A condensed phenylpropanoid glucoside and pregnane saponins from the roots of Hemidesmus indicus

Article abstract of DOI:10.1007/s11418-012-0659-6

From the roots of Hemidesmus indicus, one new condensed phenylpropanoid glucoside and three new pregnenolone glycosides, named hemidesmosides A-C, were isolated along with one known related compound, plocoside A.

Full text of DOI:10.1007/s11418-012-0659-6

J Nat Med (2013) 67:137–142
DOI 10.1007/s11418-012-0659-6
ORIGINAL PAPER
A condensed phenylpropanoid glucoside and pregnane
saponins from the roots of Hemidesmus indicus
•
Zhimin Zhao Katsuyoshi Matsunami
•
•
•
•
Hideaki Otsuka Nisha Negi Ashok Kumar
Devendra Singh Negi
Received: 15 October 2011 / Accepted: 8 March 2012 / Published online: 29 March 2012
Ó The Japanese Society of Pharmacognosy and Springer 2012
Abstract From the roots of Hemidesmus indicus, one
new condensed phenylpropanoid glucoside and three new
pregnenolone glycosides, named hemidesmosides A–C,
were isolated along with one known related compound,
plocoside A.
stems [3] and roots [4] has revealed the presence of tri-
terpenes [3, 4] and pregnane glycosides [5–7]. This paper
deals with reinvestigation of the constituents of roots of
H. indicus.
Keywords Hemidesmus indicus ꢀ Asclepiadaceae ꢀ
Condensed phenylpropanoid ꢀ Pregnenolone saponin
Results and discussion
From the 1-BuOH-soluble fraction of a MeOH extract of
the roots of H. indicus, one condensed phenylpropanoid (1)
and three pregnane glycosides, named hemidesimosides
(A–C) (2–4), were isolated, along with one known com-
pound, plocoside A (5) [8]. Their structures were eluci-
dated by analyses of spectroscopic data.
Introduction
Hemidesmus indicus (Linne) Robert Brown (Asclepiada-
ceae) is a well known ayurvedic plant called Indian sar-
saparilla or sarsaparilla, and is used as a tonic, alterative,
demulcent, diaphoretic, diuretic and blood purifier [1, 2].
H. indicus grows wild in South Asia, including India, Sri
Lanka and Myanmar. It is a slender, laticiferous, twining,
sometimes prostrate or semi-erect shrub, and its roots are
woody and aromatic. It is one of the Rasayama plants of
Ayurveda, as its effect is anabolic. Chemical analysis of its
Compound 1, [a]D ?69.7, was isolated as an amorphous
powder and its elemental composition was determined to
be C16H22O8 by high-resolution (HR)–electrospray ioni-
zation (ESI)–mass spectrometry (MS). The IR spectrum
exhibited absorptions for hydroxy groups (3390 cm-1) and
an aromatic ring (1606 and 1520 cm-1). The presence of
the aromatic ring was also supported by absorptions in the
1
UV spectrum at 282 and 230 nm. In the H-NMR spectra,
distinct signals for three aromatic protons (dH 6.95, 6.82
and 6.77) coupled in an ABX system, an anomeric proton
(dH 4.57, 1H, d, J = 8 Hz), a doublet methyl (dH 1.00, 3H,
d, J = 6 Hz), and a singlet methyl (dH 3.86) were
observed. The 13C-NMR spectrum exhibited six signals
assignable to a hexose moiety, six signals for the aromatic
ring, and four signals for a methyl, two oxygenated
methines and a methoxy group. On 1H–1H correlation
spectroscopy (COSY), seven sequential protons from H-10
to H2-60 were found to be correlated and also from the
methyl signal (H3-9) to the methine signal (H-7). Since
coupling constants of H-100 to H-500 showed that these
protons were all obviously in the axial positions, the hexose
Z. Zhao ꢀ K. Matsunami ꢀ H. Otsuka (&)
Graduate School of Biomedical Sciences, Hiroshima University,
1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
e-mail: hotsuka@hiroshima-u.ac.jp
Z. Zhao
School of Pharmaceutical Sciences, Sun Yat-Sen University,
132 East Outer Ring Road, Guangzhou University City,
510006 Guangzhou, People’s Republic of China
N. Negi ꢀ A. Kumar ꢀ D. S. Negi
Department of Chemistry, HNB Garhwal University (A Central
University), Srinagar (Garhwal), Uttarakhand 246 174, India
123

138
J Nat Med (2013) 67:137–142
21
O
was expected to be glucopyranose. However, the hetero-
nuclear single quantum correlation spectrum (HSQC),
together with the COSY spectrum, revealed that the C-2
position was fairly shifted downfield (dC 81.1) when
compared with the common signal (ca. 75 ppm) for glu-
copyranose. In the heteronuclear multiple bond correlation
spectrum (HMBC), the doublet methyl protons (dH 1.00)
were correlated with two methine carbons (dC 85.4 and
78.0), and the methine proton (dH 4.11) on C-7 showed
correlation cross-peaks with C-1, C-2, C-6 and C-8 (dC
130.6, 112.4, 122.1 and 78.0, respectively). These corre-
lations together with other HMBC data allowed assignment
of the structure of the aglycone moiety as 3-methoxy-4-
hydroxyphenylpropane with two oxygen atoms at the C-7
and C-8 positions (Fig. 2). Based on the HMBC correlation
between the H-10 proton and C-8, the glucosidic connection
was determined to be at this position. From the results of
the HR-ESI–MS requiring 6° of unsaturation and the
HMBC correlation cross-peak of H-7 with C-2 (dC 81.1), a
cyclic system must be formed between C-7 and C-20
through an ether bond. Judging from the correlations
between H-7 and H-20, and H-8 and H-10 on phase-sensitive
(PS) nuclear Overhauser exchange spectroscopy (NOESY),
the absolute configurations at C-7 and C-8 were assigned as
S, assuming that of glucose was the D-series. Therefore, the
structure of compound 1 was elucidated to be as shown in
Fig. 1. Similar and related compounds to 1 have been
isolated from several plant sources, Illicium oligandrum
[9], Myrica rubra [10], and Melia toosendan [11].
OH
18
20
O
O
HO
HO
17
1'
O
11
19
OR2
9
1
H
14
7
2
1
2
H
H
1
R1 R2 R3
OCH3
R1O
H
S1 S3
2
S1 S3 Glc(1""")
3
4
6
7
H
-
H
S2 S3
OH
S2
S1 Glc
H
O
S3
CH3
CH3
R3O
O
O
O
O
S1
S3
1"'
1'
1'
1""
HO
OH
OH
O
O
HO
CH3
CH3
HO
OH
OCH3
OCH3
O
O
O
AcO
O
1"
1"
OCH3
OCH3
1""'
OH
HO
HO
OAc
OH
Fig. 1 Structures of isolated and reference compounds
Fig. 2 HMBC correlations for
compound 1
OH
O
HO
O
HO
O
OCH3
OH
Hemidesmoside A (2), [a]D -18.2, was isolated as an
amorphous powder and its elemental composition was
determined to be C57H90O27 by HR-ESI–MS. The IR
spectrum exhibited strong absorption bands at 3384 cm-1
for hydroxy groups and at 1746 cm-1 for a ketonic func-
tional group. In the 1H-NMR spectrum, three singlet
methyl signals (dH 0.63, 0.88 and 2.34) and five anomeric
proton signals (dH 4.77, 4.88, 5.16, 5.23 and 5.27) were
observed (Table 2). Two further singlet methyls at dH 1.93
and 2.14 were expected to be for two acetyl groups,
judging from the presence of two methyl carbon signals at
dC 20.6 and 21.0, and carbonyl signals at dC 169.8 and
170.8 in the 13C-NMR spectrum. The 13C-NMR spectrum
exhibited the presence of five corresponding anomeric
carbon signals (dC 96.5, 103.0, 103.4, 104.9 and 106.7),
and six signals were assignable as those of a terminal
glucopyranose (Table 3). One of the anomeric protons
appeared at dH 5.23 as a doublet of doublet (J = 9, 2 Hz)
signal, implying the presence of a 2-deoxy sugar unit,
which is frequently found in saponins isolated from Ascl-
methylenes, four methines and two oxygenated methines,
two quatenary carbons, one trisubstitued double bond and
one carbonyl carbon. When the 13C-NMR spectrum of 2
was compared with those of known pregnane glycosides,
those for two sugar units, and rings A and B were essen-
tially superimposable on those of a 3b,16a-dihydroxypreg-
5-en-20-one derivative (6) isolated from Streptocaulon
tomentosums (Asclepiadaceae) [12], and those of three
sugar units, and rings C and D were with those of stel-
matocryptonoside C (7) isolated from Stelmatocrypton
khasianum (Asclepiadaceae) [13] (Table 2). In the HMBC
spectrum, the anomeric proton of 2,4-diacetyldigitalose
showed a correlation cross-peak with C-3 of cymaropyra-
nose and that of cymaropyranose with C-3 of the aglycone,
whereas the anomeric proton of the terminal glucopyranose
was correlated with C-2 of an inner glucopyranose, whose
anomeric proton was correlated with C-6 of the innermost
glucopyranose (Fig. 3). Attachment of the innermost glu-
copyranose was also established by the HMBC correlation
of H-1000 (dH 4.88) with C-17 (dC 72.2) (Fig. 3). Compound
2 was then hydrolyzed under acidic conditions to give
D-digitalose, D-cymarose and D-glucose [14].The structure
of hemidesmoside A (2) was therefore elucidated to be
1
epiadaceous plants. In the H–1H COSY and HSQC spec-
tra, protons and carbons of the sugar rings were assigned as
shown in Fig. 3, and the remaining 21 13C-NMR spectral
signals consisted of those of three methyls, seven
123

J Nat Med (2013) 67:137–142
139
13C: 100 MHz, 1H:
1H
O
Table 1 NMR spectroscopic data for
400 MHz, CD3OD)
1 (
13C
O
1
2
3
4
5
6
7
8
130.6
112.4
149.0
148.0
116.1
122.1
85.4
–
6.95, d, 2
–
O
–
O
HO
HO
CH3
O
6.77, d, 8
6.82, dd, 8, 2
4.11, br d, 10
O
O
O
OH
OH
HO
O
CH3
78.0
3.89,
HO
OH
1H-1H COSY
OCH3
O
overlapped
O
CH3COO
9
17.2
99.7
81.1
75.2
72.0
79.9
62.7
1.00, d, 6
OCH3
OH
10
20
30
40
50
60
4.57, d, 8
HO
H
C
3.13, dd, 9, 8
3.60, dd, 9, 9
3.41, dd, 9, 9
3.48, m
OCOCH3
OH
HMBC
Fig. 3 1H–1H COSY and diagnostic HMBC correlations for hemi-
desmoside A (2). Dual arrows denote that HMBC correlations were
observed in both directions
3.73, dd, 12, 5
3.90, dd, 12, 2
3.86, s
3b,16a-dihydroxypreg-5-en-20-one 3-O-b-D-(200,400-di-O-
acetyl-b-D-digitalopyranosyl)(100?40)cymaropyranoside, 16-
O-b-D-glucopyranosyl(100000?20000)-O-b-D-glucopyranosyl
(10000?6000)-O-b-D-glucopyranoside, as shown in Fig. 1.
Hemidesmoside B (3), [a]D -34.1, was isolated as an
amorphous powder and its elemental composition was
determined to be C63H100O32 by HR-ESI–MS. In the
13C-NMR spectrum, signals for two sets of terminal
glucopyranoses were observed and one of the C-6 signals of
glucose moieties was shifted downfield. Thus, hemidesmo-
side B (3) was an analogous compound to the preceding
hemidesmoside A (2), except for the presence of one more
glucopyranose unit. The attachment position of the new
glucopyraose unit was confirmed by the HMBC correlation
between the anomeric proton (dH 5.01) and C-60000 (dC
69.5). The structure of hemidesmoside B (3) was therefore
elucidated to be 3b,16a-dihydroxypreg-5-en-20-one 3-b-D-
(200,400-di-O-acetyl-b-D-digitalopyranosyl)(100?40)cymar-
opyranoside 16-O-b-D-glucopyranosyl(100000?20000)-O-b-D-
–OCH3
56.6
those of the inner b-D-cymaropyranosyl moiety were the same
as those of the inner 2-deoxy sugar of 4 (Table 3). In the
HMBC spectrum, the anomeric proton of the outer sugar unit
was correlated with C-40 of the inner 2-deoxy sugar and that of
the inner sugar unit with C-3 of the aglycone. The structure of
hemidesmoside C (4) was therefore elucidated to be 3b,
16a-dihydroxypreg-5-en-20-one 3-O-b-D-(b-D-oleandropyr-
anosyl)(100?40)cymaropyranoside, 16-O-b-D-glucopyranosyl
(100000?20000)-O-b-D-glucopyranosyl(10000?6000)-b-D-glucopy-
ranoside.
Experimental
Optical rotations were measured on a JASCO P-1030
polarimeter. IR spectra were measured on a Horiba FT-710
spectrophotometer. H- and 13C-NMR spectra were taken
1
glucopyranosyl, (100000?60000)-O-b-D-glucopyranosyl(10000
6000)-O-b-D-glucopyranoside, as shown in Fig. 1.
?
on JEOL JNM a-400 (H at 400 MHz and C at 100 MHz)
and ECA-600 (H at 600 MHz and C at 150 MHz) spec-
trometers with tetramethylsilane as an internal standard.
Positive-ion HR-MS were taken with an Applied Biosys-
tems QSTAR XL system ESI (Nano Spray)-MS.
Hemidesmoside C (4), [a]D -34.9, was also isolated as
an amorphous powder and its elemental composition was
determined to be C53H86O24 by HR-ESI–MS. The 13C-
NMR spectroscopic signals for three glucopyranose moi-
eties, and the rings C and D region were superimposable on
those of hemidesmoside A (2), and two anomeric protons at
dH 4.78 and 5.28 appeared as doublet of doublets. Thus,
two 2-deoxy sugars must be involved in sugar linkage on
the hydroxy group at the C-3 position. The 13C-NMR data
for the b-D-oleandropyranosyl moiety were essentially the
same as those of the outer 2-deoxy sugar of 4, whereas
A highly porous synthetic resin (Diaion HP-20) was
purchased from Mitsubishi Kagaku (Tokyo, Japan). Silica
gel CC and reversed-phase [octadecylsilanized silica gel
(ODS)] open CC were performed on silica gel 60 (Merck,
Darmstadt, Germany) and Cosmosil 75C18-OPN (Nacalai
Tesque, Kyoto, Japan) [U = 50 mm, L = 25 cm, linear
gradient: MeOH–H2O (1:9, 1 L) ? (1:1, 1 L), fractions
of 10 g being collected], respectively. The droplet
123

140
J Nat Med (2013) 67:137–142
Table 2 1H-NMR spectroscopic data for hemidesmosides A–C (2–4)
(pyridine-d5)
Table 2 continued
H
2
3
4
H
1
2
3
4
100000
200000
300000
400000
500000
600000
5.27 d 8
4.03 m
4.12 m
4.14 m
3.90 m
4.37 m
4.56 m
5.16 d 8
4.03 m
5.28 d 8
4.06 m
4.12 m
4.17 m
3.91 m
4.33 m
4.58 m
0.98 m
1.70 m
1.71 m
2.12 m
0.98 m
1.71 m
1.70 m
2.08 m
1.00 m
1.73 m
1.71 m
2.13 m
4.25 m
2
4.11 m
3.95 m
3
4
3.77 dddd 11, 11, 4, 4 3.74 dddd 11, 11, 5, 5 3.80 m
4.34 dd 12, 6
4.58 m
2.32 m
2.31 m
2.35 m
1000000
2000000
3000000
4000000
5000000
6000000
5.01 d 8
4.06 m
2.50 dd 13, 4
5.29 br d 8
1.42 m
2.50 m
2.51 m
6
7
5.29 d 7
1.40 m
5.28 br d 8
1.40 m
4.20 m
1.80 m
1.78 mz
1.30 m
1.77 m
4.18 m
8
1.28 dd 11, 5
0.86 dd 11, 5
1.24 m
1.27 m
3.90 m
9
0.86 m
0.87 m
4.37 m
11
1.26 m
1.24 m
4.58 m
30-CH3O-
300-CH3O-
3.52 3H s
3.41 3H s
3.52 3H s
3.42 3H s
3.61 3H s
3.47 3H s
1.40 m
1.50 m
1.45 m
12
1.26 m
1.27 m
1.25 m
000-CH3O-
1.81 m
1.91 m
1.84 m
3
200-CH3CO- 2.14 3H s
400-CH3CO- 1.93 3H s
2.15 3H s
1.94 3H s
14
15
1.35 dd 12, 4
1.71 ddd 12, 12, 4
1.93 m
1.35 m
1.35 m
1.71 ddd 11, 11, 3
1.93 m
1.83 m
1.93 m
16
17
18
19
21
10
5.21 dd 7, 7
2.91 d 7
5.21 dd 7, 7
2.95 d 7
0.62 3H s
0.87 3H s
2.47 3H s
5.24 dd 9, 2
1.87 m
5.21 dd 8, 7
2.91 d 7
0.963 3H s
0.88 3H s
2.34 3H s
5.28 m
counter-current chromatograph (DCCC) (Tokyo Rikakikai,
Tokyo, Japan) was equipped with 500 glass columns
(U = 2 mm, L = 40 cm), the lower and upper layers of a
solvent mixture of CHCl3–MeOH–H2O–n-PrOH (9:12:8:2)
being used as the stationary and mobile phases, respectively.
Five-gram fractions were collected and numbered according to
their order of elution with the mobile phase. HPLC was per-
formed on an ODS column (Inertsil; GL Science, Tokyo,
Japan; U = 6 mm, L = 25 cm), and the eluate was monitored
with a UV detector at 254 nm and a refractive index monitor.
Standard sugars were obtained from hydrolysis of cymarin (D-
cymarose) (MP Biochemicals, Cedex, France), troleandromy-
cine (L-oleandrose) (Wako Pure Chemical Co., Kyoto, Japan)
and chartreusin (D-digitalose) (Santa Cruz Biotechnology, CA,
USA).
0.63 3H s
0.88 3H s
2.34 3H s
5.23 dd 9, 2
1.87 m
20
1.92 m
2.32 m
2.35 m
2.35 m
30
4.02 m
4.02 m
4.10 m
40
3.51 m
3.53 m
3.56 dd 9, 3
4.26 m
50
4.20 m
4.26 m
60
1.45 3H d 6
4.77 d 8
1.46 3H d 6
4.78 d 8
5.58 dd 10, 8
1.45 3H d 6
4.78 dd 8, 2
1.73 m
100
200
5.58 d 10
2.54 m
300
3.66 dd 10, 3
5.56 m
3.68 dd 10, 3
5.57 m
3.68 ddd 9, 9, 4
5.57 m
400
500
3.92 m
3.94 m
3.94 m
Plant material
600
1.31 3H d, 6
4.88 d 8
4.30 m
1.31 3H d 6
4.88 d 8
4.30 m
1.31 3H d 6
4.89 d 8
4.03 m
1000
2000
3000
4000
5000
6000
Roots of H. indicus were purchased by one of the authors
(D.S.N.) from a market in Srinagar, India.
4.19 m
4.19 m
4.18 m
4.44 m
4.42 m
4.35 m
Extraction and isolation
4.38 m
3.92 m
4.02 m
4.33 dd 11, 4
4.58 dd 11, 2
5.16 d 8
4.06 m
4.28 m
4.31 m
Air-dried roots of H. indicus (985 g) were extracted three
times with MeOH (15 L 9 2) at room temperature for
2 weeks and then the extract was concentrated to 2 L in
vacuo. The concentrated extract was washed with n-hexane
(2 L 9 2, 10.0 g) and then the MeOH layer was concen-
trated to a gummy mass. The latter was suspended in water
(1.5 L) and then extracted with EtOAc (2 L 9 2) to give
13.4 g of an EtOAc-soluble fraction. The aqueous layer
4.58 m
4.59 m
10000
20000
30000
40000
50000
60000
5.06 d 8
4.06 m
5.17 d 8
4.09 m
4.36 m
4.27 m
4.37 m
4.26 m
4.24 m
4.24 m
3.95 m
4.02 m
4.00 m
4.32 dd 11, 4
4.48 m
4.31 m
4.35 m
4.73 dd 11, 2
4.48 m
123

J Nat Med (2013) 67:137–142
141
Table 3 13C-NMR spectroscopic data for hemidesmosides A–C (2–
4) (pyridine-d5) and reference compounds
Table 3 continued
C
2a
78.4c
3a
78.2c
4b
78.2c
6
7
C
2a
3a
4b
6
7
300000
78.12
71.22
78.86
62.24
1
37.6
37.4
37.3
37.3
30.2
77.3
39.2
37.62
32.46
71.22
43.34
400000
50000000
600000
1000000
2000000
3000000
4000000
5000000
71.5
79.1
62.6e
71.4
78.9
62.8
105.5
75.4
78.4c
71.8
78.5c
62.4
58.3
57.7
71.4
78.9
62.6d
2
30.5
77.6
39.5
141.0
121.8
32.1
31.6
50.4
37.1
21.1
38.9
45.0
54.4
33.8
80.8
72.2
14.8
19.5
208.1
32.6
96.5
37.0
77.5
84.5
68.9
18.7
103.4
71.7
80.4
69.5
69.7
16.7
104.9
75.6
78.5c
71.2d
76.6
69.9
103.0
85.2
78.3c
71.3d
78.3c
62.8e
106.7
76.7
30.4
77.5
39.9
140.9
121.6
32.0
31.5
50.2
36.9
20.9
38.9
44.9
54.5
33.7
80.8
72.1
14.6
19.3
208.2
32.6
96.3
36.8
77.9c
84.3
68.8
18.4
103.1
71.7
80.2c
69.3
69.6
16.6
104.9
75.6
78.3c
70.6
76.3
69.2d
102.7
84.8
78.5c
70.7
78.4c
69.5d
106.6
76.5
30.4
77.5
39.5
140.0
121.6
32.0
31.5
50.3
36.9
20.9
38.7
44.8
54.4
33.6
80.6
72.0
14.6
19.3
207.9
32.4
96.4
37.5
78.0c
83.5
69.0
18.70
102.0
37.0
81.5
76.3
73.0
18.68
104.7
75.4
78.4c
71.1
76.4
69.7
102.9
85.0
78.23c
71.0
77.99c
62.8d
106.6
76.6
3
4
5
140.8
121.6
31.9
31.4
50.1
36.8
20.9
38.7
44.9
54.4
33.7
81.0
72.1
14.6
19.3
208.2
32.2
96.2
36.6
77.2
84.2
68.7
18.5
103.1
71.4
80.1
69.2
69.5
16.6
105.0
75.3
78.5
71.4
78.2
62.5
141.74
120.86
31.90
31.43
50.17
36.74
20.89
38.67
44.77
54.41
33.56
80.53
71.89
14.59
19.44
207.85
32.46
6
7
8
9
6000000
10
11
12
13
14
15
16
17
18
19
20
21
10
30-CH3O-
300-CH3O-
3000-CH3O-
200-CH3CO- 21.2
200-CH3CO- 169.8
400-CH3CO- 20.6
400-CH3CO- 170.8
58.5
57.9
58.8
57.0
58.2
57.6
21.0
169.7
20.4
21.0
169.7
20.4
170.6
170.6
a
At 150 MHz
b
At 100 MHz
c,d,e
Figures with the same superscripts in each column may be
interchangeable
was extracted with 1-BuOH (2 L 9 3) to give a 1-BuOH-
soluble fraction (50.4 g), and the remaining water layer
was concentrated to furnish 104 g of a water-soluble
fraction. The 1-BuOH-soluble fraction (49.7 g) was sub-
jected to Diaion HP-20 CC (U = 50 mm, L = 50 cm),
using H2O–MeOH (4:1, 2 L), (3:2, 2 L), (2:3, 2 L), and
(1:4, 2 L), and MeOH (3 L), 300 mL fractions being col-
lected. The residue (20.5 g) in fractions 4–6 from the 20 %
MeOH eluate was subjected to silica gel (450 g) CC with
increasing amounts of MeOH in CHCl3 [CHCl3 (3 L), and
CHCl3–MeOH (49:1, 3 L), (24:1, 3 L), (23:2, 3 L), (9:1,
3 L), (7:1, 3 L), (17:3, 3 L), (4:1, 3 L), (3:1, 3 L), (7:3,
3 L), and (3:2, 3 L)], 500 mL fractions being collected.
The residue (5.70 g) in fractions 16–27 was subjected silica
gel (150 g) CC with increasing amounts of MeOH in
CHCl3 [CHCl3 (1.5 L), CHCl3–MeOH (49:1, 1 L), (24:1,
1 L), (23:2, 1 L), (9:1, 1 L), (17:3, 1 L), (4:1, 1 L), (3:1,
1 L), and (7:3, 1 L)], CHCl3–MeOH–H2O (35:15:2, 1 L),
and MeOH (800 mL), 100 mL fractions being collected.
The residue (45.1 mg) in fractions 40–46 was purified by
HPLC (H2O–MeOH, 13:7) to give 4.5 mg of 1 from the
peak at 7.5 min. The residue (1.05 g) in fractions 56–75
was subjected ODS open CC and the residue (147 mg) in
fractions 103–113 was purified by HPLC (Inertsil; GL
Science, Tokyo; U = 20 mm, L = 25 cm; flow rate:
4 mL/min; H2O–MeOH 13:7) to yield 76.3 mg of 2 from
the peak at 60 min. The residue (1.47 g) in fractions 76–96
20
30
40
50
60
100
200
300
400
500
600
1000
2000
3000
4000
5000
6000
10000
20000
30000
40000
50000
60000
100000
200000
104.67
76.35
78.23
70.92
76.53
69.57
102.75
85.01
78.00
70.81
78.12
62.40
106.55
76.38
123

142
J Nat Med (2013) 67:137–142
was subjected to ODS open CC and the residue (95.3 mg)
in fractions 81–84 was purified by HPLC (H2O–MeOH,
13.7) to afford 3 (16.6 mg), 5 (34.3 mg), and 4 (6.7 mg)
from the peaks at 16, 20.5, and 23 min, respectively.
for L-oleandrose) [14]. Authentic D-digitalose and D-glucose
showed peaks at 4.7 and 11.0 min, respectively, with positive
signs on an amino column [Asahipak NH2P-50 4E,
U = 4.6 mm, L = 25 cm, CH3CN–H2O (3:1), 1 mL/min].
Authentic L-oleandrose and D-cymarose showed peaks at
9.3 min with a positive sign and 10.5 min with a negative
sign, respectively, on an ODS column [Inertsil ODS-3,
U = 4.6 mm, L = 25 cm, CH3CN–H2O (1:49), 1 mL/min].
Compound 1
Amorphous powder, [a]2D0 ?69.7 (c = 0.30, MeOH). IR mmax
(film) cm-1: 3390, 2935, 1606, 1520, 1279, 1130, 1035. UV
1
Acknowledgments The authors are grateful for access to the
superconducting NMR instrument at the Analytical Center of
Molecular Medicine of the Hiroshima University Faculty of Medicine
and an Applied Biosystem QSTAR XL system ESI (Nano Spray)-MS
at the Analysis Center of Life Science of the Graduate School of
Biomedical Sciences, Hiroshima University. The authors are also
grateful for the use of the NMR instrument (JEOL ECA-600) at the
Natural Science Center for Basic Research and Development, Hiro-
shima University. Thanks are also due to DG, Uttarakhand State
Council for Science and Technology, Government of Uttarakhand for
financial assistant.
kmax (MeOH) nm (log e): 282 (3.53), 230 (3.83). H-NMR
(400 MHz, CD3OD): Table 1. 13C-NMR (100 MHz, CD3OD):
Table 1. HR-ESI–MS (positive-ion mode) m/z: 365.1201
[M ? Na]? (Calcd for C16H22O8Na: 365.1206).
Hemidesmoside A (2)
Amorphous powder, [a]2D0 -18.2 (c = 0.26, MeOH). IR mmax
(film): 3384, 2935, 1746, 1371, 1233, 1088, 1066 cm-1. 1H-
NMR (600 MHz, pyridine-d5): Table 2. 13C-NMR
(150 MHz, pyridine-d5): Table 3. HR-ESI–MS (positive-ion
mode) m/z: 1229.5579 [M ? Na]? (Calcd for C57H90O27Na:
1229.5561).
References
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indicus (L.) R. Br. J Biol Sci 8:1–12
2. George S, Tushar KV, Unnikrishnan KP, Kashim KM, Bala-
chandran I (2008) Hemidesmus indicus (L.) R. Br. A review.
J Plant Sci 3:146–156
Hemidesmoside B (3)
Amorphous powder, [a]2D0 -34.1 (c = 0.26, MeOH). IR mmax
(film): 3395, 2934, 1747, 1370, 1232, 1065 cm-1. 1H-NMR
(600 MHz, pyridine-d5): Table 2. 13C-NMR (150 MHz,
pyridine-d5): Table 3. HR-ESI–MS (positive-ion mode)
m/z: 1391.6061 [M ? Na]? (Calcd for C63H100O32Na:
1391.6089).
3. Gupta MM, Verma RK, Misra LN (1992) Terpenoids from
Hemidesmus indicus. Phytochemistry 31:4036–4037
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Amorphous powder, [a]2D0 -34.9 (c = 0.45, MeOH). IR mmax
´
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(film): 3392, 2933, 1700, 1456, 1369, 1164, 1067 cm-1
.
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(100 MHz, pyridine-d5): Table 3. HR-ESI–MS (positive-ion
mode) m/z: 1129.5387 [M ? Na]? (Calcd for C53H86O24Na:
1129.5401).
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Sugar analysis
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About 2.0 mg of each of compounds 2, 3 and 4 was
hydrolyzed with 1 M HCl in 50 % dioxane (0.1 mL) at
80 °C for 2 h. The water layers were neutralized with
Amberlite IRA-96SB and analyzed with a chiral detector
(JASCO OR-2090plus). Hydrolyzates of compounds 2 and
3 gave peaks for D-cymarose, D-digitalose and D-glucose
with positive optical rotation signs, and that of compound 4
D-cymarose and D-glucose with positive optical rotation
signs. A peak for D-oleandrose could not be detected in the
hydrolyzate, due to a small optical rotation value ([a]D ?10.3
14. Wuts PGM, Bigelow SS (1983) Total synthesis of oleandrose and
the avermecin disaccharide, benzyl a-L-oleadrosyl-a-L-acetoxyo-
leadroside. J Org Chem 48:3489–3493
123