Y. Diab et al. / Steroids 77 (2012) 686–690
689
observed in the COSY spectrum of 1 were consistent with a 22-
methoxy-furostanol aglycone (Table 1).
(1?3)]-b-
ignated as desmettianoside A.
Desmettianoside B (2) was isolated as white amorphous solid.
Its molecular formula was assigned as C58 29 on the basis of
its HRESIMS spectrum, which exhibited a quasi-molecular ion peak
D
-glucopyranosyl-(1?4)-b-
D
-galactopyranoside and des-
The 3b and 5
the 13C NMR chemical shifts observed in rings A and B, which are
quite distinct in the case of 5 and 5b isomers [26,27]. The config-
uration of the methoxyl group at C-22 was assigned as due to the
a configurations were determined on the basis of
98
H O
a
+
1
13
a
at m/z 1281.6143 [M+Na] . The H and C NMR data of 2 included
signals indicating the same aglycone as in metabolite 1 (Table 1).
observed NOE correlation between the methoxy protons at d 3.28
and H-16 at d 4.47 [28]. The stereochemistry at C-25 was deduced
as R on the basis of the chemical shifts difference for the geminal
1
However, in contrast to 1, examination of the H NMR spectrum
of 2 revealed the presence of five anomeric protons of b-monosac-
charides resonating at d 4.87, 4.90, 5.17, 5.33, and 5.60 with J = 7.6–
7.9 Hz (Table 2). Acid hydrolysis of 2 yielded tigogenin as the arti-
factual aglycone and D-galactose and D-glucose as the sugar units.
Analysis of the NMR and MS spectra suggested that the only differ-
protons at H
that ab > 0.57 ppm is expected in 25S configuration, whereas
for 25R configuration ab < 0.48 ppm is observed [29]. On the ba-
sis of the above-mentioned data the aglycone was identified as
25R)-22 -methoxy-5 -furostan-3b,26-diol [30].
The presence of six b-monosaccharides in the molecule was de-
2
-26 (D
d
ab = d
a
b
ꢁd = 0.36 ppm). It has been reported
D
d
D
d
(
a
a
ence between compounds 1 and 2 was the absence of the terminal
1
3
b-
presence of one 4-monosubstituted b-
(Gal), one 2,3-disubstituted b- -glucopyranosyl unit (Glc-2), and
three terminal b- -glucopyranosyl units (Glc-1, Glc-3, and Glc-4).
D-xylopyranosyl unit. The C NMR chemical shifts indicated the
1
13
duced from the H and C NMR data of compound 1, which in-
cluded six doublet signals at d 4.86, 4.89, 5.11, 5.15, 5.23, and
D-galactopyranosyl unit
D
5
.58 with J = 6.3–7.8 Hz ascribed to six anomeric protons with their
corresponding carbons resonating at d 105.2, 102.7, 106.3, 105.1,
04.7, and 104.3 (Table 2). The fact that 35 signals observed in
D
The connectivity of the sugar units was deduced from the HMBC
0
1
correlations observed between H-1 of Glc-1 at d 4.87 and C-26
1
3
00
the C NMR spectrum of 1 were assigned to the sugar moiety sug-
gested that the latter consisted of one pentose and five hexose
units. Acid hydrolysis of 1 yielded an artifactual aglycone, which
was determined to be tigogenin (the corresponding cyclic spirost-
anol aglycone) by TLC analysis, and the sugar units, which were
of the aglycone at d 75.5, H-1 of Gal at d 4.90 and C-3 of the agly-
0
00
00
cone at d 77.7, H-1 of Glc-2 at d 5.17 and C-4 of Gal at d 80.5, H-
0
000
000
00000
1
of Glc-3 at d 5.33 and C-3 of Glc-2 at d 88.8, and H-1 of Glc-4
0
00
at d 5.60 and C-2 of Glc-2 at d 81.6. The nature and the sequence
of the sugars were verified by the ion peaks observed in the ESIMS
spectrum and MS–MS experiments of metabolite 2. Specifically, it
displayed a quasi-molecular ion peak at m/z 1281 [M+Na] and
fragment ion peaks at m/z 1249 [M+Na-CH
identified as
MS analyses.
D-galactose, D-glucose, and D-xylose by TLC and GC–
+
+
The sequence of the monosaccharides was determined by
extensive analyses of the MS and NMR spectra of compound 1.
The ESIMS spectrum and MS–MS experiments indicated the nature
and the sequence of the sugar units. In particular, 1 showed a qua-
3
OH] , 1119 [M+Na-
+
+
+
3
162] , 1087 [M+Na-CH OH-162] , 957 [M+Na-162-162] , 925
+
+
[M+Na-CH
[M+Na-CH
3
OH-162-162] , 795 [M+Na-162-162-162] , and 763
+
3
OH-162-162-162] . In addition, fragment ion peaks at
+
m/z 509 [162+162+162+Na] and 671 [162+162+162+162+Na]+
+
si-molecular ion peak at m/z 1413 [M+Na] and a fragment ion
+
peak at m/z 1381 [M+Na-CH
1
3
OH] . The fragment ion peaks at m/z
were observed.
281 [M+Na-132]+ and 1251 [M+Na-162] represented the
+
Accordingly, the structure of compound 2 was elucidated as
glycosidic cleavage of a terminal pentose and a terminal hexose,
respectively. Further fragment ion peaks were observed at m/z
(25R)-26-O-b-
diol 3-O-b- -glucopyranosyl-(1?2)-[b-
b- -glucopyranosyl-(1?4)-b- -galactopyranoside and designated
as desmettianoside B.
The natural origin of the aglycone part of compounds 1 and 2
through enzymatic processes in the living cells of the investigated
organism cannot be unambiguously proposed, since they could
potentially be artifacts formed from the corresponding 22-hydro-
xy-furostanols in the course of the extraction and isolation due
to their possible interconversion in the presence of MeOH and
D
-glucopyranosyl-22
a
-methoxy-5
a-furostan-3b,26-
D
D-glucopyranosyl-(1?3)]-
+
+
1
1
1
219 [M+Na-CH
3
OH-162] , 1119 [M+Na-132-162] , 1087 [M+Na-
D
D
+
+
32-162-CH
3
OH] , 1057 [M+Na-CH
32-162-162] , and 925 [M+Na-132-162-CH
fragment ion peaks were observed at m/z 509 [162+162+162+Na] ,
41 [162+162+162+132+Na] , 671 [162+162+162+162+Na] , and
03 [162+162+162+162+132+Na] .
The H NMR chemical shifts of the protons of the six sugar units
3
OH-162-162] , 957 [M+Na-
+
+
3
OH-162] . Finally,
+
+
+
6
8
+
1
were assigned by a combination of COSY and TOCSY cross-peaks
1
3
starting from the anomeric proton of each sugar unit. The
C
2
H O [31].
NMR chemical shifts of the corresponding carbons were assigned
based on the HSQC spectrum, while HMBC correlations provided
information on the glycosidic linkages. Specifically, the H and
Compounds 1 and 2 were evaluated for their molluscicidal
activity against B. alexandrina snails and exhibited high levels of
activity with LC100 values of 6 ± 0.6 and 11 ± 0.7 mg/L, respectively.
The molluscicidal activity of these bisdesmosidic furostanol sapo-
nins is in accordance with data reported for other steroidal sapo-
nins. The molluscicidal LC100 values for different saponins
isolated from other plant species varied considerably depending
upon the nature of the sugar chain, the number of sugar residues,
the sequence of the sugars, the interglycosidic linkages and the
substitution patterns of the aglycone, as well as the species of
snails [32]. Since desmettianosides A and B share the same agly-
cone moiety, it is assumed that the observed variation in their mol-
luscicidal activity results from the difference in the nature and
number of the sugar units.
1
1
3
C NMR data were indicative of the presence of a 4-monosubsti-
tuted b- -galactopyranosyl unit (Gal, d 4.89; d 102.7, 73.4, 75.7,
0.2, 75.6, 60.9), a 2,3-disubstituted b- -glucopyranosyl unit
Glc-2, d 5.15; d 105.1, 81.1, 88.7, 71.0, 77.8, 63.3), a 3-monosub-
stituted b- -glucopyranosyl unit (Glc-4, d 5.58; d 104.3, 75.3,
7.1, 69.3, 78.8, 62.3), two terminal b- -glucopyranosyl units
Glc-1, d 4.86; d 105.2, 75.3, 78.9, 72.0, 78.9, 63.1 and Glc-3, d
104.7, 75.6, 78.9, 71.7, 78.3, 62.6), and a terminal b- -xylo-
D
H
C
8
(
D
H
C
D
H
C
8
(
5
D
H
C
H
.23; d
C
D
pyranosyl unit (Xyl, d
HMBC spectrum of 1 correlations were observed between H-1 of
Glc-1 at d 4.86 and C-26 of the aglycone at d 75.4, H-1 of Gal at
H
5.11; d
C
106.3, 75.5, 78.4, 71.8, 67.3). In the
0
00
0
00
d 4.89 and C-3 of the aglycone at d 77.7, H-1 of Glc-2 at d 5.15
00
0000
000
and C-4 of Gal at d 80.2, H-1 of Glc-3 at d 5.23 and C-3 of
Acknowledgements
00000
000
Glc-2 at d 88.7, H-1 of Glc-4 at d 5.58 and C-2 of Glc-2 at d
000000
00000
8
1.1, and H-1
Therefore, compound 1 was identified as (25R)-26-O-b-
glucopyranosyl-22 -methoxy-5 -furostan-3b,26-diol 3-O-b-
xylopyranosyl-(1?3)-b- -glucopyranosyl-(1?2)-[b-
of Xyl at d 5.11 and C-3 of Glc-4 at d 87.1.
The authors thank Fayoum University for a postdoctoral
D
D
-
-
research studies scholarship awarded to Y.D., PNG Gerolymatos
for a postdoctoral research studies scholarship awarded to E.I.,
and Mr. Ahmed Salah (Fayoum University, Egypt) for his assistance
a
a
D
D-glucopyranosyl-