Resin glycosides from Ipomoea pes-caprae

Article abstract of DOI:10.1021/np070040h

Ipomoea pes-caprae (beach morning-glory; "rinonina" for the herbal drug in Mexico) is prescribed by traditional healers to moderate "heat" in an infected kidney. The hexane-soluble extract from the aerial parts of this medicinal plant, through preparative-scale recycling HPLC, yielded six new lipophilic oligosaccharides of jalapinolic acid: pescaproside B (1) and pescapreins V-IX (2-6). The previously known pescaproside A (7), pescapreins I-IV (8-11), and stoloniferin III (12) were also identified in the analyzed material by means of HPLC comparison with authentic samples. The glycosidic acid structure for all pentasaccharides was confirmed as simonic acid B. Pescaproside B (1), an acylated glycosidic acid methyl ester, is structurally related to pescaprein III (10). Pescapreins V (2) and VI (3) are diasteroisomeric tetraglycosidic lactones of operculinic acid C. Both of these compounds contain (2S)-methylbutyric and n-dodecanoic acids as their esterifying residues. Pescapreins VII (4) and IX (6) are pentasaccharides that contain an n-decanoyl group as their esterifying fatty acid residue instead of the n-dodecanoyl found in pescapreins I (8) and IV (11). Pescaprein VIII (5) represents an isomer of pescaprein II (9) containing an n-dodecanoyl unit as the esterifying residue at position C-4 of the third rhamnose moiety and a 2-methylpropanoyl at C-2 of the second rhamnose. High-field NMR spectroscopy and FAB mass spectrometry were used to characterize all new isolated compounds.

Full text of DOI:10.1021/np070040h

9
74
J. Nat. Prod. 2007, 70, 974-978
Resin Glycosides from Ipomoea pes-caprae†
Carolina Escobedo-Mart ´ı nez and Rogelio Pereda-Miranda*
Departamento de Farmacia, Facultad de Qu ´ı mica, UniVersidad Nacional Aut o´ noma de M e´ xico, Ciudad UniVersitaria,
Mexico City 04510 D.F., Mexico
ReceiVed January 25, 2007
Ipomoea pes-caprae (beach morning-glory; “ri n˜ onina” for the herbal drug in Mexico) is prescribed by traditional healers
to moderate “heat” in an infected kidney. The hexane-soluble extract from the aerial parts of this medicinal plant,
through preparative-scale recycling HPLC, yielded six new lipophilic oligosaccharides of jalapinolic acid: pescaproside
B (1) and pescapreins V-IX (2-6). The previously known pescaproside A (7), pescapreins I-IV (8-11), and stoloniferin
III (12) were also identified in the analyzed material by means of HPLC comparison with authentic samples. The
glycosidic acid structure for all pentasaccharides was confirmed as simonic acid B. Pescaproside B (1), an acylated
glycosidic acid methyl ester, is structurally related to pescaprein III (10). Pescapreins V (2) and VI (3) are diasteroisomeric
tetraglycosidic lactones of operculinic acid C. Both of these compounds contain (2S)-methylbutyric and n-dodecanoic
acids as their esterifying residues. Pescapreins VII (4) and IX (6) are pentasaccharides that contain an n-decanoyl group
as their esterifying fatty acid residue instead of the n-dodecanoyl found in pescapreins I (8) and IV (11). Pescaprein
VIII (5) represents an isomer of pescaprein II (9) containing an n-dodecanoyl unit as the esterifying residue at position
C-4 of the third rhamnose moiety and a 2-methylpropanoyl at C-2 of the second rhamnose. High-field NMR spectroscopy
and FAB mass spectrometry were used to characterize all new isolated compounds.
1
Ipomoea pes-caprae (L.) R. Br. (Convolvulaceae) is used in
infusions for urinary or kidney complaints, hypertension, and
scrofula and in decoctions to treat functional digestive disorders,
internal pain, colic, lumbago, arthritis, rheumatism, dysentery, and
2
3
inflammatory conditions. This herbal drug is called “ri n˜ onina”
in Mexico, reflecting the belief of traditional healers that it
4
moderates the “heat” in an infected kidney. The dried whole plant
is found readily in herbal markets and stores throughout the country.
Our previous research has been focused on the resin glycosides
of the Convolvulaceae family, whose structural diversity holds
considerable potential for medicinal usage.5 This paper describes
the isolation and characterization of six new oligosaccharides from
the hexane-soluble resin obtained from “ri n˜ onina”: pescaproside
B (1) and pescapreins V-IX (2-6). Several NMR techniques and
FABMS were used to characterize the structure of these complex
glycolipids. The known compounds pescaproside A (7), pescapreins
I-IV (8-11), and stoloniferin III (12) were also identified by
HPLC.7
,6
Hexane-soluble extracts of the crude drug “ri n˜ onina”, purchased
from a herbal store located in downtown Mexico City, were
compared by C18 reversed-phase HPLC (CH CN-MeOH, 9:1) with
3
7
reference solutions of authentic samples. This analysis allowed
for the detection of resin glycosides 8-12 (t 11-17 min) as well
as for a mixture of highly lipophilic compounds (t 32-45 min).
R
R
The total extract was fractionated by column chromatography on
silica gel. The major fractions, rich in these low-polarity resin
glycosides, were purified further by passing through activated
charcoal to eliminate pigmented residues. These processes were
followed by recycling preparative-scale HPLC, resulting in the
isolation of six new glycolipids (1-6).
5 8
difference of 84 mass units (C H O) between this natural product
and the previously reported pescaproside A (7; m/z 1197;
-
[
M - H] ) corroborated the presence of R-methylbutyric acid as
8
1
an additional ester group on the oligosaccharide core. From the H
NMR spectrum (Table 1), the esterified position at C-4 of the third
rhamnose moiety (Rha′′-1, δ 5.53) was identified through the strong
Results and Discussion
7
deshielding effect (δ∆ 1.3) relative to the nonesterified equivalent
on compound 7. The triplet-like signal for the methylene protons
at C-2 (δ 2.3) and the methyl ester group (δ 3.6) of the aglycon
confirmed its acyclic structure. HPLC analysis of several hexane-
soluble crude extracts prepared from commercial samples of this
herbal drug confirmed the presence of compounds 1 and 7 as natural
constituents. This type of non-macrocyclic resin glycoside has been
identified in only two other plant species apart from that used in
Pescaproside B (1) gave a quasimolecular ion at m/z 1281
-
[
M - H] (C64
113
H O25) in the negative-ion FABMS; the observed
†
This work was taken in part from the Ph.D. thesis of C. Escobedo-
Mart ´ı nez.
*
To whom correspondence should be addressed. Tel: +52-55-5622-
5
288. Fax: +52-55-5622-5329. E-mail: pereda@servidor.unam.mx.
1
0.1021/np070040h CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/19/2007

Resin Glycosides from Ipomoea pes-caprae
Journal of Natural Products, 2007, Vol. 70, No. 6 975
additional ester linkage (2S-methylbutyric acid), that is, C-4 of Rha′′
and C-3 of Rha′, in pescapreins V (2) and VI (3), respectively.
Pescaprein VII (4) afforded a [M - H]- peak at m/z 1137
C H O23) in contrast to the ion at m/z 1165 for its homologue 8
56 97
(
(
pescaprein I), indicating a difference of two methylene groups
-
between these compounds. Pescaprein VIII (5; [M - H] ion at
m/z 1235, C62
IX (6) showed also the [M - H] ion at m/z 1235 (C62
H
107
O
24) is an isomer of pescaprein II (9). Pescaprein
-
107 24
H O )
and a difference of 28 amu in relation to pescaprein IV (11) and
confirmed the presence of an n-decanoyl group as its acylating
residue. All compounds displayed the same fragmentation pattern
7
previously described for the pescaprein series, and the resulting
diagnostic peaks were useful to confirm the position for the
esterifying moieties; for example, the ion at m/z 907 [M - H -
-
C
12
H22O - C
H
6 10
O
4
]
indicated that the n-dodecanoyl residue is
located at C-4 of the third rhamnose moiety (Rha′′), and therefore
the isobutyroyl residue should be placed at C-2 of the second
rhamnose (Rha′).
Pescapreins VII-IX (4-6) showed similar H and 13C NMR
1
spectra (Tables 1 and 2) and displayed diagnostic signals for the
7
pescaprein series, thus confirming their pentasaccharide core as
simonic acid B, which has also been identified previously in the
7
the present investigation. The first of these yielded cuscutic acids
13
14
15
resin glycosides of I. batatas, I. stolonifera, and I. murucoides.
The features registered were (a) the site of lactonization at C-3
77.9) of the second saccharide (Rha) established by the
significant downfield shift observed for its geminal proton (δ 5.60);
b) a triplet-like signal for the methylene group at C-2 (δ 2.35)
for the fatty acid esterifying the hydroxyl group on position C-2 of
the third saccharide unit (Rha′: δ 5.79; δ 73); and (c) signals
A-D isolated from the seeds of Cuscuta chinensis, a member of
the Convolvulaceae family. The second yielded crypthophilic acids
A-C from the aerial parts of Scrophularia crypthophila, a species
taxonomically unrelated to Convolvulaceae but belonging instead
to the Scrophulariaceae.10
Negative-ion FAB mass spectra generated by pescapreins V (2)
and VI (3) were found to be very similar, with a pseudomolecular
M - H] ion at m/z 1103, and therefore these constituents
represented diastereoisomeric tetrasaccharides of molecular formula
9
(δ
C
(
H
H
C
attributable to the nonequivalent protons of the methylene group
at C-2 in the aglycon moiety that confirmed their macrocyclic
-
[
7
lactone-type structure: δH2a 2.91-2.94 (broad triplet-like doublet
1
57 100
C H O20. The H NMR spectrum of 2 showed signals of four
anomeric protons and, when compared with the values previously
reported for operculinic acid C,11 exhibited the acylation shifts for
of doublet of doublets) and δH2b 2.24-2.25 (doublet of doublet of
doublets). Furthermore, compound 5 showed a diagnostic signal at
δ 2.63 (1H, septet) for one isobutyroyl residue that esterified
5,12
H-3 of Rha, H-2 of Rha′, and H-4 of Rha′′. HMBC studies using
C
position C-2 at Rha′ (δ 73). Compound 6 also showed the
2,3
long-range heteronuclear coupling correlation ( JCH) provided
evidence for the location of each ester substituent at the oligosac-
charide core. Thus, the site of lactonization by the aglycon
C-1 174.9) could be placed at C-3 of the second saccharide (Rha)
at C-3 (δ 5.59); a methylbutyroyl group (δC-1 176.4) was located
at C-4 of Rha′′ (δ 5.82); and the additional acyl substituent
n-dodecanoic acid; δC-1 173.2) exhibited a JCH coupling with the
H-2 signal of Rha′ (δ 5.78).
characteristic triplet-like signal for the H-2 methylene equivalent
protons of the hexanoyl group at δ 2.38. From the TOCSY
1
experiment, edited H NMR subspectra for each individual monosac-
(δ
charide moiety were obtained for all oligosaccharides and permitted
the assignment of their resonances (Table 1). Homonuclear spin-
decoupling experiments were carried out to verify coupling
constants. The anomeric configuration in each sugar unit was
H
H
3
(
H
1
deduced from a 2D JCH NMR experiments. For D-sugars in the
1
From the H NMR spectrum of pescaprein VI (3), the three
acylation sites were identified at H-3 of Rha, H-3 of Rha′, and
H-2 of Rha′′. However, a significant signal overlap in the proton
4
C conformation, the R-anomeric configuration (â-equatorial C-H
1
1
bond) has a JCH value of 170 Hz, which is 10 Hz higher than that
ca. 160 Hz) for the â-anomer (R-axial C-H bond). From the
(
3
region δ 5.6-5.8 hampered JCH complete assigments for these
13
anomeric signals in the C NMR spectra of the glycosidic acids
signals. Careful analysis of the negative FABMS generated by both
tetrasaccharides 2 and 3 confirmed the substitution pattern proposed
for each glycolipid. The initial loss of the esterifying group at the
i.e., simonic acid B and operculinic acid C), 1JCH values for fucose
(
(
159 Hz) supported its found â-anomeric configuration. The
1
R-configuration was deduced for the rhamnose units ( JCH ) 170-
1
terminal saccharide unit (Rha′′) afforded a peak at m/z 921 in
5
72 Hz). All monosaccharides were in their naturally occurring
-
pescaprein VI (3) representing [M - H - C12
H
22O] , while the
form (D-fucose and L-rhamnose), as confirmed by optical rotation
measurements of the acid-liberated individual monosaccharides.
-
5 8
same elimination afforded a peak at m/z 1019 [M - H - C H O]
in pescaprein V (2). The loss of the terminal deoxyhexose afforded
The results of the present investigation suggest that convolvu-
laceous plants may have the ability to synthesize an array of
amphipatic oligosaccharides from such simple building blocks as
sugars and fatty acids. The highly elaborated chemical diversity of
this plant family, as this study illustrates, may confer selective
biological properties for which these species have long been used
in traditional agriculture and medicinal practices.5
-
the peak at m/z 873 [1019 - 146 (methylpentose unit)] in 2 and
at m/z 775 [921 - 146 (methylpentose unit)] in 3. Thus, these
peaks suggested that an n-dodecanoyl residue should be placed at
C-2 of Rha′′ in compound 3. The pattern for the nonequivalent
protons at C-2 of the aglycon in the H NMR spectrum (Table 1)
-
1
of pescapreins V (2) and VI (3) showed a multiplet in the form of
a broad triplet-like signal (ddd) for the deshielding resonance
(
δ 2.71 for 2 and δ 2.29 for 3) and a clear doublet of doublet of
Experimental Section
doublets for the shielded proton (δ 2.23 for 2 and δ 2.14 for 3).
This pattern was similar to that previously observed in the
pentasaccharides 8-11, which are macrocyclic lactones with the
esterification by the aglycon at C-3 of Rha.7 Therefore, the
remaining esterified position represented the location for the
General Experimental Procedures. All melting points were
determined on a Fisher-Johns apparatus and are uncorrected. Optical
rotations were measured with a Perkin-Elmer model 241 polarimeter.
1
13
H (500 MHz) and C (125.7 MHz) NMR experiments were conducted

9
76 Journal of Natural Products, 2007, Vol. 70, No. 6
Escobedo-Mart ´ı nez and Pereda-Miranda
Table 1. 1H NMR Spectroscopic Data for 1-6 (500 MHz)a
protonb
1
2
3
4
5
6
fuc-1
2
3
4
4.80-4.83*
4.79 d (8.0)
4.79 d (7.5)
4.80 d (7.9)
4.81 d (7.9)
4.79 d (7.8)
4.52 dd (9.5, 8.0)
4.16 dd (9.5, 3.5)
3.94 d (3.5)
4.53 dd (9.5, 8.0)
4.19 dd (9.5, 3.5)
3.92 d (3.5)
4.54 dd (9.5, 7.5)
4.20 dd (9.5, 3.5)
3.93 d (3.5)
4.52 dd (9.6, 7.9)
4.18 dd (9.6, 3.4)
3.91 d (3.4)
4.52 dd (9.5, 7.9)
4.20 dd (9.5, 3.3)
3.91 d (3.3)
4.50 dd (9.4, 7.8)
4.20 dd (9.4, 3.4)
3.90 d (3.4)
5
3.80 q (6.5)
3.83 q (6.5)
3.83 q (6.5)
3.81 q (6.4)
3.82 q (6.5)
3.80 q (6.4)
6
1.53 d (6.5)
1.53 d (6.5)
1.52 d (6.5)
1.51 d (6.4)
1.51 d (6.5)
1.50 d (6.4)
rha-1
6.28 d (1.0)
6.38 bs
6.4 d (1.5)
6.33 d (1.6)
6.33 d (1.3)
6.32 d (1.6)
2
3
4
5
4.64 dd (3.0, 1.0)
4.33 dd (9.5, 3.0)
4.31 dd (9.5, 9.5)
4.91 dq (9.0, 6.0)
1.62 d (6.0)
5.29 bs
5.26 dd (2.3, 1.5)
5.68 dd (9.8, 2.3)
4.70 dd (9.8, 9.8)
5.08 dq (9.8, 6.1)
1.55 bs
5.30 dd (2.3, 1.6)
5.61 dd (9.8, 2.3)
4.65 dd (9.8, 9.8)
5.00 dq (9.8, 6.4)
1.55 d (6.4)
5.31 dd (2.8, 1.3)
5.61 dd (9.6, 2.8)
4.63 dd (9.6, 9.6)
5.01 dq (9.6, 6.3)
1.57 d (6.3)
5.29 d (2.8, 1.6)
5.60 dd (9.8, 2.8)
4.62 dd (9.8, 9.8)
5.00 dq (9.8, 6.2)
1.56 d (6.2)
5.60 dd (9.5, 2.5)
4.59 dd (9.5, 9.5)
5.02 dq (9.5, 6.5)
1.60 d (6.5)
6
rha′-1
6.11 d (2.0)
5.56 d (1.5)
5.90 d (1.0)
5.66 d (1.7)
5.66 d (1.7)
5.64 d (1.3)
2
3
4
5
6.04 dd (3.5, 2.0)
4.67 dd (9.5, 3.5)
4.30 dd (9.5, 9.5)
4.35 dq (9.5, 6.0)
1.73 d (6.0)
5.78 dd (2.8, 1.5)
4.59 dd (9.5, 2.8)
4.25 dd (9.5, 9.5)
4.36 dq (9.5, 6.0)
1.67 d (6.0)
4.75 bs
5.82 dd (3.4, 1.7)
4.50 dd (9.1, 3.4)
4.27-4.34*
5.82 dd (3.0, 1.7)
4.51 dd (9.5, 3.0)
4.26 (9.5, 9.5)
4.33 dq (9.5, 6.1)
1.59 d (6.1)
5.81 dd (2.4, 1.3)
4.49 dd (9.3, 2.4)
4.25-4.32*
5.70 dd (9.5, 3.0)
4.57 dd (9.5, 9.5)
4.34 dq (9.5, 6.3)
1.54 d (6.3)
4.27-4.34*
4.25-4.32*
6
1.57 d (6. 0)
1.58 d (6.3)
rha′′-1
5.92 bs
6.15 d (1.5)
5.50 d (1.5)
5.92 bs
5.91 bs
5.90 bs
2
3
4
5
4.64 dd (3.5, 1.5)
4.42 dd (9.8, 3.0)
5.53 dd (9.8, 9.8)
4.37 dq (9.8, 6.0)
1.40 d (6.0)
4.77 dd (3.0, 1.5)
4.18 dd (9.5, 3.0)
5.82 dd (9.5, 9.5)
4.41 dq (9.5, 6.5)
1.44 d (6.5)
5.71 dd (3.0, 1.5)
4.48 dd (9.3, 3.0)
4.13 dd (9.3, 9.3)
4.29 dq (9.3, 6.3)
1.63 d (6.3)
4.65 dd (3.3, 1.4)
4.36 dd (9.2, 3.3)
4.26 dd (9.2, 9.2)
4.27-4.34*
4.62 dd (3.3, 2.0)
4.42 dd (9.8, 3.3)
5.76 dd (9.8, 9.8)
4.33 dq (9.3, 6.3)
1.37 d (6.3)
4.62 bs
4.36 dd (9.8, 3.4)
5.79 dd (9.8, 9.8)
4.34 dq (9.6, 6.2)
1.39 d (6.3)
6
1.65 d (6.1)
rha′′′-1
5.62 bs
5.58 d (1.5)
5.56 bs
5.55 d (1.1)
2
3
4
5
4.80-4.83 bs*
4.48 dd (9.3, 3.3)
4.23 dd (9.3, 9.3)
4.27 dq (9.3, 6.0)
1.58 d (6.5)
4.52 dd (3.3, 1.5)
4.53 dd (9.3, 3.3)
4.24 dd (9.3, 9.3)
4.27-4.34*
4.78 bs
4.77 bs
4.52 dd (9.5, 3.2)
4.24 dd (9.5, 9.5)
4.29 dq (9.5, 6.2)
1.70 d (6.2)
4.51 dd (9.3, 3.3)
4.25-4.32*
4.25-4.32*
1.69 d (6.1)
6
1.71 d (6.1)
jal-2a
2.33 t (7.5)
2.23 ddd (14.0,
2.14 ddd (14.5,
7.0, 2.5)
2.29 ddd (14.5,
12.5, 2.0)
3.91 m
0.96 t (7.3)
2.24 ddd (15.7,
7.7, 2.1)
2.91 ddd (15.7,
12.5, 1.8)
3.86 m
0.94 t (7.0)
2.25 ddd (14.0,
6.5, 2.6)
2.94 ddd (14.0,
11.6, 1.7)
3.87 m
0.95 t (6.9)
2.63 sept (7.0)
1.19 d (7.0)
1.16 d (7.0)
2.24 m
7
.0, 3.0)
c
c
c
c
c
2b
2.71 ddd (14.0,
2.0, 1.7)
2.91 ddd (13.1,
12.5, 1.8)
3.85 m
1
1
1
1
6
4.01 m
0.94 t (7.0)
3.87 m
1.2 t (7.0)
0.98 t (7.1)
iba-2
3
3′
mba-2
2.49 tq (7.0, 6.5)
1.20 d (7.0)
0.93 t (7.5)
2.53 tq (7.0, 6.5)
1.22 d (7.0)
0.96 t (7.5)
2.51 tq (7.0, 6.5)
1.20 d (7.0)
0.91 t (7.5)
2
3
-Me
-Me
hexa-2
6
deca-2
2.38 t (7.3)
0.93 t (7.1)
2.35 t (7.5)
0.77 t (7.2)
2.35 t (7.4)
0.86 t (7.2)
10
dodeca-2
2.32 t (7.5)
0.87 t (7.0)
2.34 ddd (15.5,
2.42 ddd (14.0,
6.5, 6.5)
2.77 ddd (14.0,
6.5, 6.0)
2.36 t (7.3)
0.87 t (7.1)
8
2
8
.5, 8.5)
.25 ddd (15.5,
.0, 8.0)
12
0.87 t (7.0)
0.86 t (7.1)
a
5 5
Data recorded in C D N. Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is given in parentheses (Hz). Chemical shifts
marked with an asterisk (*) indicate overlapped signals. Spin-coupled patterns are designated as follows: s ) singlet, bs ) broad singlet, d )
1
1
b
doublet, t ) triplet, m ) multiplet, q ) quartet, sept ) septet. All assignments are based on H- H COSY and TOCSY experiments. Abbreviations:
fuc ) fucose; rha ) rhamnose; jal ) 11-hydroxyhexadecanoyl; iba ) 2-methylpropanoyl; mba ) 2-methylbutanoyl; hexa ) hexanoyl; deca )
c
decanoyl; dodeca ) dodecanoyl. Broad triplet-like signal.
on a Bruker DMX-500 instrument. The NMR techniques were
performed according to previously described methodology.12 Negative-
ion LRFABMS and HRFABMS were recorded using a matrix of
triethanolamine on a JEOL SX-102A spectrometer. The instrumentation
used for HPLC analysis consisted of a Waters (Millipore Corp., Waters
Chromatography Division, Milford, MA) 600E multisolvent delivery
system equipped with a refractive index detector (Waters 410). GC-
MS was performed on a Hewlett-Packard 5890-II instrument coupled
to a JEOL SX-102A spectrometer. GC conditions: HP-5MS (5%
phenyl)-methylpolysiloxane column (30 m × 0.25 mm, film thickness
(50 g) was archived at the Departamento de Farmacia, Facultad de
Qu ´ı mica, UNAM. Macroscopic anatomical features enabled the drug
to be identified by one of the authors (R.P.-M.) as Ipomoea pes-caprae
through comparison with a voucher specimen collected on the Mexican
Pacific coast (R. Bye 17707) and deposited in the Ethnobotanical
Collection of the National Herbarium (MEXU), Instituto de Biolog ´ı a,
UNAM.
7
Extraction and Isolation. The whole plant (1 kg) was powdered
and extracted exhaustively by maceration at room temperature with
hexane to give, after removal of the solvent, a dark green syrup
(41.8 g). This crude extract was subjected to column chromatography
0
.25 µm); He, linear velocity 30 cm/s; 50 °C isothermal for 3 min,
linear gradient to 300 °C at 20 °C/min; final temperature hold, 10 min.
3
over silica gel (650 g) using a gradient of CHCl in hexane followed
MS conditions: ionization energy, 70 eV; ion source temperature,
by CHCl -Me CO (7:3). A total of 85 fractions (250 mL each) were
3
2
-
1
2
80 °C; interface temperature, 300 °C; scan speed, 2 scans s ; mass
collected and combined in 12 fractions (I-XII), which were partially
purified by passing through activated charcoal to eliminate pigmented
residues. Each fraction was analyzed by HPLC on a Symmetry C18
column (Waters; 5 µm, 4.6 × 250 mm) with an isocratic elution of
range, 33-880 amu.
Plant Material. The herbal drug “ri n˜ onina” was purchased in June
2004 at the herbal store “La Magnolia” in Mexico City. A small sample

Resin Glycosides from Ipomoea pes-caprae
Journal of Natural Products, 2007, Vol. 70, No. 6 977
Table 2. 13C NMR Spectral Data of Compounds 1-6 (125.7
injection: 500 µL). The elution was isocratic with CH
3
CN-MeOH
MHz)a
(9:1) using a flow rate of 9 mL/min. Eluates across the peaks with t
R
carbonb
of 32.91 (peak I), 38.44 (peak II), and 44.65 (peak III) were collected
by the technique of heart cutting and independently reinjected to be
recycled in order to achieve total homogeneity after 20 consecutive
cycles employing the same isocratic elution for peaks I and II. The
1
2
3
4
5
6
fuc-1
2
3
4
5
6
rha-1
2
3
4
5
6
rha′-1
2
3
4
5
6
rha′′-1
2
3
4
5
6
101.2
74.8
76.7
73.5
71.3
17.2
101.3
72.9
80.6
79.7
66.9
18.9
99.2
72.6
79.8
79.6
68.2
18.7
103.7
72.7
70.2
74.8
68.1
17.9
104.9
72.6
72.7
73.7
73.7
19.1
174.0
34.5
77.8
14.5
101.5
73.2
76.8
73.5
71.3
17.2
100.3
69.6
77.9
70.9
67.7
19.0
100.7
74.3
79.2
80.8
68.5
18.7
103.8
72.3
70.2
75.1
68.0
18.0
101.7
73.0
76.9
73.6
71.2
17.2
100.3
69.1
79.0
75.7
67.5
19.3
102.3
70.3
75.7
77.9
69.1
18.5
100.6
73.6
70.1
73.6
70.7
18.3
101.6
73.4
76.6
73.5
71.2
17.2
100.3
69.8
77.9
77.5
67.9
19.2
99.1
73.0
79.8
78.6
68.5
18.6
103.7
72.5
70.7
74.0
70.7
18.3
104.4
72.6
72.5
73.5
70.7
18.8
174.9
33.7
79.4
14.4
101.6
73.4
76.6
73.6
71.3
17.2
100.3
69.8
77.8
78.0
67.9
19.2
99.2
73.0
80.3
79.2
68.1
18.7
103.9
72.6
70.2
74.9
68.3
17.8
104.3
72.7
72.5
73.8
70.8
18.8
174.9
33.7
79.4
14.3
176.7
34.5
19.3
19.1
101.6
73.4
76.7
73.6
70.8
17.2
100.3
69.8
77.9
79.2
68.3
19.2
99.2
73.0
79.0
79.6
68.0
18.8
103.7
72.7
69.8
75.1
68.1
17.9
104.4
72.6
71.3
73.8
70.2
18.8
174.9
33.7
79.3
14.5
solvent system used for recycling peak III was CH
These techniques afforded pure compounds 4 (6.4 mg) and 12
3
CN-MeOH (7:3).
(
(
15.0 mg) from peak I; 2 (1.0 mg) and 3 (0.8 mg) from peak II; and 1
12.5 mg), 7 (23.3 mg), and 10 (7.6 mg) from peak III.
Pescaproside B (1): white powder; mp 87-88 °C; [R] -35 (c 0.1
D
MeOH); 1H and C NMR, see Tables 1 and 2; HRFABMS m/z
13
-
1
281.7572 [M - H] (calcd for C64
H
113
O
25 requires 1281.7570).
Pescaprein V (2): white powder; mp 140-142 °C; [R]
MeOH); H and C NMR, see Tables 1 and 2; negative FABMS m/z
D
-57 (c 0.1
1
13
-
-
-
1103 [M - H] , 1019 [M - H - C
5
H
8
O] , 921 [M - H - C12
H22O] ,
-
873 [1019 - C
6
H
10
O
4
] , 691, 545, 417, 271; HRFABMS m/z 1103.6730
-
[M - H] (calcd for C57
99
H O20 requires 1103.6729).
Pescaprein VI (3): white powder; mp 136-138 °C; [R] -17
D
1
13
(c 0.1 MeOH); H and C NMR, see Tables 1 and 2; negative FABMS
-
-
m/z 1103 [M - H] , 921 [M - H - C12
H
22O] , 837, 775 [921 -
-
-
C
H O
6 10 4
] , 691, 545, 417, 271; HRFABMS m/z 1103.6728 [M - H]
(calcd for C57
99
H O20 requires 1103.6729).
Pescaprein VII (4): white powder; mp 134-136 °C; [R]
D
-72
1
13
(
c 0.1 MeOH); H and C NMR, see Tables 1 and 2; negative FABMS
-
-
-
4
m/z 1137 [M - H] , 983 [M - H - C10
H
18O] , 837 [983 - C
H
6 10
O
] ,
-
5
C
45, 417, 271; HRFABMS m/z 1137.6421 [M - H] (calcd for
rha′′′-1
2
3
4
5
6
jal-1
2
56
H
97
O
23 requires 1137.6420).
Pescaprein VIII (5): white powder; mp 110-112 °C; [R]
D
-17
1
13
(
c 0.1 MeOH); H and C NMR, see Tables 1 and 2; negative FABMS
-
-
m/z 1235 [M - H] , 1089 [M - H - C
6
H
10
O
4
] , 1053 [M - H -
] , 837, 545, 417, 271; HRFABMS
24 requires 1235.7152).
-53
(c 0.1 MeOH); H and C NMR, see Tables 1 and 2; negative FABMS
-
-
C
12
H
22O] , 907 [1053 - C
6
H
10
O
4
-
174.9
34.2
79.3
14.6
174.4
34.6
79.8
14.6
m/z 1235.7154 [M - H] (calcd for C62
H
107
O
Pescaprein IX (6): white powder; mp 123-124 °C; [R]
D
1
13
1
1
1
6
-
-
m/z 1235 [M - H] , 1137 [M - H - C
6
H
10O] , 1081 [M - H -
] , 837, 545, 417, 271; HRFABMS
24 requires 1235.7152).
iba-1
2
3
C
H
18O] , 991 [1137 - C
-
H
6 10
O
-
4
10
-
m/z 1235.7153 [M - H] (calcd for C62
107
H O
Alkaline Hydrolysis of Resin Glycoside Mixture. A solution of
the crude fractions IV and V (80 mg) in 5% KOH-H O (5 mL) was
refluxed at 95 °C for 3 h. The reaction mixture was acidified to pH 4.0
and extracted with CHCl (30 mL). The organic layer was washed with
O, dried over anhydrous Na SO , and evaporated under reduced
3
′
2
mba-1
2
176.3
41.6
11.8
17.0
176.4
41.6
11.8
17.0
176.1
41.4
11.7
16.8
3
2
3
-Me
-Me
H
2
2
4
pressure. The aqueous phase was extracted with n-BuOH (20 mL) and
hexa-1
2
6
deca-1
2
173.0
34.4
14.3
173.5
34.7
14.3
concentrated to dryness. The residue from the organic phase was directly
+
R
analyzed by GC-MS: 2-methylpropanoic acid (t 4.12 min): m/z [M]
8
8 (10), 73 (27), 60 (3), 55 (5), 45 (7), 43 (100), 41 (40), 39 (10), 29
173.0
34.4
14.3
+
(
6), 27 (24); 2-methylbutanoic acid (t
R
7.2 min): m/z [M] 102 (3), 87
9.5 min):
m/z [M] 116 (2), 99 (3), 87 (21), 73 (63), 60 (100), 41 (16), 39 (7);
1
2
(33), 74 (100), 57 (50), 41 (28), 39 (8); n-hexanoic acid (t
R
+
dodeca-1
2
172.9
34.4
114.3
173.2
34.4
14.3
173.0
34.3
14.3
172.9
34.4
14.4
+
n-decanoic acid (t
129 (62), 115 (15), 112 (12), 87 (20), 73 (100), 60 (90), 57 (40), 55
(45), 43 (30), 41 (35), 39 (6); and n-dodecanoic acid (t 17.8 min):
R
14.6 min): m/z [M] 172 (2), 155 (3), 143 (12),
1
2
a
R
5 5
Data recorded in C D N. Chemical shifts (δ) are in ppm relative
+
m/z [M] 200 (15), 183 (2), 171 (18), 157 (40), 143 (10), 129 (48),
to TMS. All assignments are based on HMQC and HMBC experiments.
b
115 (20), 101 (15), 85 (33), 73 (100), 60 (80), 57 (30), 55 (47), 43
Abbreviations: fuc ) fucose; rha ) rhamnose; jal ) 11-hydroxy-
hexadecanoyl; iba ) 2-methylpropanoyl; mba ) 2-methylbutanoyl;
hexa ) hexanoyl; deca ) decanoyl; dodeca ) dodecanoyl.
(44), 41 (30). The residue extracted (35 mg) from the aqueous phase
was subjected to preparative HPLC on a Waters µBondapak NH
column (7.8 × 300 mm; 10 µm). The elution was isocratic with CH
CN-H
of 500 µL (35 mg/mL). This procedure yielded operculinic acid C
(5.2 mg, t
2
3
-
CH
3
CN-MeOH (9:1), a flow rate of 0.7 mL/min, and a sample injection
2
O (4:1), using a flow rate of 3 mL/min, and a sample injection
1
1
of 10 µL (1 mg/mL). This analysis allowed the comparison with
reference solutions of the previously reported resin glycosides from
) 8.7 min) and simonic acid B13 (18.6 mg, t
) 14.6 min),
R
R
7
this species, confirming the detection of the following compounds:
which were identified by comparison of physical constants and NMR
data with published values.
stoloniferin III (12, t
pescaprein III (10, t
pescaproside A (7, t
fractions VII-IX (4.6 g). The eluate across the peaks with t
min (13 mg) was collected by the technique of heart cutting and
independently reinjected in the apparatus operated in the recycle mode.8
These techniques afforded pure compound 6 (3 mg) after 20 consecutive
cycles employing the same isocratic elution. The eluate with t
12.8 mg) represented a complex mixture of related oligosaccharides,
which was further recycled (35 consecutive times) to afford pure
compound 5 (2 mg). The crude fractions IV and V were combined
R
11.8 min), pescaprein II (9, t
16.2 min), pescaprein IV (11, t
37.0 min), and pescaprein I (8, t
R
13.5 min),
17.5 min),
39.5 min) in
of 12.9
R
R
Sugar Analysis. A solution of fractions IV and V (15 mg) in 4 N
HCl (10 mL) was heated at 90 °C for 2 h. The reaction mixture was
diluted with H O (5 mL) and extracted with Et O (30 mL). The aqueous
2 2
phase was neutralized with 1 N KOH, extracted with n-BuOH (30 mL),
and concentrated to give a colorless solid. The residue was dissolved
R
R
R
in CH
column for carbohydrate analysis (µBondapak NH
O (17:3), a flow rate
3
CN-H
2
O and directly analyzed by HPLC: Waters standard
R
15.1
2
; 3.9 × 300 mm,
(
10 µm), using an isocratic elution of CH
3
CN-H
2
of 1 mL/min, and a sample injection of 20 µL (sample concentration:
5 mg/mL). Coelution experiments with standard carbohydrate samples
(
740 mg) and subjected to preparative HPLC on a reversed-phase C18
allowed the identification of L-rhamnose (t
(t ) 9.8 min). Each of these eluates were individually collected,
R
R
) 7.2 min) and D-fucose
column (7 µm, 19 × 300 nm), using a flow rate of 9 mL/min (sample

9
78 Journal of Natural Products, 2007, Vol. 70, No. 6
Escobedo-Mart ´ı nez and Pereda-Miranda
concentrated, and dissolved in H
stirring the solutions for 2 h at room temperature: L-rhamnose [R]598
8, [R]578 +8, [R]546 +9, [R]436 +15, [R]365 +21(c 0.1, H O); D-fucose
2
O. Optical activity was recorded after
(2) (a) Mart ´ı nez, M. Las Plantas Medicinales de M e´ xico; Ediciones
Botas: Mexico City, 1989; p 486. (b) Cano Asseleih, L. M. Flora
Medicinal de Veracruz. InVentario Etnobot a´ nico; Universidad Ve-
racruzana: Xalapa, Mexico, 1997; pp 184-185.
(3) “Ri n˜ onina”, coming from the Spanish word “ri n˜ o´ n”, meaning kidney,
is the Mexican name for this herbal tea used for the treatment of
“mal de or ´ı n” (bladder and kidney ailments).
+
[
2
R]598 +81, [R]578 +83, [R]546 +94, [R]436 +155, [R]365 +236 (c 0.1,
H
2
O).
Alkaline Hydrolysis of Pescaproside B (1). A solution of compound
1
(10 mg) was submitted to an alkaline hydrolysis following the same
procedures described above. The organic layer residue from extraction
(4) In Mexico, traditional healers classify illnesses and herbal remedies
as “hot” or “cold”. A hot-cold imbalance must be redressed by the
ingestion of contrary elements. For the hot-cold dichotomy, see:
(a) L o´ pez Austin, A. The Human Body and Ideology. Concepts of
the Ancient Nahuas; University of Utah Press: Salt Lake City, 1988;
pp 270-282. (b) Ortiz de Montellano, B. R. Aztec Medicine, Health,
and Nutrition; Rutgers University Press: New Brunswick, NJ, 1990;
pp 213-235.
was directly analyzed by GC-MS, and two peaks were detected:
+
2
5
2
1
-methylbutanoic acid (t
R
7.2 min) [m/z [M] 102 (3), 87 (33), 74 (100),
+
R
7 (50), 41 (28), 39 (8)] and n-dodecanoic acid (t 17.8 min) [m/z [M]
00 (15), 183 (2), 171 (18), 157 (40), 143 (10), 129 (48), 115 (20),
01 (15), 85 (33), 73 (100), 60 (80), 57 (30), 55 (47), 43 (44), 41
(30)]. The preparation and identification of 4-bromophenyacyl (2S)-
2
-methylbutyrate [40-42 °C; [R] +18 (c 1.0, MeOH)] from pesca-
D
(
5) Pereda-Miranda, R.; Bah, M. Curr. Top. Med. Chem. 2003, 3, 111-
131.
proside B (1) were performed according to previously reported
procedures. The aqueous phase-soluble residue was methylated with
8
(
6) Pereda-Miranda, R.; Fragoso-Serrano, M.; Escalante-S a´ nchez, E.;
Hern a´ ndez-Carlos, B.; Linares, E.; Bye, R. J. Nat. Prod. 2006, 69,
1460-1466.
CH
2 2
N to yield 3.5 mg of a white powder. The physical and
1
13
spectroscopic constants ( H and C NMR) registered for this derivative
1
3
were identical in all aspects to those previously reported for simonic
acid B methyl ester: mp 113-115 °C; [R] -82.5 (c 1.0, MeOH);
HRFABMS m/z 1015.5322 [M - H] (calcd for C47 23 requires
015.5325).
(
(
(
7) Pereda-Miranda, R.; Escalante-S a´ nchez, E.; Escobedo-Mart ´ı nez, C.
D
-
J. Nat. Prod. 2005, 68, 226-230.
83
H O
8) Pereda-Miranda, R.; Hern a´ ndez-Carlos, B. Tetrahedron 2002, 58,
1
3145-3154.
9) Du, X.-M.; Kohinata, K.; Kawasaki, T.; Guo, Y.-T.; Miyahara, K.
Acknowledgment. This research was partially supported by Consejo
Phytochemistry 1998, 48, 843-850.
Nacional de Ciencia y Tecnolog ´ı a (45861-Q). C.E.-M. is grateful to
CONACyT for a graduate student scholarship. Thanks are due to G.
Duarte and M. Guzm a´ n (USAI, Facultad de Qu ´ı mica) for the recording
of mass spectra. We thank Dr. M. Fragoso-Serrano (Facultad de
Qu ´ı mica, UNAM) for HPLC technical assistance.
(
10) C¸ alı s¸ , I.; Sezgin, Y.; D o¨ nmez, A. A.; R u¨ edi, P.; Tasdmir, D. J. Nat.
Prod. 2007, 70, 43-47.
(
11) Ono, M.; Kawasaki, T.; Miyahara, K. Chem. Pharm. Bull. 1989, 37,
3209-3213.
(
(
12) Bah, M.; Pereda-Miranda, R. Tetrahedron 1996, 52, 13063-13080.
13) Noda, N.; Yoda, S.; Kawasaki, T.; Miyahara, K. Chem. Pharm. Bull.
References and Notes
1992, 40, 3163-3168.
(
1) Ipomoea pes-caprae, an herbaceous pantropical trailing vine, colo-
nizes sand dunes and is called railroad vine, bay hops, and beach
morning-glory in Florida and Georgia (bay winters in Bahamas) and
(14) Noda, N.; Takahashi, N.; Kawasaki, T.; Miyahara, K.; Yang, C.-R.
Phytochemistry 1994, 36, 365-371.
(15) Ch e´ rigo, L.; Pereda-Miranda, R. J. Nat. Prod. 2006, 69, 595-599.
“
bejuco de playa” in Cuba, the Antilles, and the Caribbean coasts of
Mexico and Central and South America.
NP070040H