+
The HRESI-MS data of steroidal glycoside 1 showed the pseudomolecular ion at m/z 979.4545 [M+Na] , suggesting that it had
the molecular formula C47 20 (Calcd. for C47 20Na 979.4539) and accounted for 12 indices of hydrogen deficiency. The IR
spectrum indicated the presence of hydroxy (3441 cm ), carbonyl (1735 cm ), and olefinic (1645 cm ) functionalities. The H
NMR spectrum (400 MHz, in C N) showed two tertiary methyl groups at 0.78 (s, 3H, H-19) and 1.55 (s, 3H, H-21) and two
olefinic protons at 5.33 (d, 1H, J = 5.0 Hz, H-6) and 6.48 (s, 1H, H-18). The C NMR spectrum showed an ester carbonyl
signal at δ 175.8 (C-14), four olefinic carbon signals at δ 120.8 (C-6), 141.0 (C-5), 114.8 (C-13) and 144.2 (C-18), and two
tertiary methyl signals at δ 18.2 (C-19) and 25.2 (C-21). Comparison of the H and C NMR data with that of stauntoside O [8]
suggested that 1 had the aglycone of glaucogenin C, which was confirmed by 1D and 2D NMR data (Fig. 2). The H NMR
spectrum of 1 showed four anomeric proton signals at δ 4.81 (d, 1H, J = 7.7 Hz), 4.91 (d, 1H, J = 7.8 Hz), 5.11 (d, 1H, J = 9.3
Hz), and 5.50 (d, 1H, J = 9.1 Hz), and three secondary methyl proton signals at δ 1.38 (d, 3H, J = 6.0 Hz), 1.46 (d, 3H, J = 5.4
Hz), and 1.55 (d, 3H, J = 6.0 Hz), indicating that it had four sugar units, including three 6-deoxysugars. The 13C NMR spectrum
displayed four anomeric carbon signals at δ 102.7 (C-1′), 99.3 (C-1″), 100.1 (C-1″′), and 106.9 (C-1″″) and three methyl carbon
signals at δ 19.0 (C-6′), 18.9 (C-6″), and 18.8 (C-6′″), which confirmed that it was a tetraglycoside, including three 6-
deoxysugars. The composition of sugar units was identified to be thevetose, digitoxose, cymarose, and glucose by comparison of
H
72
O
72
H O
-1
-1
-1
1
5
D
5
H
1
3
H
C
C
1
13
C
1
H
H
C
C
1
13
its H and C NMR data with that of stauntoside L [9], which was confirmed by mild acid hydrolysis and subsequent silica gel
column chromatography purification. The D-configuration of glucose was determined by acid hydrolysis and appropriate
derivatization of the resulting sugar [10]. The D-configurations of cymarose, thevetose, and digitoxose were determined by
comparison of their specific rotation with that of literatures [5, 11-15] after 24 h equilibration in an aqueous solution. The
3
orientation of all the sugar units was identified by their large J values of JH-1′, H-2′ (J 7 Hz). The HMBC correlations (Fig. 2)
from δ
5.50 (H-4″ of digitoxose) to δ
correlation (Fig. 2) between 4.81 (d, 1H, J = 7.7 Hz, H-1 of thevetose) and
H
4.91 (H-1 of glucose) to δ
C
83.2 (C-4 of cymarose), δ
83.2 (C-4′ of thevetose) suggested that the sugar units were 14 linkages. The HMBC
78.7 (C-3) indicated that the sugar chain was
H
5.11 (H-1 of cymarose) to δ
C
83.4 (C-4″ of digitoxose), and
δ
H
C
H
C
attached to C-3 position of glaucogenin C. Thus, steroidal glycoside 1 was identified to be glaucogenin C 3-O-(1→4)-β-D-
1
glucopyranosyl-β-L-cymaropyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-thevetopyranoside. Full assignment of its H and
1
3
1
13
1
1
C NMR data was achieved by analyses of H, C NMR, H- H COSY, HSQC, and HMBC spectra (Table 1).
Steroidal glycoside 2 had the molecular formula C47 18 by its pseudomolecular ion at m/z 949.4760 [M+Na] (Calcd. for
18Na 949.4773) in its HRESI-MS and accounted for 11 indices of hydrogen deficiency. The IR spectrum showed the
+
74
H O
C
47
74
H O
-1
-1
1
absorption bands for hydroxy (3457 cm ) and olefinic (1646 cm ) functionalities. The H NMR spectrum (300 MHz in CDCl
displayed two tertiary methyl signals at δ 0.91 (s, 3H, H-19) and 1.44 (s, 3H, H-21), one olefinic proton signal at δ 5.39 (d, 1H,
J = 4.9 Hz, H-6), two oxygen-substituted methine proton signals at δ 3.49 (m, 1H, H-3) and 4.69 (q, 1H, J = 3.7 Hz, H-16), and
two oxygen-substituted methylene proton signals at δ 4.16 (d, 1H, J = 10.9 Hz, H-15a) / 3.72 (t, 1H, J = 4.0 Hz, H-15b) and
3
)
H
H
H
H
1
3
4
6
.48 (d, 1H, J = 9.0 Hz, H-18a) / 3.75 (m, 1H, H-18b). The C NMR spectrum showed two olefinic carbon signals at δ
) and 139.3 (C-5), two oxygenated methine carbon signals at δ 79.0 (C-3) and 80.7 (C-16), two oxygenated methylene carbon
75.3 (C-15) and 77.9 (C-18), and two tertiary methyl carbon signals at δ 18.3 (C-19) and 22.5 (C-21). Comparison
of its H and C NMR data with that of hirundigosides E-J [7] suggested that the aglycone of steroidal glycoside 2 was
hirundigenin, which was confirmed by the presence of hemiketal carbon signal at 108.3 (C-14). The anomeric proton signals at
4.33 (d, 1H, J = 7.8 Hz), 4.75 (dd, 1H, J = 7.5, 1.5 Hz), 4.78 (dd, 1H, J = 7.4, 1.5 Hz), and 4.95 (d, 1H, J = 3.2 Hz) suggested
C
122.6 (C-
C
signals at δ
C
C
1
13
C
δ
H
1
that 2 was a tetraglycoside in the H NMR spectrum, which was confirmed by the anomeric carbon signals at δ
C
100.8 (C-1′), 99.3
C-1″), 98.3 (C-1″′), and 100.7 (C-1″″) in the C NMR spectrum. The four sugar units were 6-deoxysugars by the presence of
four secondary methyl signals at 1.23 (d, 3H, J = 5.8 Hz, H-6) / δ 17.8 (C-6′), 1.21 (d, 3H, J = 5.6 Hz, H-6) / 18.0 (C-6″),
.16 (d, 3H, J = 6.3 Hz, H-6″′) / 18.4 (C-6″′), and 1.26 (d, 3H, J = 6.8 Hz, H-6″″) / 17.2 (C-6″″) in its H and C NMR spectra.
The composition of sugar units was identified to be 3-demethyl-thevetose, digitoxose, cymarose, and diginose by comparison of
1
3
(
H
C
1
13
1
1
13
its H and C NMR data with that of stauntoside A and hirundigoside G [7, 16]. Mild acid hydrolysis of 2 and subsequent silica
gel column chromatography purification led to obtain the monosugars, which confirmed the deduction mentioned above. The L-
configuration of diginose and D-configurations of 3-demethyl-thevetose, digitoxose, and cymarose were identified by comparing
their specific rotation with literatures [5, 11, 13-15]. The orientation of anomeric proton for diginose was identified to be by the
small value of JH-1′, H-2′ (J = 3.2 Hz). The orientations of 3-demethyl-thevetose, digitoxose, and cymarose were determined by
their large splitting values of JH-1′, H-2′ (J = 7.5, 7.8, and 9.5 Hz, respectively). The 14 linkages of sugar units were identified by
3
3
the HMBC correlations (Fig. 2) from δ
C-4″), and from δ 4.78 (H-1″ of digitoxose) to δ
4.33 (H-1′ of 3-demethyl-thevetose) and δ 79.0 (C-3) attached the sugar chain to the C-3 position of aglycone. Thus, steroidal
glycoside was identified to be hirundigenin 3-O-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-
digitoxopyranosyl-(1→4)-β-D-3-demethyl-thevetopyranoside, and named Hirundigoside K. Full assignment of H and C NMR
H
4.95 (H-1″″ of diginose) to δ
C
81.7 (C-4″′), from δ
H
4.75 (H-1″′ of cymarose) to δ 82.0
C
(
H
C
87.0 (C-4′ of 3-demethyl-thevetose). The HMBC correlation (Fig. 2) between
δ
H
C
2
1
13
1
13
1
1
data was achieved by analyses of H, C-NMR, H- H COSY, HSQC, and HMBC spectra (Table 1).
Steroidal glycoside 3 had a molecular formula C41 13 by the pseudomolecular ion at m/z 787.4249 [M+Na] (Calcd. for
13Na 787.4245) and accounted for 10 indices of hydrogen deficiency. The IR spectrum showed the absorption bands for
+
64
H O
C
41
H O
64
-1
-1
1
hydroxy (3442 cm ) and methyl (2939 cm ) functionalities. The H NMR spectrum (300 MHz, in C
5
D
5
N) showed the presence
of one olefinic proton at δ 5.44 (m, 1H, H-6) and three tertiary methyl protons at δ 1.00 (s, 3H, H-19), 1.11 (s, 3H, H-18), and
H
H