m
1.79 (br d J = 13.5)
1.80 br s
35.0
1.17 m
1.79 m
2.05 br s
2.11 m
3.90 m
35.0
1.27 m
1.84 (br d J = 13.5)
2
30.1
30.1
30.1
1.77 m
3
4
77.0
34.5
3.93 m
76.9
34.5
77.0
34.5
3.94 (d, J = 5.5)
2.01 br t J = 10.5
2.00 br t J = 10.5
2.08 m
1.99 m
5
39.2
30.4
1.32 m
2.09 br d J = 5.0
5.35 br s
39.1
30.2
1.24 m
39.2
30.1
1.28 m
1.77 m
5.35 br s
6
1.87 br s
7
120.9
136.3
144.2
36.1
120.6
136.5
144.1
36.1
5.32 br s
120.8
136.0
144.2
36.2
8
9
10
11
12
118.5
40.4
5.41 (br d J = 6.0)
2.34 br d J = 17
2.52 br d J = 7.0
119.0
40.3
5.48 br s
2.36 br s
2.40 br s
118.5
41.7
5.46 br s
2.19 m
13
14
15
16
17
18
19
20
21
42.5
53.3
23.0
26.8
51.7
11.6
19.4
49.7
178.7
42.9
51.7
23.0
26.0
49.5
12.4
19.5
40.9
174.4
43.7
49.4
36.1
74.5
62.6
13.5
19.5
41.9
63.6
2.08 br d J = 5.0
1.69 br t J = 12
2.00 m
2.31 br s
1.72 br t J = 10.5
1.99 m
2.75 m
2.11 m
4.58 br s
2.25 m
2.52 br s
2.05 m
0.81 s
0.74 s
0.67 s
0.82 s
0.83 s
0.82 s
2.48 br t J = 6.5
2.55 br s
1.90 br s
3.84(dd, J = 9.5, 4.5)
4.14 (br, d, J = 9.5)
c
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Copyright ꢀ 2008 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2009, 47, 179–183

Spectral Assignments and Reference Data
Table 1. (Continued)
1
2
3
Position
22
δ(C)
δ(H)
δ(C)
δ(H)
δ(C)
δ(H)
27.8
2.01 m
2.03 m
22.6
1.87 m
21.8
1.79 m
23
24
25
26
27
28
29
1ꢁ
30.2
76.5
33.9
17.8
17.8
81.3
16.1
102.2
74.7
78.5
71.7
78.5
62.7
2.11 (br d J = 8.0)
21.9
88.6
34.5
17.2
17.5
78.6
14.9
102.3
75.0
78.1
71.7
78.6
62.9
2.11 (br d J = 8.0)
30.0
76.8
33.8
17.8
18.1
80.7
15.7
102.3
75.0
78.4
71.7
78.6
62.7
2.11 m
2.16 m
1.09 d J = 7.0
1.22 d J = 7.0
4.30 m
2.10 m
0.91 d J = 6.0
1.01d J = 6.0
2.17 m
1.09 d J = 7
1.21d J = 7
4.37 m
4.28 t J = 8.0
1.41 d J = 6.5
5.00 d J = 8.0
4.00 br t J = 7.5
3.97 m
1.38 d J = 6.5
5.00 d J = 7.5
4.00 br t J = 8.5
3.97 br s
1.50 d J = 6.5
5.01 d J = 7.5
2ꢁ
3.99 (br dd, J = 8, 6.5)
3.93 m
3ꢁ
4ꢁ
4.20 m
4.29 br d J = 8.0
4.20 t J = 8.5
4.17 br d J = 8.0
4.22 t J = 9.0
5ꢁ
3.97 m
6ꢁ
4.34 (dd J = 10.5, 5.5)
4.53 m
4.40 (dd J = 10.5, 5.0)
4.52 (br d J = 11)
4.84 d J = 7.5
4.03 br t J = 8.0
3.99 m
4.31 (br, d, J = 8.0)
4.51 (br, d, J = 10)
4.93 d J = 7.5
1ꢁꢁ
2ꢁꢁ
3ꢁꢁ
4ꢁꢁ
5ꢁꢁ
6ꢁꢁ
103.7
75.3
78.6
71.7
78.5
62.8
4.92 d J = 7.5
4.04 br t J = 8.0
4.28 t J = 6.5
4.29 t J = 9.0
4.22 br t J = 8.0
4.39 (dd, J = 10.5, 5.0)
4.57 br d J = 10
103.0
75.3
78.5
71.8
78.8
63.1
103.4
75.3
78.5
71.8
78.8
62.9
4.03 br t J = 8.0
3.98(d, J = 8.5)
4.27(t, J = 8.0)
4.26 (t, J = 8.0)
4.41 (d, J = 5.0)
4.57 (br d J = 9.5)
4.15 t J = 10.5
4.28 br t J = 8.0
4.34 (dd, J = 10.5, 4.5)
4.57 br d J = 12
a The experiments were carried out at 500 MHz for 1H and 125 MHz for 13C in C5D5N and TMS as reference (0.00 ppm).
the upfield shift of the signal ascrible to C-21 (δ 174.4 in 2 vs δ
178.7 in 1) (Table 1), indicating C-24 was linked to C-21 by ester
bonds. The cross peaks were observed between H-1ꢁ (δ 5.00) and
C-3 (δ 76.9), H-1ꢁꢁ (δ 4.84) and C-28 (δ 78.6) in the HMBC spectrum
(Fig. 2), suggesting two D-glucopyranosyl units were linked at C-3
and C-28 respectively as that of 1. Their β-linkage were shown by
the coupling constant values (both J = 7.5 Hz) of two anomeric
protons at δ 5.00 and 4.84. All proton and carbon signals were
assigned by NMR, HMQC and HMBC data.
In the ROESY spectrum of 2, significant cross peaks between
H-3 (δ 3.90) and H-5 (δ 1.24), H-17 (δ 2.52) and H-14 (δ 2.31),
H-20 (δ 2.55) and H-18 (δ 0.74) were observed. So, compound 2
was determined as 3β, 24, 28-trihydroxy-stigmastane-7(8), 9(11)-
dien-21, 24-lactone-3, 28-dioxy-β-D-glucopyranoside, named as
Vernonioside S2.
Figure 3. Structure and the key HMBC correlations of compound 3.
Compound 3, was obtained as a colorless powder. Its molecular
formula C41H68O15 was deduced by HRFAB–MS [M + Na]+ m/z
821.4282, and the degree of unsaturation was 8. The UV spectrum
of 3 showed absorption at 242.2 and 235.4 nm. The IR spectrum
displayed the presence of hydroxyls (3415 cm−1) and double
bonds (1458, 1379 cm−1).
The analysis of the NMR data of 3 revealed the same aglycon as
in 1 and two sugar chains comprised of two glucosides (Table 1)
and its aglyone possessed 3, 28-dihydroxy-stigmastane-7 (8), 9
(11)-dien. Comparison of the 13C NMR spectrum of 3 with that of
1 showed the absence of carboxyl group at δ 178.7. Differences
were also observed in the 13C NMR of aglycone of 3 added an
oxymethylene and an oxymethine at δ 63.6 (C-21) and 74.5 (C-16)
than that of 1, suggesting two hydroxyl was linked at C-21 and
C-16 respectively, which was approved by the correlation peaks
between C-21 (δ 63.6) and H-17 (δ 2.05), C-20 (δ 41.9); C-14 (δ 49.4)
and H-16 (δ 4.58) in the HMBC spectrum of (Fig. 3) 3.
The 1H NMR and 13C NMR of 3 (Table 1) gave two sets of
D-glucose signals, which were supported by GC analysis after
the acid hydrolysis experiment. The coupling constant values of
both anomeric protons (δ 5.01, 4.93) were 7.5 Hz, suggesting that
the compound 3 was β-D-pyranglucosides. Similarly, two β-D-
glucose were connected with C-3 and C-28 respectively, which
were confirmed by the correlation peaks between H-1ꢁ (δ 5.01)
and C-3 (δ 77.0), H-1ꢁꢁ (δ 4.93) and C-28 (δ 80.7) in the HMBC of 3.
All proton and carbon signals were assigned by 1H NMR, 13C NMR,
HMQC and HMBC data.
c
Magn. Reson. Chem. 2009, 47, 179–183
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M. Suo and J. Yang
The relative stereochemistry of 3 was determined by evaluation
ofthecross-peakintheROESYspectrumof3,thestrongcorrelation
peaks between H-3 (δ 3.94) and H-5 (δ 1.28), H-17 (δ 2.05) and H-14
(δ 2.75), H-16 (δ 4.58) and H-12 (δ 2.19) were observed. Thus,
compound 3 was elucidated as 3β, 16α, 21, 24, 28-penta hydroxy-
stigmastane-7 (8), 9 (11)-dien–3, 28–dioxy-β-D-glucopyranoside,
named as Vernonioside S3.
for each of 200 F1 increments, 4096 data points in F2, the same as
those of 1 except for AQ 0.207 s.
The pulse conditions for (3) were as follows: for the 1H NMR and
13C NMRspectrum: the same as those of 1; for the HMQC spectrum:
two sets of 256 time increments were obtained in the phase-
sensitive mode with 48 transients obtained per time increment,
the same as those of 1 except for SW 5050.8 Hz, 2D SW 19 431.6 Hz;
for the HMBC spectrum: 80 scans for each of 320 F1 increments,
2048 data points in F2, SF 499.745, AQ 0.204 s, TE 298.1 K, RD
1.000 s, SW 5030.5 Hz, 2D SW 19 431.6 Hz; for the ROESY spectrum:
eight scans for each of 200 F1 increments, 2048 data points in
F2, the same as those of 1 except for AQ 0.207 s, RD 1.600 s, MT
0.800 s, SW 4952.0 Hz, 2D SW 4952.0 Hz.
Experimental
Methods
Melting points were determined on an X4 micro melting point
apparatus and uncorrected. Optical rotations were measured on
a Perkin-Elmer 341 digital polarimeter (Perkin-Elmer, Norwalk, CT,
US) at 589 nm. UV spectra were recorded on Hitachi UV–2201
spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were
recorded in KBr discs on an Impact 410 FTIR spectrophotometer.
1H NMR, 13C NMR, DEPT, HMQC, HMBC and ROESY spectra of
compound 1, 2 and 3 were recorded on a Varian Inova-500
spectrometer with a 5-mm inverse probe at room temperature.
The compound 1, 2 and 3 were dissolved separately in 1-ml
pyridine-d5, and transferred to a 5-mm NMR tube. All chemical
shifts are in ppm (δ), using TMS as internal standard and coupling
constants(J)areinhertz.FAB–MSandEIMSwereobtainedonaVG-
Autospec- 3000 spectrometer (Thermo Electron, Manchester, UK).
GC analysis was carried out on Agilent 6890N gas chromatography
using an HP-5 capillary column (28 m × 0.32 mm i.d.); detection,
FID; detector temperature, 260 ◦C; column temperature, 180 ◦C;
carrier gas, N2; flow rate, 40 ml/min. Silica gel (100−200, 300−400
mesh) and silica gel GF254 sheets (both from Qingdao Haiyang
Chemical Group Co., Qingdao, Shandong Province, China) were
used for column chromatography and thin layer chromatography
(TLC), respectively.
Plant material
The stems of V. cumingiana were collected from Nanning, Guangxi
Province, people’sRepublicofChina, inSeptember2003. Theplant
was identified by the Advisor Chao-Liang Zhang in the Guangxi
Subinstitute of Medicinal Plant Development, Chinese Academy
of Medical Sciences and Peking Union Medical College. A voucher
sample (NO. YA-02-0726) was deposited in the Herbarium of
Institute of Medicinal Plant Development, Chinese Academy of
Medical Science and Peking Union Medical College.
Extraction and isolation
The air-dried and pulverized stem of V. cumingiana Benth. (8.0 kg)
was extracted two times with 95% EtOH for 1.5 h under re-
flux condition, and the extract (1.10 kg) was chromatographed
with silica gel column (100−200 mesh), eluting with petroleum
ether, chloroform, EtOAc, MeOH and H2O respectively. The EtOAc
fraction (205 g) was chromatographed on silica gel (300−400
mesh) column, eluting with gradient CHCl3−MeOH to afford
40 fractions. Fraction 25−40 (1.0 g) were combined and chro-
matographed on silica gel (300−400 mesh) with gradient sol-
vent (CHCl3:MeOH : H2O) and 200-mg eluent (CHCl3:MeOH : H2O
= 5 : 1 : 0.1) was obtained. Then the eluent was further chro-
matographed on ODS gel with MeOH–H2O as eluting solvent to
yield compound 3 (18 mg) (MeOH:H2O = 2 : 5). Fraction 8 (69 mg)
was chromatographed over ODS gel to yield compound 1 (50 mg)
(MeOH : H2O = 1 : 2) and 2 (25 mg) (MeOH : H2O = 2 : 3).
The pulse conditions for (1) were as follows: for the 1H NMR
spectra: spectrameter frequency (SF) 499.745 MHz, acquisition
time (AQ) 1.865 s, number of repetition (NR) 16, temperature (TE)
298.1 K, relaxation delay (RD) 4.000 s, flip angle (FA) 87.2◦, spectral
width (SW) 7998.4 Hz; for the 13C NMR spectrum: SF 125.7 MHz, AQ
1.000 s,NR1728,TE298.1K,RD1.000 s,FA72.1◦,SW31421.8 Hz;for
theHMQCspectrum:theHMQCexperimentforsingle-bond1H,13
C
chemical shift correlation spectra utilized GARP, 13C decoupling.
Two sets of 128 time increments were obtained in the phase-
sensitive mode with 48 transients obtained per time increment,
SF 499.745 MHz, AQ 0.203, TE 298.1, RD 1.000 s, SW 5039.4 Hz, 2D
SW 23 809.5 Hz; for the HMBC spectrum: the HMBC experiment
was performed with 32 scans for each of 320 F1 incerements, 2048
data points in F2, SF 499.745, AQ 0.189 s, TE 298.1 K, RD 1.000 s,
SW 5417.9 Hz, 2D SW 23 809.5 Hz; for the ROESY spectrum: the
ROESY experiment was performed with 64 scans for each of 256
F1 increments, 4096 data points in F2, SF 499.745, AQ 0.206 s, TE
298.1 K, RD 1.600 s, mixing time (MT) 0.800 s, SW 4948.4 Hz, 2D SW
4948.4 Hz.
Vernonioside S1 (1) colorless powder (MeOH), [α]2D0 +7.62
(c 0.92, MeOH);UV (MeOH) λmax (logε): 242.4 (2.70), 235.4 (2.67),
IR (KBr) cm−1: 3406, 2943, 2873, 1699; 1H NMR and 13C NMR data
(Table 1); HR–FAB–MS (positive) m/z 821.4282 [M + Na]+ (calcd
for C41H66O15Na, 821.4299).
VernoniosideS2(2)colorlesspowder(MeOH),[a]2D0 −6.31(c0.63,
MeOH); UV (MeOH) λmax (logε): 242.8 (3.14), 235.6 (3.10); IR (KBr)
cm−1: 3410, 2960, 2877, 1718; 1H NMR and 13C NMR data (Table 1);
FAB–MS (positive) m/z 781[M + H]+, HR–FAB–MS (positive) m/z
803.4163 [M + Na]+ (calcd for C41H64O14Na, 803.4193).
Vernonioside S3 (3) colorless powder (MeOH), [a]2D0 +10.14 (c
0.69, MeOH); UV (MeOH) λmax (logε): 242.2 (2.97), 235.4 (2.93); IR
The pulse conditions for (2) were as follows: for the 1H NMR and
13C NMR spectra: the same as those of 1; for the HMQC spectrum:
two sets of 256 time increments were obtained in the phase-
sensitive mode with 64 transients obtained per time increment,
the same as those of 1 except for AQ 0.210 s, SW 4978.3 Hz, 2D SW
23 809.5 Hz; for the HMBC spectrum: 96 scans for each of 320 F1
incerements, 2048 data points in F2, the same as those of 1 except
for AQ 0.208 s, SW 4925.2 Hz; for the ROESY spectrum: eight scans
(KBr) cm−1: 3415, 2927, 2875, 1637, 1458, 1379, 1078, 1034; H
1
NMR and 13C NMR data (Table 1); HR–FAB–MS (positive) m/z [M
+ Na]+ 821.4282 (calcd for C41H68O15Na, 821.4299).
Acid hydrolysis of 1-3
Compounds 1–3 (each 2 mg) were refluxed with 10% HCl in aq.
MeOH (3 ml) for 6 h. Each reaction mixture was diluted with H2O
c
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Magn. Reson. Chem. 2009, 47, 179–183

Spectral Assignments and Reference Data
and neutralized with Ag2CO3. The neutral hydrolysate revealed References
the presence of glucose by TLC detection comparison with an
authentic sample.
[1] Zhonghuabencao Editorial Board, Zhonghuabencao, vol. 7, Shanghai
Scientific and Technological Press: Shanghai, 1999, p. 1001.
[2] R. Sanogo, M. P. Germano, N. D. Tommasi, Phytochemistry 1998, 47,
73.
[3] M. Jisaka, H. Ohigashi, T. Takagaki, Phytochemisry 1993, 34, 409.
[4] D. Ponglux, S. Wongseripipatana, N. Aimi, Chem. Pharm. Bull. 1992,
40, 553.
[5] H. Ohigashi, M. Jisaka, T. Takagaki, Agric. Biol. Chem. 1991, 55, 1201.
[6] D. Valeria, G. P. Luigi, M. R. Luigi, D. Cecil, L. Claudi, J. Nat. Prod. 1992,
55, 311.
[7] L. Tang, Y. Jiang, H. T. Chang, M. B. Zhao, P. F. Tu, J. R. Cui, R. Q. Wang,
J. Nat. Prod. 2005, 68, 1169.
Sugar identification by hydrolysis and GC analysis
Compounds 1–3 (each 3 mg) was heated in 2 ml of 10%
HCl–dioxane (1 : 1) at 80 ◦C for 4 h. After the dioxane was removed,
the solution was extracted with EtOAc (2 ml × 3). The aqueous
fractions were evaporated and the residues were prepared to their
derivatives for GC analysis according to the methods described in
theliterature.[7] TheD-glucosewasconfirmedbythecomparisonof
itsretentiontime(tR, 12.50 min)withthatofauthenticstandards.[8]
[8] S. Hara, H. Okabe, K. Mihashi, Chem. Pharm. Bull. 1986, 34, 1843.
Acknowledgement
The authors thank the National Natural Science Foundation of
People’s Republic of China for financial support (No. 20432030).
c
Magn. Reson. Chem. 2009, 47, 179–183
Copyright ꢀ 2008 John Wiley & Sons, Ltd.
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