Two new steroidal saponins from the processed Polygonatum kingianum

Article abstract of DOI:10.1002/hlca.200900308

Two new spirostanol saponins, kingianoside I (1) and kingianoside K (2), corresponding to (3β,23S,25R)-23-hydroxy-12-oxospirost-5-en-3-yl 4-O-β-D-glucopyranosyl-β-D-galactopyranoside (1) and (3β,25R)-7-oxospirost-5-en-3-yl α-L-arabinofuranosyl-(1→4)-[6- deoxy-a-l-mannopyranosyl-(1→ 2)]-β-D-glucopyranoside (2), along with 13 known compounds, daucosterol, (25R)-kingianoside G, (25RS)-kingianoside A, pratioside D1, (25RS)-pratioside D₁, (25S)-kingianoside C, kingianoside C, ginsenoside Rb₁, saponins Tb and Pb, dioscin, gracillin, and saponin Pa, were isolated from the processed rhizomes of Polygonatum kingianum. The structures of the new compounds were elucidated by detailed spectroscopic analyses, including 1D- and 2D-NMR techniques, and chemical methods. Compound 2 contains a novel unusual spirostanol saponin aglycone. Ginsenoside Rb₁ and saponin Tb were isolated for the first time from the genus Polygonatum. The 13 known compounds were detected for the first time in the processed Polygonatum kingianum.

Full text of DOI:10.1002/hlca.200900308

1
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Helvetica Chimica Acta – Vol. 93 (2010)
Two New Steroidal Saponins from the Processed Polygonatum kingianum
a
b
a
b
a
a
a
by He-Shui Yu ) ), Bai-Ping Ma* ), Xin-Bo Song* ), Li-Ping Kang ), Tao Zhang ), Jing Fu ),
a
a
a
b
a
c
Yang Zhao ), Cheng-Qi Xiong ), Da-Wei Tan ), Li-Juan Zhang ), Jie Zhang ), and Kate Yu )
a) Beijing Institute of Radiation Medicine, No. 27 Tai-Ping Road, Haidian District, Beijing 100850,
P. R. China (phone: þ 86-10-68210077 ext. 930265; fax: þ 86-10-68214653; e-mail: ma_bp@sohu.com)
b
)
Tianjin University of Traditional Chinese Medicine, No. 88 Yuquan Road, Nankai District,
Tianjin 300193, P. R. China (phone: þ 86-22-27494976; fax: þ 86-22-27460231;
e-mail: songxinbo@tjutcm.edu.cn)
c) Waters Corporation, Milford, MA 01757, U.S.A.
Two new spirostanol saponins, kingianoside I (1) and kingianoside K (2), corresponding to
3b,23S,25R)-23-hydroxy-12-oxospirost-5-en-3-yl 4-O-b-d-glucopyranosyl-b-d-galactopyranoside (1)
(
and (3b,25R)-7-oxospirost-5-en-3-yl a-l-arabinofuranosyl-(1 ! 4)-[6-deoxy-a-l-mannopyranosyl-(1 !
)]-b-d-glucopyranoside (2), along with 13 known compounds, daucosterol, (25R)-kingianoside G,
25RS)-kingianoside A, pratioside D , (25RS)-pratioside D , (25S)-kingianoside C, kingianoside C,
2
(
1
1
ginsenoside Rb , saponins Tb and Pb, dioscin, gracillin, and saponin Pa, were isolated from the processed
1
rhizomes of Polygonatum kingianum. The structures of the new compounds were elucidated by detailed
spectroscopic analyses, including 1D- and 2D-NMR techniques, and chemical methods. Compound 2
contains a novel unusual spirostanol saponin aglycone. Ginsenoside Rb and saponin Tb were isolated for
1
the first time from the genus Polygonatum. The 13 known compounds were detected for the first time in
the processed Polygonatum kingianum.
Introduction. – The rhizomes of Polygonatum kingianum Coll. et Hemsl.
(
Liliaceae), one of the original plants commonly known as Huang-jing in traditional
Chinese medicine, are used as a tonic remedy to treat lung troubles and ringworm [1].
Clinically, the processed (with yellow rice wine) products are routinely used since it is
widely believed that this process would mitigate side-effects and enhance the effect of
the tonic remedy.
The chemical constituents of rhizomes of several Polygonatum species have been
studied by several groups [2 – 6], but no systematic study on the chemical constituents
of the processed products has been reported so far. Previous phytochemical
investigations on the fresh rhizomes of P. kingianum have resulted in the isolation of
some steroidal saponins [7 – 9]. Our preliminary phytochemical studies on processed P.
kingianum indicated that the levels of certain steroidal saponins were higher in
processed P. kingianum than those in fresh P. kingianum, whereas the levels of some
other chemical constituents were decreased. In addition, the UPLC/MS profile of the
processed P. kingianum revealed a few constituents that have not been reported before
[
10]. As a result, our detailed chemical investigation on steroidal saponins of the
processed P. kingianum led to the isolation of two new steroidal saponins, along with 13
known compounds. Compound 2, kingianoside K, was found to possess a novel
aglycone (with a C¼C bond at C(5) and an oxo group in the B ring on the aglycone)
ꢀ
2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 93 (2010)
1087
rarely encountered in steroidal saponins. Ginsenoside Rb and saponin Tb were
1
reported from the genus Polygonatum for the first time. In this article, we describe the
isolation and structure elucidation of two new steroid saponins based on extensive
spectral analyses, including 1D- and 2D-NMR spectral data, and chemical evidence.
Results and Discussion. – The crude extract of the processed P. kingianum was
chromatographed on macroporous resin, silica gel, and octadecyl silane (ODS), and
further purified by semi-preparative HPLC to afford 15 compounds. Compounds 1 and
2
were found to be new spirostanol saponins, and their structures were elucidated by
1D- and 2D-NMR in combination with MS studies. The other 13 compounds are known
saponins, and they were identified by comparison of their physicochemical properties
and the NMR data with those reported in the literature.
Kingianoside I (1) was obtained as a white amorphous powder. The HR-ESI-MS
(
negative-ion mode) of 1 indicated a molecular formula C H O , which was derived
39
60 15
ꢀ
from the quasi-molecular-ion peak at m/z 767.3850 ([M ꢀ H] ) and confirmed by the
13
C-NMR data. The FAB-MS (positive-ion mode) of 1 also showed characteristic
þ
þ
fragment ion peaks at m/z: 769.3 ([M þ H] ), 751.2 ([M þ H ꢀ 18] ), 607.2 ([M þ H ꢀ
þ
þ
þ
1
1
62] ), 589.2 ([M þ H ꢀ 162 ꢀ 18] ), 445.2 ([M þ H ꢀ 162 ꢀ 162] ), 427.2 ([M þ H ꢀ
þ þ
62 ꢀ 162 ꢀ 18] ), 409.2 ([M þ H ꢀ 162 ꢀ 162 ꢀ 18 ꢀ 18] ), suggesting the presence of
two hexose units and two OH groups in the molecule. Compound 1 was hydrolyzed
with acid to afford d-galactose and d-glucose. This was identified by comparison with
authentic samples on TLC and GC analysis. Based on H- and C-NMR (Table), and
1
13
2D-NMR analyses, the structure of 1 was elucidated as (3b,23S,25R)-23-hydroxy-12-
oxospirost-5-en-3-yl 4-O-b-d-glucopyranosyl-b-d-galactopyranoside.
1
The H-NMR spectrum of 1 revealed the presence of two singlet Me signals at d(H)
0
6
.84 (s, Me(19)) and 1.26 (s, Me(18)), and two doublet Me signals at d(H) 0.71 (d, J ¼
.6, Me(27)) and 1.40 (d, J ¼ 6.6, Me(21)), which were assigned to characteristic
spirostanol saponin Me groups. Furthermore, the signal of an olefinic H-atom at d(H)
5
.24 (br. s, HꢀC(6)) was readily assigned, as well as signals for two anomeric H-atoms
at d(H) 4.85 (d, J ¼ 7.2) and 5.28 (d, J ¼ 8.4). The J values (> 7 Hz) of two anomeric H-
atoms confirmed the b-orientation at the anomeric centers for both hexoses [11][12].
13
The C-NMR spectrum of 1 showed two anomeric C-atom signals at d(C) 102.9 and

1
088
Helvetica Chimica Acta – Vol. 93 (2010)
1
07.2, in addition, two olefinic C-atom signals at d(C) 140.7 and 121.4. Comparison of
13
the C-NMR data of 1 with those of (25R)-kingianoside G [9] indicated that the
aglycone of 1 is the same as in (25R)-kingianoside G, namely (23S,25R)-3b,23-
dihydroxy-12-oxospirost-5-en. The complete assignment of the glycosidic NMR signals
1
1
was performed by analyses of H, H-COSY, HSQC, and HMBC data. In the HMBC
spectrum of 1 (Fig.), the correlations between the C-atom signal d(C) 67.4 (C(23)) and
d(H) 1.78 (HꢀC(24)) and 2.03 – 2.10 (HꢀC(20)) confirmed the location of one OH
group at C(23). Furthermore, the HMBC cross-peaks d(H) 4.85 (Gal HꢀC(1)/d(C)
7
7.7 (C(3)) and d(H) 5.28 (Glc HꢀC(1)/d(C) 80.0 (Gal C(4)) were observed, which
confirmed the sugar sequence and the glycosylation position.
Figure. Key HMBCs (H ! C) for compounds 1 and 2
Kingianoside K (2) was obtained as a white amorphous powder. The HR-ESI-MS
þ
(
positive-ion mode) of 2 exhibited the peak ([M þ Na] ) at m/z 891.4354 consistent
with the molecular formula C H O . Furthermore, the FAB-MS (positive-ion mode)
44
68 17
þ
of 2 also showed the characteristic fragment-ion peaks at m/z: 869.4 ([M þ H] ), 723.2
þ
þ
þ
(
[M þ H ꢀ 146] ), 591.1 ([M þ H ꢀ 146 ꢀ 132] ), 429.0 ([M þ H ꢀ 146 ꢀ 132 ꢀ 162] ),
þ
and 411.0 ([M þ H ꢀ 146 ꢀ 132 ꢀ 162 ꢀ 18] ), corresponding to loss of one deoxy-
hexosyl, one pentosyl, and one hexosyl moiety, respectively. On the basis of extensive
1
13
H- and C-NMR (Table), and 2D-NMR analyses, the structure of 2 was determined
as (3b,25R)-7-oxospirost-5-en-3-yl a-l-arabinofuranosyl-(1 ! 4)-[6-deoxy-a-l-manno-
pyranosyl-(1 ! 2)]-b-d-glucopyranoside.
1
The H-NMR spectrum of 2 showed the presence of two singlet Me signals at d(H)
1
.11 (s, Me (19)) and 0.83 (s, Me (18)), two doublet Me signals at d(H) 0.67 (d, J ¼ 6.6,
Me (27)) and 1.13 (d, J ¼ 6.6, Me (21)), an olefinic H-atom signal at d(H) 5.77 (br. s),

Helvetica Chimica Acta – Vol. 93 (2010)
1089
Table. 1H- and 13C-NMR Data ((D )Pyridine) of Compounds 1 and 2. d in ppm, J in Hz.
5
1
2
d(H)
d(C)
d(H)
d(C)
CH (1)
1.42 – 1.47 (m), 0.86 – 0.88 (m)
2.03 – 2.05 (m), 1.60 – 1.63 (m)
37.0
30.0
0.97 (d, J ¼ 13.8), 1.67 – 1.69 (m)
1.84 (dd, J ¼ 10.8, 10.8),
36.5
29.8
2
CH (2)
2
2
.09 (d, J ¼ 9.6)
HꢀC(3)
3.80 – 3.86 (m)
2.65 – 2.67 (m), 2.32 – 2.35 (m)
77.7
39.0
3.89 – 3.92 (m)
2.75 – 2.80 (m), 2.91 – 2.93 (m)
77.7
38.9
CH (4)
2
C(5)
140.7
121.4
31.7
165.2
126.3
200.9
HꢀC(6)
5.24 (br. s)
1.46 ), 1.77 – 1.84 (m)
5.77 (br. s)
a
CH (7) or
2
C(7)
CH (8)
1.80a)
30.8
52.3
37.5
37.5
2.40 (d, J ¼ 4.8)
1.35 – 1.40 (m)
45.0
49.9
38.8
21.1
2
HꢀC(9)
1.28 – 1.32 (m)
C(10)
CH (11)
2.49 (dd, J ¼ 15.0, 13.2),
1.42 – 1.44 (m), 1.44a)
2
2.26 (dd, J ¼ 15.0, 6.0)
C(12) or
212.7
1.02 – 1.04 (m), 1.60 – 1.65 (m)
38.7
CH (12)
2
C(13)
55.5
56.0
31.7
80.3
53.6
16.0
18.7
36.4
13.6
111.8
67.4
41.2
50.0
34.4
81.4
61.9
16.5
17.1
42.0
15.1
109.3
31.9
HꢀC(14)
1.45a)
1.48 – 1.50 (m)
CH (15)
2.09 – 2.13 (m), 1.65 – 1.70 (m)
4.57 – 4.60 (m)
2.89 (dd, J ¼ 9.0, 7.2)
1.26 (s)
1.71 – 1.77 (m), 3.26 – 3.30 (m)
4.63 – 4.66 (m)
2
HꢀC(16)
HꢀC(17)
Me(18)
a
1.77 )
0.83 (s)
1.11 (s)
1.92 – 1.95 (m)
1.13 (d, J ¼ 7.2)
Me(19)
0.84 (s)
HꢀC(20)
2.03 – 2.10 (m)
1.40 (d, J ¼ 7.2)
Me(21)
C(22)
3.86a)
1.63 ), 1.65 )
a
a
HꢀC(23) or
CH (23)
2
a
a
CH (24)
2.10 ), 1.78 )
38.7
31.6
66.1
1.51 – 1.59 (m)
29.3
30.6
66.8
2
a
a
HꢀC(25)
1.82 )
1.57 )
CH (26)
3.54 (dd, J ¼ 3.0, 11.4),
3.46 – 3.49 (m), 3.54 – 3.56 (m)
0.67 (d, J ¼ 4.8)
2
3
.48 (dd, J ¼ 10.4, 10.4)
Me(27)
Gal
0.71 (d, J ¼ 6.0)
16.9
17.3
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
4.85 (d, J ¼ 7.8)
102.9
73.5
75.4
80.0
76.0
61.0
4.93 (d, J ¼ 7.8)
4.20 – 4.27 (m)
4.27 )
100.4
77.9
77.1
76.9
77.0
62.5
4.38 (dd, J ¼ 7.8, 9.6)
a
4.20 – 4.24 (m)
4.69 (d, J ¼ 3.0)
4.29 – 4.35 (m)
3.79 (d, J ¼ 7.8)
3.99 – 4.08 (m)
a
a
CH (6)
4.20 – 4.24 (m), 4.61 – 4.64 (m)
4.22 ), 4.29 )
2
Glc
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
5.28 (d, J ¼ 7.8)
107.2
75.2
78.7
72.3
78.5
63.2
4.13 (dd, J ¼ 7.8, 8.4)
a
4.23 )
4.06 (dd, J ¼ 9.6, 9.6)
a
4.01 )
4.20 ), 4.58 )
a
a
CH (6)
2

1
090
Helvetica Chimica Acta – Vol. 93 (2010)
Table (cont.)
1
2
d(H)
d(C)
d(H)
d(C)
Rha
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
6.29 (br. s)
4.76 (m)
101.8
72.4
72.8
74.1
69.5
18.7
4.54 – 4.56 (m)
a
4.35 )
4.94 (d, J ¼ 7.8)
1.73 (d, J ¼ 7.2)
Ara
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
5.91 (s)
109.6
82.7
77.9
86.8
61.5
4.84 – 4.89 (m)
a
4.85 )
a
4.86 )
a
a
CH (5)
4.17 ), 4.27 )
2
a) Overlapped with other signals.
and three anomeric H-atom signals at d(H) 4.93 (d, J ¼ 7.8), 6.29 (br. s), and 5.91 (s).
The C-NMR spectrum of 2 displayed three anomeric C-atom signals at d(C) 100.4,
01.8, and 109.6, two olefinic C-atom signals at d(C) 165.2 and 126.3, and a ketone C-
atom signal at d(C) 200.9. The complete assignment of the glycosidic NMR signals was
13
1
1
1
confirmed by analyses of H, H-COSY, HSQC, and HMBC data. Evaluation of spin-
spin couplings and chemical shifts allowed the identification of one b-glucopyranosyl
(Glc), one a-rhamnopyranosyl (Rha), and one a-arabinofuranosyl unit (Ara). In the
HMBC spectrum, the C-atom signal at d(C) 165.2 showed long-range correlations with
H-atom signals at d(H) 2.75 – 2.80 (HꢀC(4)) and 1.11 (Me(19)), indicating a C¼C
bond between C(5) and C(6) on the aglycone. Furthermore, the C-atom signal at d(C)
2
00.9 showed long-range correlations with the H-atom signals at d(H) 1.49 (HꢀC(14))
and 2.40 (HꢀC(8)). This demonstrated that the oxo group is at C(7). The chemical
structure of the steroidal saponin with a C¼C bond at C(5) and an oxo group within
ring B in the aglycone is rare in natural products. The HMBC cross-peaks d(H) 4.93
(
Glc HꢀC(1))/d(C) 77.7 (C(3)), d(H) 5.91 (Ara HꢀC(1))/d(C) 76.9 (Glc C(4)), and
d(H) 6.29 (Rha HꢀC(1))/d(C) 77.9 (Glc C(2)) were observed, which confirmed the
sugar sequence and the glycosylation position.
Comparison of the physicochemical properties and NMR data with those reported
in the literature allowed us to identify the 13 known compounds as daucosterol [13],
(25R)-kingianoside G [9], (25RS)-kingianoside A [9], pratioside D1 [4], (25RS)-
pratioside D1 [9], (25S)-kingianoside C [7], kingianoside C [7], ginsenoside Rb [14],
1
saponins Tb [15] and Pb [5], dioscin [5], gracillin [5], and saponin Pa [5]. Ginsenoside
Rb and saponin Tb are reported for the first time from the genus Polygonatum. All 13
1
known compounds are reported from the processed P. kingianum for the first time.

Helvetica Chimica Acta – Vol. 93 (2010)
1091
This work was supported by the National Natural Science Foundation of China (30600822) and the
Major Program of Municipal Natural Science Foundation of Beijing (7090001). The authors are grateful
to Mrs. Yan Xue and Mr. He-Bing Chen of the National Center of Biomedical Analysis for recording the
NMR and mass spectra.
Experimental Part
General. Macroporous resin SP825 (Mitsubishi Chemical, Japan), silica gel (SiO ; Qingdao Haiyang
2
Chemical Co., Ltd., P. R. China), and ODS silica gel (120 ꢂ, 50 mm, YMC) were used for
chromatography. HPLC was performed on Agilent 1100 system (pump, quaternary pump. detector,
RID and DAD, USA), Apollo C18 (Alltech, 8.0 mm i.d. ꢁ250, ODS, 10 mm, USA), and YMC-Pack ODS-
A C18 (YMC, 4.6 mm i.d. ꢁ250 mm, ODS, 5 mm, Japan). Gas chromatographic (GC) analysis: Agilent
6
0
890 gas chromatograph, equipped with a H2 flame ionization detector; HP-5 cap. column (30 m ꢁ
.25 mm ꢁ 0.25 mm; Agilent, USA). Optical rotations: Perkin-Elmer 343 polarimeter. NMR Spectra:
UNITY
1
13
Varian
INOVA 600 (at 599.8 ( H) and 150.8 MHz ( C)); chemical shifts given in d [ppm] with TMS
as internal standard. FAB-MS: Micromass Zabspec. HR-ESI-MS: 9.4 T Q-FT-MS Apex Qe (Bruker Co.).
Plant Material. The material was collected from Fenggang County of Guizhou Province, P. R. China,
in December 2006, and was identified as rhizomes of Polygonatum kingianum Coll. et Hemsl. by L.-J. Z.
of the Tianjing University of Traditional Chinese Medicine. The samples were processed according to the
procedures of the Chinese pharmacopoeia. A voucher specimen (No. 061201) was deposited with the
Herbarium of Beijing Institute of Radiation Medicine, Beijing.
Extraction and Isolation. The decoction pieces of processed Polygonatum kingianum (5.5 kg) were
extracted for three times with 45% acetone. The combined extract was concentrated under reduced
pressure to give 72.0 g of residue. The extract was fractionated by CC on a macroporous resin (SP825)
with a gradient mixture of Me CO/H O (10 :90, 40 :60, and 80 :20) to give three fractions, Frs. A – C.
2
2
Fr. A was further purified on a macroporous resin (SP825) column with a gradient mixture of Me CO/
2
H O (25 :75, 30 :70, and 60 :40) to give three fractions, Frs. A (1.8 g), A (0.9 g), and A (4.0 g). Fr. A
1
2
1
2
3
(
(
1.8 g) was subjected to CC (SiO ; CHCl /MeOH/H O 12 :1:0.01 ! 2 :1:0.01) to yield daucosterol
2
3
2
Frs. A -36 – A -42; 49.4 mg), and Frs. A -162 – A -212 were separated by semi-prep. HPLC with MeCN/
1
1
1
1
H O 32 :68, to give compound 1 (37.4 mg) and (25R)-kongianoside G (48.5 mg). Fr. A (0.9 g) was
2
2
submitted to CC (SiO ; CHCl /MeOH/H O 6 :1:0.01 ! 2 :1:0.01) to afford ginsenoside Rb (Frs. A -
2
3
2
1
2
4
2
6 – A -58; 15.4 mg). Fr. B (3.0 g) was subjected to CC (SiO ; CHCl /MeOH/H O 10 :1 :0.01 !
2
2
3
2
:1 :0.01) to yield (25RS)-kingianoside A (Frs. B-41 – B-45; 22.4 mg) and (25RS)-pratioside D
1
(
Frs. B-76 – B-83; 36.7 mg), and Frs. B-72 – B-75 were separated by semi-prep. HPLC with MeCN/H O
2
3
0 :70 to give pratioside D (8.3 mg); Frs. B-85 – B-92 were further separated by semi-prep. HPLC with
1
MeCN/H O 22 :78 to give (25S)-kingianoside C (5.3 mg) and kingianoside C (6.7 mg). Fr. C (2.4 g) was
2
subjected to CC (ODS SiO ; MeCN/H O 45 :55, 48 :52, and 52 :48) to give saponin Pb (Frs. C-173 and C-
2
2
1
74; 26.3 mg) and dioscin (Frs. C-192 – C-196; 10.3 mg); Frs. C-76 – C-84 were further separated by semi-
prep. HPLC with MeCN/H O 46 :54 to afford compound 2 (10.4 mg) and saponin Tb (18.3 mg). Finally,
2
Frs. C-181 – C-186 were further purified by semi-prep. HPLC with MeCN/H O 49 :51 to yield gracillin
2
(
8.3 mg) and saponin Pa (16.7 mg).
Kingianoside I (¼(3b,23S,25R)-23-Hydroxy-12-oxospirost-5-en-3-yl 4-O-b-d-Glucopyranosyl-b-d-
2
0
1
13
galactopyranoside; 1). White amorphous powder. [a] ¼ ꢀ43.6 (c ¼ 0.046, pyridine). H- and C-NMR:
D
þ
þ
þ
Table. FAB-MS (pos.): 769.3 ([M þ H] ), 751.2 ([M þ H ꢀ 18] ), 607.2 ([M þ H ꢀ 162] ), 589.2
þ
þ
þ
(
(
[M þ H ꢀ 162 ꢀ 18] ), 445.2 ([M þ H ꢀ 162 ꢀ 162] ), 427.2 ([M þ H ꢀ 162 ꢀ 162 ꢀ 18] ), 409.2
þ ꢀ ꢀ
[M þ H ꢀ 162 ꢀ 162 ꢀ 18 ꢀ 18] ). HR-ESI-MS (neg.): 767.3850 ([M ꢀ H] , C H O15 ; calc. 767.3859).
39
59
Kingianoside K (¼(3b,25R)-7-Oxospirost-5-en-3-yl a-l-Arabinofuranosyl-(1 ! 4)-[6-deoxy-a-l-
2
0
mannopyranosyl-(1 ! 2)]-b-d-glucopyranoside; 2). White amorphous powder. [a] ¼ ꢀ113.8 (c ¼
D
1
13
þ
0
1
1
.065, pyridine). H- and C-NMR: Table. FAB-MS (pos.): 869.4 ([M þ H] ), 723.2 ([M þ H ꢀ
þ
þ
þ
46] ), 591.1 ([M þ H ꢀ 146 ꢀ 132] ), 429.0 ([M þ H ꢀ 146 ꢀ 132 ꢀ 162] ), 411.0 ([M þ H ꢀ 146 ꢀ
þ
þ
þ
17
32 ꢀ 162 ꢀ 18] ). HR-ESI-MS (pos.): 891.4354 ([M þ Na] , C H NaO ; calc. 891.4359).
44
68
Acid Hydrolysis. Each of 1 and 2 (ca. 2.0 mg) was treated in 1m HCl (dioxane/H O 1:1; 2 ml) at 1008
2
for 1.5 h. The mixture was neutralized with silver carbonate, and the solvent was thoroughly driven out

1
092
Helvetica Chimica Acta – Vol. 93 (2010)
under N overnight. The residue was extracted with CHCl and H O. Then, in monosaccharide mixture,
2
3
2
glucose and galactose of 1, and glucose, rhamnose, and arabinose of 2 were detected by TLC on a
cellulose plate with BuOH/AcOEt/C H N/H O 6 :1:5 :4 for development and aniline-o-phthalic acid for
5
5
2
detection, comparing with the authentic samples: glucose (R 0.46), rhamnose (R 0.69), arabinose (R
f
f
f
0
.48), and galactose (R 0.39). Furthermore, the sugar residue in pyridine (1 ml) was added to l-cysteine
f
methyl ester hydrochloride (3.0 mg) and kept at 608 for 1 h. Then, HMDS-TMCS (hexamethyldisila-
zane-trimethylchlorosilane; 0.6 ml) was added to the mixture, and the mixture was kept at 608 for 0.5 h.
The supernatant (1.0 ml) was analyzed by GC under the following conditions: Agilent Technologies 6890
GC was the equipment carrying H flame ionization detector and HP-5 cap. column (30 m ꢁ 0.25 mm ꢁ
2
0
.25 mm); column temp.: 1808/2508; programmed increase: 38/min; carrier gas: N (1 ml/min); injection
2
and detector temp.: 2508; injection volume: 4.0 ml, split ratio: 1:50. The retention times of the derivatives
for the standards were: t : 17.949 min (d-glucose derivative), 18.388 min (l-glucose derivative),
R
1
8.571 min (d-galactose derivative), 19.181 min (l-galactose derivative), 13.728 min (d-arabinose
derivative), 13.001 min (l-arabinose derivative), and 14.532 min (l-rhamnose derivative). The retention
times of the derivatives of d-glucose and d-galactose for compound 1 were 17.940 and 18.567 min, resp.
The derivatives of d-glucose, l-rhamnose, and l-arabinose for compound 2 were observed at 17.941,
1
4.528, and 13.006 min, resp.
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Received August 27, 2009