New triterpenoid and ergostane glycosides from the leaves of Hydrocotyle umbellata L.

Article abstract of DOI:10.1002/hlca.201100109

Two new triterpenoid glycosides, together with two new ergostane glycosides, umbellatosides A-D (1-4, resp.), have been isolated from the leaves of Hydrocotyle umbellata L. Their structures were established by 2D-NMR spectroscopic techniques (¹H,¹H-COSY, TOCSY, NOESY, HSQC, and HMBC) and mass spectrometry as 3β,22β-dihydroxy-3-O-[α-L- rhamnopyranosyl-(1→2)-β-D-glucuronopyranosyl]olean-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (1), 3-O-[α-L-rhamnopyranosyl- (1→2)-β-D-glucuronopyranosyl]oleanolic acid 28-O-β-D- glucopyranosyl ester (2), (3β,11α,26)-ergosta-5,24(28)-diene-3,11,26- triol 3-O-(β-D-glucopyranosyl)-11-O-(α-L-rhamnopyranosyl)-26-O- β-D-glucopyranoside (3), and (3β,11α,21,26)-ergosta-5,24(28)- diene-3,11,21,26-tetrol 3-O-(β-D-glucopyranosyl)-11-O-(α-L- rhamnopyranosyl)-26-O-β-D-glucopyranoside (4). Copyright

Full text of DOI:10.1002/hlca.201100109

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Helvetica Chimica Acta – Vol. 94 (2011)
New Triterpenoid and Ergostane Glycosides from the Leaves of Hydrocotyle
umbellata L.
by Adakarleny Sosaa)b)c), Carmelo Rosquetea), Luis Rojasb), Laurent Pouyse´guc), Ste´phane
Quideauc), Thomas Paululatd), Anne-Claire Mitaine-Offera), and Marie-Aleth Lacaille-Dubois*a)
a
´
´
´ ˆ
) Laboratoire de Pharmacognosie, Unite de Molecules dꢀInteret Biologique, UMIB UPRES-EA 3660,
´
´
Faculte de Pharmacie , Universite de Bourgogne, F-21079 Dijon Cedex
(phone: þ 33-380393229; fax: þ 33-380393300; e-mail: m-a.lacaille-dubois@u-bourgogne.fr)
b
c
´
) Laboratorio de Productos Naturales, Facultad de Ciencias, Universidad de Los Andes. Merida 5101,
Venezuela
´
´
´
) Universite de Bordeaux, Institut des Sciences Moleculaires (CNRS-UMR 5255) and Institut Europeen
de Chimie et Biologie, 2 rue Robert Escarpit, F-33607 Pessac Cedex
d) Organische Chemie II, Naturwissenschaftlich-Technische Fakultꢁt Universitꢁt Siegen,
Adolf-Reichwein-Strasse 2, D-57076 Siegen
Two new triterpenoid glycosides, together with two new ergostane glycosides, umbellatosides A – D
(1 – 4, resp.), have been isolated from the leaves of Hydrocotyle umbellata L. Their structures were
established by 2D-NMR spectroscopic techniques (1H,1H-COSY, TOCSY, NOESY, HSQC, and HMBC)
and mass spectrometry as 3b,22b-dihydroxy-3-O-[a-l-rhamnopyranosyl-(1 ! 2)-b-d-glucuronopyrano-
syl]olean-12-en-28-oic acid 28-O-b-d-glucopyranosyl ester (1), 3-O-[a-l-rhamnopyranosyl-(1 ! 2)-b-d-
glucuronopyranosyl]oleanolic acid 28-O-b-d-glucopyranosyl ester (2), (3b,11a,26)-ergosta-5,24(28)-
diene-3,11,26-triol 3-O-(b-d-glucopyranosyl)-11-O-(a-l-rhamnopyranosyl)-26-O-b-d-glucopyranoside
(3), and (3b,11a,21,26)-ergosta-5,24(28)-diene-3,11,21,26-tetrol 3-O-(b-d-glucopyranosyl)-11-O-(a-l-
rhamnopyranosyl)-26-O-b-d-glucopyranoside (4).
Introduction. – The genus Hydrocotyle (Apiaceae) is constituted by aquatic or semi-
aquatic plants particularly well represented in temperate areas and higher tropical
regions [1]. Some species were reported to have antioxidant and antitumoral properties
[2][3]. Triterpene saponins were isolated from several species of this genus [4 – 7].
From H. umbellata, a herbaceous plant native to North America and parts of South
America, this compound type has not been reported so far. In our continuing search for
bioactive saponins from tropical plants, we report here the isolation and structure eluci-
dation of two new triterpene saponins and two new ergostane glycosides, named umbella-
tosides A – D (1 – 4, resp.), from the 70% MeOH extract of the leaves of H. umbellata.
Results and Discussion. – The BuOH fraction of the MeOH/H2O 70 :30 extract of
the leaves of H. umbellata was submitted to multiple chromatographic steps involving
vacuum-liquid chromatography (VLC) on reversed-phase RP18 silica gel, and
medium-pressure liquid chromatography (MPLC) on normal silica gel and RP18
silica gel to yield umbellatosides A – D (1 – 4, resp.). Their structures were determined
mainly by spectroscopic methods including 600-MHz 1D- and 2D-NMR experiments
(1H,1H-COSY, TOCSY, NOESY, HSQC, and HMBC) in combination with HR-ESI-
MS and FAB-MS. Compounds 1 – 4 were isolated as amorphous powders.
ꢂ 2011 Verlag Helvetica Chimica Acta AG, Zꢃrich

Helvetica Chimica Acta – Vol. 94 (2011)
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Umbellatoside A (1) exhibited in the HR-ESI-MS (positive-ion mode) a pseudo-
molecular-ion peak at m/z 979.4883 ([M þ Na]þ) consistent with the molecular
formula C48H76O19. The FAB-MS of 1 (negative-ion mode) displayed a pseudo-
molecular-ion peak at m/z 955 ([M ꢀ H]ꢀ), followed by fragment-ion peaks at m/z 793
([M ꢀ H ꢀ 162]ꢀ), 647 ([M ꢀ H ꢀ 162 ꢀ 146]ꢀ), 633 ([M ꢀ H ꢀ 146 ꢀ 176]ꢀ), and 471
([M ꢀ H ꢀ 162 ꢀ 146 ꢀ 176]ꢀ) suggesting the elimination of one hexosyl, one desoxy-
hexosyl, and one hexosyluronic moiety, respectively. The aglycone of 1 was identified as
3b,22b-dihydroxyolean-12-en-28-oic acid by comparison of its NMR spectral data,
1
based on correlations observed in the H,1H-COSY, NOESY, HSQC, and HMBC
spectra, with those reported in the literature [8].
1
The H- and 13C-NMR spectra of 1 exhibited seven characteristic Me signals at
d(H) 0.87 (s, Me(25)), 0.95 (s, Me(24)), 1.01 (s, Me(29)), 1.06 (s, Me(30)), 1.12 (s,
Me(26)), 1.23 (s, Me(23)), and 1.84 (s, Me(27)), and six quaternary C-atom resonances
at d(C) 30.8 (C(20)), 37.0 (C(10)), 39.5 (C(4)), 40.1 (C(8)), 42.0 (C(14)), and 49.1
(C(17)), indicating the presence of a triterpenoid skeleton (see Table 1). Most of the
1H- and 13C-NMR signals assigned from extensive analysis of 2D-NMR spectra were
superimposable with those of 3b-hydroxyolean-12-en-28-oic acid except the presence
of an O-bearing moiety with a signal at d(H) 5.29 and d(C) 74.4. The deshielded H-
atom at d(H) 5.29 had a correlation with d(H) 1.78 (HaꢀC(21)) and 2.53 (HbꢀC(21)) in
the 1H,1H-COSY spectrum, indicating a OH group at C(22), and the HMBC between

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Helvetica Chimica Acta – Vol. 94 (2011)
d(H) 5.29 (HꢀC(22)) and d(C) 41.3 (C(18)) was also observed. The a-equatorial
orientation of the H-atom at C(22) was deduced from the ROESY cross-peaks d(H)
5.29 (HꢀC(22))/d(H) 2.40 (HbꢀC(16)), and d(H) 2.40 (HbꢀC(16))/d(H) 1.01 (Me(29))
indicating the b-axial orientation for OH at C(22). This was supported by the absence
of ROESY cross-peaks between d(H) 5.29 (HꢀC(22)) and d(H) 1.06 (Me(30)), and
between d(H) 5.29 (HꢀC(22)) and d(H) 3.52 (HꢀC(18)). These data allowed us to
characterize the aglycone of 1 as 3b,22b-dihydroxyolean-12-en-28-oic acid [8].
Compound 1 was shown to contain three sugar residues on the basis of the HSQC
spectrum. The three anomeric H-atom NMR signals at d(H) 4.78 (d, J ¼ 7.0), 6.27 (s),
and 6.30 (d, J ¼ 8.0) correlated with three anomeric C-atom signals at d(C) 106.5, 102.3,
and 95.8, respectively. Complete assignments of sugar H-atoms were achieved by
1
TOCSY and H,1H-COSY plots, while the C-atoms were assigned from HSQC and
HMBC spectra. Evaluation of spin-spin couplings and chemical shifts obtained from
the 2D-NMR spectra allowed the identification of one a-rhamnopyranosyl (Rha), one
b-glucopyranosyl (Glc), and one b-glucopyranosyluronic acid (Glc A) unit [9]. The
3
relatively large J(1,2) value (7.0 – 8.0 Hz) of the anomeric H-atom signals of Glc and
GlcA indicated a b-anomeric orientation. The large 1J(HꢀC(1),C(1)) value of the Rha
(167 Hz) confirmed that the anomeric H-atom was equatorial, indicating an a-pyranoid
anomeric form. The common d-configuration for Glc and Glc A, and the l-
configuration for Rha were determined by acid hydrolysis and GC analysis (see Exper.
Part). Analysis of HMBC connectivities between sugar residues and the aglycone
indicated that 1 was a bisdesmoside. The cross-peak in the HMBC spectrum between
d(H) 4.78 (Glc A HꢀC(1)) and d(C) 89.2 (Agly C(3)) showed that the b-
glucuronopyranosyl moiety was linked to the aglycone at C(3). Additional HMBCs
were observed between d(H) 6.27 (br. s, Rha HꢀC(1)) and d(C) 79.3 (Glc A C(2))
establishing the sequence at C(3) as a-l-rhamnopyranosyl-(1 ! 2)-b-d-glucuronopyr-
anosyl. The remaining sugar (terminal Glc) is linked to the aglycone by an ester linkage
at C(28), ascertained by the HSQC at d(H)/d(C) 6.30/95.8 and confirmed by the
HMBC d(H) 6.30 (d, J ¼8.0, Glc HꢀC(1))/d(C) 175.9 (Agly C(28)). All these data
were consistent with the structure of 1-O-[(3b,22b)-3-{[2-O-(6-deoxy-a-l-mannopyr-
anosyl)-b-d-glucopyranuronosyl]oxy}-22-hydroxy-28-oxoolean-12-en-28-yl]-b-d-gluco-
pyranose for 1, named umbellatoside A.
The HR-ESI-MS of umbellatoside B (2) showed a quasi-molecular-ion peak at m/z
963.4924 ([M þ Na]þ) consistent with the molecular formula C48H76O18. The FAB-MS
(negative-ion mode) of 2 displayed a pseudo-molecular ion peak at m/z 939 ([M ꢀ
H]ꢀ), and fragment-ion peaks at m/z 777 ([M ꢀ H ꢀ 162]ꢀ), 631 ([M ꢀ H ꢀ 162 ꢀ
146]ꢀ), 617 ([M ꢀ H ꢀ 146 ꢀ 176]ꢀ), and 455 ([M ꢀ H ꢀ 162 ꢀ 146 ꢀ 176]ꢀ), suggesting
the elimination of one hexosyl, one desoxyhexosyl, and one hexosyluronic acid moiety,
respectively. The aglycone of 2 was identified as oleanolic acid by comparison of its
1
NMR spectral data, based on correlations observed in the H,1H-COSY, NOESY,
HSQC, and HMBC spectra, with those reported in the literature (Table 1) [10].
Most of the H- and C-atom NMR signals of 2 (Table 1) were superimposable with
those of 1, except the signals at C(22). The absence of a signal at d(H) 5.29/d(C) 74.4
observed in 1 (C(22)) and the presence of a correlation in the HSQC spectrum d(H)
1.76, 1.84/d(C) 32.5 in 2 confirmed the presence of a CH2(22) group instead of a
secondary alcoholic function. The absolute configuration of the sugars was determined

Helvetica Chimica Acta – Vol. 94 (2011)
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Table 1. 1H- and 13C-NMR Data ((D5)Pyridine) of 1 and 2. d in ppm, J in Hz.
1
2
d(H)
d(C)
d(H)
d(C)
CH2(1)
CH2(2)
HꢀC(3)
C(4)
0.88a)
1.80, 2.24
38.7
26.5
89.2
39.5
55.8
18.5
0.84, 1.37
38.6
26.0
89.2
39.4
55.8
18.5
1.79, 2.22 – 2.28 (m)
3.33 (br. d, J ¼ 8.53)
–
3.33 (br. d, J ¼ 9.7)
–
HꢀC(5)
0.80 (br. d, J ¼ 11.3)
0.75 (br. d, J ¼ 11.3)
CH2(6)
1.25 – 1.32 (m),
1.28, 1.46
1.47 (br. d, J ¼ 11.7)
CH2(7)
C(8)
1.38 – 1.42 (m), 1.56 – 1.63 (m)
33.4
40.1
47.0
37.0
23.8
1.34, 1.48 (br. d, J ¼ 5.6)
33.1
39.9
48.0
36.9
23.8
–
1.78
–
–
HꢀC(9)
1.59 (t, J ¼ 9.3)
C(10)
–
CH2(11)
1.94 – 2.00 (m)
1.97 (br. d, J ¼ 12.1),
2.10 (t, J ¼ 13.0)
HꢀC(12)
C(13)
C(14)
5.61 (br. s)
–
–
122.7
144.4
42.0
5.44 (br. s)
122.9
144.1
42.1
–
–
CH2(15)
1.28, 2.43
35.9
1.18 (br. d, J ¼ 12.9),
2.32 – 2.37 (m)
1.85 – 1.92 (m)
–
28.0
CH2(16)
C(17)
HꢀC(18)
CH2(19)
C(20)
CH2(21)
HꢀC(22) or
CH2(22)
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
C(28)
2.09 – 2.15 (m), 2.36 – 2.41 (m)
–
3.52 (dd, J ¼ 13.1, 4.0)
1.38 – 1.41 (m), 2.82 (t, J ¼ 12.9)
–
32.2
49.1
41.3
47.0
30.8
36.1
74.4
24.0
47.0
41.7
46.8
30.8
34.0
32.5
3.20 (dd, J ¼ 13.5, 3.8)
1.29, 1.78
–
1.78, 2.53 (br. d, J ¼ 12.1)
1.12, 1.37
1.76, 1.84
5.29 (br. s)
1.23 (s)
0.95 (s)
0.87 (s)
1.12 (s)
1.84 (s)
–
28.2
17.0
15.7
17.6
27.2
175.9
33.2
24.6
1.26 (s)
0.96 (s)
0.84 (s)
1.08 (s)
1.30 (s)
–
28.2
16.9
15.5
17.5
26.1
176.4
33.1
23.7
Me(29)
Me(30)
Glc A
1.01 (s)
1.06 (s)
0.95 (s)
0.92 (s)
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
C(6)
4.78 (d, J ¼ 7.0)
106.5
79.3
75.9
71.2
78.8
170.0
4.77 (d, J ¼ 7.0)
106.5
79.2
75.9
71.2
78.9
170.0
4.02
4.00
4.28
4.24
–
4.01 – 4.04 (m)
3.99
4.29
4.27
–
Glc
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
6.30 (d, J ¼ 8.0)
4.13 (t, J ¼8.1)
4.28
4.17 – 4.22 (m)
3.99
95.8
74.1
76.6
72.2
75.8
62.3
6.30 (d, J ¼ 8.1)
4.18 (t, J ¼8.5)
4.26
4.18
3.99
95.7
74.1
76.5
72.2
75.8
62.3
4.37 (dd, J ¼ 11.7, 4.4),
4.38 (dd, J ¼ 12.0, 4.2),
4.43 (br. d, J ¼ 9.7)
4.44 (br. d, J ¼ 9.7)

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Helvetica Chimica Acta – Vol. 94 (2011)
Table 1 (cont.)
1
2
d(H)
d(C)
d(H)
d(C)
Rha
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
6.27 (br. s)
4.70 (br. s)
4.58 (br. d, J ¼ 8.1)
4.26
5.04
1.70 (d, J ¼ 4.8)
102.3
72.5
72.7
74.3
69.5
18.7
6.27 (br. s)
4.70 (br. d)
102.3
72.5
72.7
74.3
69.5
18.7
4.60 (br. d, J ¼ 8.1)
4.26
5.00 – 5.06 (m)
1.69 (d, J ¼ 5.6)
a) Overlapped signals are reported without multiplicity.
as described above (see also the Exper. Part). On the basis of these results, the structure
of 2 was elucidated as 1-O-[(3b)-3-{[2-O-(6-deoxy-a-l-mannopyranosyl)-b-d-gluco-
pyranuronosyl]oxy}-28-oxoolean-12-en-28-yl]-b-d-glucopyranose, named umbellato-
side B.
Umbellatoside C (3) exhibited in the HR-ESI-MS (positive-ion mode) the [M þ
Na]þ peak at m/z 923.4975 consistent with the molecular formula C46H76O17. FAB-
MS (negative-ion mode) of 3 displayed a pseudo-molecular-ion peak at m/z 899 ([M ꢀ
H]ꢀ), followed by fragment-ion peaks at m/z 753 ([M ꢀ H ꢀ 146]ꢀ), 737 ([M ꢀ H ꢀ
162]ꢀ), and 575 ([M ꢀ H ꢀ 162 ꢀ 162]ꢀ), suggesting the elimination of two hexosyl
and one desoxyhexosyl moiety, respectively. The basic skeleton of 3 was determined as
ergostane by comparison of its NMR spectral data, based on correlations observed in
1
the H,1H-COSY, NOESY, HSQC, and HMBC spectra, with those reported in the
literature [11].
The HSQC spectrum of the aglycone moiety of 3 (Table 2) displayed four Me
signals at d(H) 0.53 (s)/d(C) 12.8 (Me(18)), d(H) 0.84 (d, J ¼ 6.5)/d(C) 18.8 (Me(21)),
d(H) 1.01 (s)/d(C) 12.3 (Me(19)), d(H) 1.27 (d, J ¼ 6.9)/d(C) 17.6 (Me(27)), and an
olefinic signal at d(H) 5.26/d(C) 120.3. Signals for two secondary alcoholic functions at
d(H) 3.96/d(C) 76.4 and d(H) 4.20/d(C) 75.8, of one primary alcoholic function at
d(H) 3.59 and 4.28/d(C) 74.6, four quaternary C-atom signals at d(C) 35.6 (C(10)), 42.8
(C(13)), 152.9 (C(24)), and 136.9 (C(5)), and characteristic signals of an alkene CH2
moiety (d(H) 4.91 and 4.94/d(C) 109.0) were also observed. These signals suggested
that the aglycone part of 3 was closely related to a methylidenecholesterol (ergosta-
5,24(28)-dien-3b-ol) [12][13] with two additional OH functions. In the tetracyclic
system of the aglycone part, the presence of a secondary alcoholic function at C(11) was
deduced by correlations between the CH2 signal at d(H) 1.32 and 2.65 (CH2(12)), the
CH H-atom signals at d(H) 1.98 (HꢀC(9)) and d(C) 75.8 (C(11)) observed in the
HMBC spectrum of 3 (Fig.). The b-axial orientation of the H-atom at C(11) was
deduced from the ROESY cross-peaks d(H) 0.53 (s, Me(18))/4.20 (HꢀC(11)), d(H)
1.01 (HꢀC(19))/4.20 (HꢀC(11)), d(H) 2.65 (HbꢀC(12))/4.20 (HꢀC(11)), indicating the
a-orientation of HO at C(11). In the side chain, the presence of a primary alcoholic
function at C(26) was deduced from the correlations observed in the HMBC spectrum
between d(H) 3.59 (HaꢀC(26)) and d(C) 40.4 (C(25)), 152.9 (C(24)), and 17.6 (C(27)),

Helvetica Chimica Acta – Vol. 94 (2011)
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Table 2. 1H- and 13C-NMR Data ((D5)Pyridine) of 3 and 4. d in ppm, J in Hz.
3
4
d(H)
d(C)
d(H)
d(C)
CH2(1)
CH2(2)
HꢀC(3)
CH2(4)
C(5)
HꢀC(6)
CH2(7)
HꢀC(8)
HꢀC(9)
C(10)
1.16a), 2.83 – 2.89 (m)
1.73 – 1.76 (m), 2.04
3.96
38.7
29.9
76.4
35.1
136.9
120.3
30.0
40.8
54.8
35.6
75.8
45.7
42.8
55.2
23.3
28.0
55.9
12.8
12.3
35.9
18.8
1.12 – 1.18 (m), 2.83
1.68 – 1.72 (m), 2.03
3.96
1.44, 2.04
38.7
29.9
76.5
35.2
137.1
120.3
30.0
40.8
55.0
35.7
75.6
45.0
42.8
51.1
23.2
27.4
55.3
13.1
12.3
43.2
62.3
1.44 (d, J ¼ 11.7), 2.04
–
–
5.26 (br. s)
1.66 – 1.72 (m), 2.04
1.26
1.98
5.27 (br. s)
1.68 – 1.72 (m), 2.03
1.23 – 1.29 (m)
1.94 – 2.00 (m)
–
4.23
1.49, 2.85
–
1.77 – 1.83 (m)
1.45, 1.52 – 1.56 (m)
1.40, 1.90 – 1.95 (m)
1.96
0.61 (s)
1.00 (s)
–
HꢀC(11)
CH2(12)
C(13)
4.18 – 4.22 (m)
1.32, 2.65
–
1.87 – 1.93 (m)
1.37 – 1.42 (m), 1.48 – 1.52 (m)
1.23, 1.82 – 1.87 (m)
1.22
0.53 (s)
1.01 (s)
1.33
0.84 (d, J ¼ 6.5)
HꢀC(14)
CH2(15)
CH2(16)
HꢀC(17)
Me(18)
Me(19)
HꢀC(20)
Me or
CH2OH(21)
CH2(22)
CH2(23)
C(24)
HꢀC(25)
CH2(26)
Me(27)
CH2(28)
Glc I
1.63
3.80 (dd, J ¼ 10.5, 5.2), 3.99
1.19, 1.55 – 1.57 (m)
1.97, 2.14 – 2.19 (m)
–
34.6
31.9
152.9
40.4
74.6
17.6
2.18 – 2.24 (m), 2.32 – 2.38 (m)
1.80, 2.10
–
35.1
32.3
153.2
40.5
74.6
17.6
2.65
2.67 – 2.73 (m)
3.59 (t, J ¼ 8.9), 4.28
1.27 (d, J ¼ 6.9)
4.91 (br. s), 4.94 (br. s)
3.59 (t, J ¼ 8.5), 4.28
1.25 (d, J ¼ 6.9)
4.91 (br. s), 4.94 (br. s)
109.0
108.9
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
5.01 (d, J ¼ 7.6)
4.04 (t, J ¼ 8.1)
4.28
4.26
3.98
101.9
75.2
78.6
71.8
78.4
62.9
5.00 (d, J ¼ 7.5)
4.03 (t, J ¼ 8.1)
4.28
4.26
3.96
101.9
75.2
78.6
71.8
78.4
62.8
4.40 (dd, J ¼ 11.7, 4.8),
4.39 – 4.41 (m),
4.54 – 4.59 (m)
4.57 (dd, J ¼ 10.1, 3.0)
Glc II
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
4.87 (d, J ¼ 7.6)
4.04 (t, J ¼ 8.1)
4.28
4.24
3.96
105.2
75.4
78.6
71.8
78.6
62.9
4.86 (d, J ¼ 7.6)
4.03 (t, J ¼ 8.1)
4.22
4.20
3.94
105.2
75.4
78.5
71.7
78.4
62.9
4.38 (dd, J ¼ 11.7, 4.8),
4.36 – 4.40 (m),
4.52 – 4.56 (m)
4.56 (dd, J ¼ 10.1, 3.0)
Rha
HꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
Me(6)
5.55 (br. s)
4.48 (br. s)
4.45(dd, J ¼ 5.6, 2.8)
4.29
4.29
1.65 (d, J ¼ 5.2)
99.5
73.4
72.9
73.9
71.3
18.5
5.60 (br. s)
4.48 (br. s)
4.39 – 4.45 (m)
4.27
4.28
1.62 (d, J ¼ 5.2)
99.3
73.4
72.8
73.9
71.3
18.4
a) Overlapped signals are reported without multiplicity.

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Helvetica Chimica Acta – Vol. 94 (2011)
Figure. HMBCs of the aglycone of 3
between d(H) 4.28 (HbꢀC(26)) and d(C) 152.9 (C(24)) and 17.6 (C(27)), and the
reverse correlations between d(H) 1.27 (Me(27)) and d(C) 74.6 (C(26)). Furthermore,
additional HMBCs between the signals at d(H) 0.84 (Me(21)) and d(C) 35.9 (C(20))
and d(C) 34.6 (C(22)), between those at d(H) 1.97 (HaꢀC(23)) and d(C) 34.6 (C(22)),
between those at d(H) 2.16 (HbꢀC(23)) and d(C) 152.9 (C(24)), between those at d(H)
4.91 (HaꢀC(28)) and d(C) 152.9 (C(24)), 31.9 (C(23)), and 40.4 (C(25)) confirmed the
different functionalities of the side chain. Therefore, the aglycone of 3 was elucidated
as ergosta-5,24(28)-diene-3b,11b,26-triol, which has, to the best of our knowledge,
never been described.
The HSQC spectrum of 3 displayed three anomeric H-atom signals at d(H) 4.87 (d,
J ¼ 7.6), 5.01 (d, J ¼ 7.6), and 5.55 (br. s), which were correlated with three anomeric C-
atom signals at d(C) 105.2, 101.9, and 99.5, respectively, indicating the presence of three
sugar units. The evaluation of chemical shifts and spin-spin couplings obtained from the
2D-NMR data allowed the identification of two b-glucopyranosyl units (Glc I, Glc II),
and one a-rhamnopyranosyl unit (Rha) [9]. The d-configuration for Glc and l-
configuration for Rha was determined by acid hydrolysis and GC analysis (see Exper.
Part). The relatively large 3J(1,2) values (7.6 Hz) for Glc I and Glc II indicated their b-
anomeric orientation. The large 1J (HꢀC(1),C(1)) values of the Rha (167 Hz)
confirmed that the anomeric H-atom was equatorial indicating a a-pyranoid anomeric
form. The correlation in the HMBC spectrum between d(H) 4.87 (Glc II HꢀC(1)) and
d(C) 74.6 (Agly C(26)), and the reverse correlation between d(H) 3.59 (Agly HꢀC(26)
and d(C) 105.2 (Glc II C(1)) established that Glc II was at C(26) of the aglycone. The
HMBC between d(H) 5.01 (Glc I HꢀC(1)) and d(C) 76.4 (Agly C(3)), and the reverse
correlation between d(H) 3.96 (Agly C(3)) and d(C) 101.9 (Glc I C(1)) evidenced that
Glc I was at C(3). The HMBC between d(H) 5.55 (Rha HꢀC(1)) and d(C) 75.8 (Agly
C(11)) established the position of Rha as C(11); this was confirmed by the ROESY
cross-peak d(H) 5.55 (Rha HꢀC(1))/d(H) 4.20 (Agly HꢀC(11)).
Based on the above results, the structure of compound 3 was elucidated as (3b,11a)-
11-[(6-deoxy-a-l-mannopyranosyl)oxy]-26-(b-d-glucopyranosyloxy)ergosta-5,24(28)-
dien-3-yl b-d-glucopyranoside, named umbellatoside C. The presence of tridesmosidic
ergostane-type saponins in the genus Hydrocotyle was not previously reported.
Umbellatoside D (4) exhibited in the HR-ESI-MS (positive-ion mode) an [M þ
Na]þ peak at m/z 939.4935, consistent with the molecular formula C46H76O18. FAB-
MS (negative-ion mode) of 4 displayed a pseudo-molecular-ion peak at m/z 915 ([M ꢀ
H]ꢀ), and fragment-ion peaks at m/z 769 ([M ꢀ H ꢀ 146]ꢀ), 753 ([M ꢀ H ꢀ 162]ꢀ) and
591 ([M ꢀ H ꢀ 162 ꢀ 162]ꢀ), suggesting the elimination of one desoxyhexosyl and two

Helvetica Chimica Acta – Vol. 94 (2011)
1857
hexosyl moieties, respectively. The comparison of the 1H- and 13C-NMR chemical shifts
of 4 assigned from extensive 2D-NMR analysis with those of 3 (Table 2) revealed that
4 differed from 3 only by presence of an OH group at C(21) of the aglycone. The
absence of the Me signal observed in 3 (d(H) 0.84 (d, J ¼ 6.5)/d(C) 18.8 (Me (21))),
and the presence of a correlation d(H) 3.80 and 3.99/d(C) 62.3 in the HSQC spectrum
of 4 suggest the presence of a primary alcoholic function at C(21) in 4. This proposal
was confirmed from the ROESY cross-peaks of d(H) 3.80 and 3.99 (CH2(21))/d(H)
1.63 (HꢀC(20)), and d(H) 3.99 (HꢀC(21))/d(H) 2.35 (HꢀC(22)). The 1H- and
13C-NMR data of the oligosaccharide moiety of 4, assigned from the 2D-NMR
analyses, were almost superimposable with those of 3, indicating that 4 possessed the
same glycosidic substituents, a-rhamnopyranosyl at C(11), and b-glucopyranosyl
moieties at C(3) and C(26). The d-configuration for Glc and l-configuration for Rha
were determined by acid hydrolysis and GC analysis (see Exper. Part). Thus, the
compound 4 was elucidated as (3b,11a)-11-[(6-deoxy-a-l-mannopyranosyl)oxy]-26-
(b-d-glucopyranosyloxy)-21-hydroxyergosta-5,24(28)-dien-3-yl b-d-glucopyranoside,
named umbellatoside D.
Experimental Part
General. Medium-pressure liquid chromatography (MPLC): silica gel 60 (SiO2, 15 – 40 mm; Merck),
reversed-phase (RP) silica gel RP-18 (25 – 40 mm; Merck), Gilson M 305 pump; Bꢀchi glass column
(460 ꢁ 25, 460 ꢁ 15, and 230 ꢁ 15 mm), Bꢀchi precolumn (110 ꢁ 15 mm). Vacuum-liquid chromatog-
raphy (VLC): RP silica gel RP-18 (25 – 40 mm; Merck). TLC and HPTLC: silica gel 60 F254 (Merck),
solvent system: CHCl3/MeOH/H2O 60 :32 :6.5; detection: Komarowsky reagent, a 5 :1 mixture of p-
hydroxybenzaldehyde (2% in MeOH) and 50% EtOH/H2SO4. GC: Thermoquest gas chromatograph
using a DB-1701 cap. column (30 m ꢁ 0.25 mm, i.d.; J & W Scientific), with detection by FID; initial
temp. maintained at 808 for 5 min and then raised to 2708 at the rate of 158/min; carrier gas: He. NMR
Spectra: Varian VNMR-S 600 MHz spectrometer equipped with 3-mm triple resonance inverse and 3 mm
dual broadband probeheads; in 150 ml of (D5)pyridine; solvent signals as internal standard ((D5)pyri-
dine: d(H) ¼ 7.21, d(C) ¼ 123.5), all spectra recorded at T¼ 358; pulse sequences taken from Varian pulse
sequence library (gCOSY; gHSQCAD and gHMBCAD with adiabatic pulses CRISIS-HSQC and
CRISIS-HMBC); TOCSY spectra acquired using DIPSI spin-lock and 150 ms missing time; mixing time
in ROESY experiments: 300 ms; carbon type (Me, CH2, CH): DEPT experiments. FAB-MS: neg.-ion
mode; JEOL SX 102 spectrometer; glycerol as matrix; in m/z. HR-ESI-MS (pos.): Micromass Q-TOF-1
apparatus; in m/z.
Plant Material. The leaves of H. umbellata L. were provided from Botanical Garden of San Juan de
´
Lagunillas, in April 2008 (Merida, Venezuela), and identified by Er. Juan Carmona, Herbarium MERF
of Faculty of Pharmacy and Bioanalysis, Universidad de Los Andes. A voucher specimen (N8 113) was
deposited with the Herbarium MERF, Venezuela.
Extraction and Isolation. Powdered leaves (73 g) of H. umbellata were refluxed with MeOH/H2O
70 :30 (3 ꢁ 2 l) for 1 h. After evaporation of the solvent in vacuum, the resulting MeOH/H2O extract
(12.7 g) was suspended in H2O (200 ml), and partitioned successively with CH2Cl2 (3 ꢁ 300 ml) and
BuOH (sat. with H2O; 3 ꢁ 200 ml), yielding, after evaporation of the solvents, the corresponding CH2Cl2
(1 g) and BuOH (7.69 g) fractions. The BuOH residue was submitted to VLC (RP-18; MeOH/H2O
0 :100, 50 :50, 100 :0): Fractions VLC (1), VLC (2), and VLC (3) after evaporation. VLC (2) (914.3 mg)
was submitted to MPLC (system A: RP-18, MeOH/H2O 30 ! 100%): 1 (8.9 mg). VLC (3) (446 mg) was
subjected to MPLC (system B: silica gel 15 – 40 mm, CHCl3/MeOH/H2O 60 :32 :6.5): Frs. 1 – 18. Fr. 15
was submitted to MPLC (system A): 2 (13 mg). Fr. 9 was purified by MPLC (system C: RP-18, MeOH/
H2O 40 ! 100%): 3 (12.5 mg). Fr. 11 was submitted to MPLC (system A): 4 (11.6 mg).
Umbellatoside A (¼1-O-[(3b,22b)-3-{[2-O-(6-Deoxy-a-l-mannopyranosyl)-b-d-glucopyranurono-
syl]oxy}-22-hydroxy-28-oxoolean-12-en-28-yl]-b-d-glucopyranose; 1). White amorphous powder.

1858
Helvetica Chimica Acta – Vol. 94 (2011)
[a]2D0 ¼ þ18.5 (c ¼ 0.20, MeOH). 1H- ((D5)pyridine, 600 MHz) and 13C-NMR ((D5)pyridine, 150 MHz):
see Table 1. FAB-MS (neg.; glycerol matrix): 955 ([M ꢀ H]ꢀ), 793 ([M ꢀ H ꢀ 162]ꢀ), 647 ([M ꢀ H ꢀ
162 ꢀ 146]ꢀ), 633 ([M ꢀ H ꢀ 146 ꢀ 176]ꢀ), 471 ([M ꢀ H ꢀ 162 ꢀ 146 ꢀ 176]ꢀ). HR-ESI-MS (pos.):
979.4883 ([M þ Na]þ, C48H76NaO1þ9 ; calc. 979.4878).
Umbellatoside B (¼1-O-[(3b)-3-{[2-O-(6-Deoxy-a-l-mannopyranosyl)-b-d-glucopyranuronosyl]-
oxy}-28-oxoolean-12-en-28-yl]-b-d-glucopyranose; 2). White amorphous powder. [a]2D0 ¼ þ17.5 (c ¼ 0.25,
1
MeOH). H- ((D5)pyridine, 600 MHz) and 13C-NMR ((D5)pyridine, 150 MHz): see Table 1. FAB-MS
(neg.; glycerol matrix): 939 ([M ꢀ H]ꢀ), 777 ([M ꢀ H ꢀ 162]ꢀ), 631 ([M ꢀ H ꢀ 162 ꢀ 146]ꢀ), 617 ([M ꢀ
H ꢀ 146 ꢀ 176]ꢀ), 455 ([M ꢀ H ꢀ 162 ꢀ 146 ꢀ 176]ꢀ). HR-ESI-MS (pos.): 963.4924 ([M þ Na]þ,
C48H76NaOþ18 ; calc. 963.4929).
Umbellatoside C (¼(3b,11a)-11-[(6-Deoxy-a-l-mannopyranosyl)oxy]-26-(b-d-glucopyranosyloxy)-
ergosta-5,24(28)-dien-3-yl b-d-Glucopyranoside; 3). White amorphous powder. [a]2D0 ¼ þ30.0 (c ¼ 0.30,
1
MeOH). H- ((D5)pyridine, 600 MHz) and 13C-NMR ((D5)pyridine, 150 MHz): see Table 2. FAB-MS
(neg.; glycerol matrix): 899 ([M ꢀ H]ꢀ), 753 ([M ꢀ H ꢀ 146]ꢀ), 737 ([M ꢀ H ꢀ 162]ꢀ), 575 ([M ꢀ H ꢀ
162 ꢀ 162]ꢀ). HR-ESI-MS (pos.): 923.4975 ([M þ Na]þ, C46H76NaO1þ7 ; calc. 923.4980).
Umbellatoside D (¼(3b,11a)-11-[(6-Deoxy-a-l-mannopyranosyl)oxy]-26-(b-d-glucopyranosyl-
oxy)-21-hydroxyergosta-5,24(28)-dien-3-yl b-d-Glucopyranoside; 4). White amorphous powder.
[a]2D0 ¼ þ25.0 (c ¼ 0.33, MeOH). 1H- ((D5)pyridine, 600 MHz) and 13C-NMR ((D5)pyridine,
150 MHz): see Table 2. FAB-MS (neg.; glycerol matrix): 915 ([M ꢀ H]ꢀ), 769 ([M ꢀ H ꢀ 146]ꢀ), 753
([M ꢀ H ꢀ 162]ꢀ), 591 ([M ꢀ H ꢀ 162 ꢀ 162]ꢀ). HR-ESI-MS (pos.): 939.4935 ([M þ Na]þ,
C46H76NaOþ18 ; calc. 939.4929).
Acid Hydrolysis and GC Analysis. Each compound (3 mg) was hydrolyzed with 2n aq. CF3COOH
(5 ml) for 3 h at 958. After extraction with CH2Cl2 (3 ꢁ 5 ml), the aq. layer was repeatedly evaporated to
dryness with MeOH until neutral, and then analyzed by TLC (SiO2; CHCl3/MeOH/H2O 8 :5 :1) by
comparison with authentic samples. Furthermore, the residue of sugars was dissolved in anh. pyridine
(100 ml), and l-cysteine methyl ester hydrochloride (0.06 mol/l) was added. The mixture was stirred at
608 for 1 h, then 150 ml of HMDS (¼ hexamethyldisilazane)/Me3SiCl 3 :1 was added, and the mixture was
stirred at 608 for another 30 min. The precipitate was centrifuged off, and the supernatant was
concentrated under N2. The residue was partitioned between hexane and H2O (0.1 ml each), and the
hexane layer (1 ml) was analyzed by GC [14]. d-Glucose, d-glucuronic acid, and l-rhamnose were
detected from 1 and 2 by co-injection of the hydrolysate with standard silylated samples with tR values of
18.58, 15.40, and 13.18 min, resp. Identification of d-glucose and l-rhamnose was carried out for 3 and 4,
to give peaks at tR18.62 and 13.16 min, resp.
The authors are grateful to FONACIT-PCP (Project of postgraduate cooperation), Fran-
`
`
ceꢀVenezuela, and the Ministere FranÅais des Affaires Etrangeres for financial support.
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Received March 14, 2011