Mammalian multidrug resistance lipopentasaccharide inhibitors from Ipomoea alba seeds

Article abstract of DOI:10.1021/np300414d

As part of an ongoing project to identify inhibitors of multidrug efflux pumps, three new resin glycosides, albinosides I-III (1-3), were isolated from a CHCl₃-soluble extract from the seeds of moon vine (Ipomoea alba). Their structures were established through NMR spectroscopy and mass spectrometry as partially acylated branched pentasaccharides derived from three new glycosidic acids, named albinosinic acids A-C (4-6). The same oligosaccharide core formed by two d-quinovose, one d-glucose, and two l-rhamnose units was linked to either convolvulinolic or jalapinolic acid for 1 and 3, respectively. They were partially esterified with (2R,3R)-3-hydroxy-2-methylbutanoic, acetic, or 2-methyl-2-butenoic acid. Compound 2 has two d-quinovose and three l-rhamnose units, linked to convolvulinolic acid, and its esterifying residues were characterized as two units of 2-methyl-2-butenoic acid. The aglycone lactonization site was located at C-2 of the terminal rhamnose unit (Rha) for 1, at C-3 of the terminal rhamnose unit (Rha') for 2, and at C-3 of the second saccharide unit (Glc) for 3. Reversal of multidrug resistance by this class of plant metabolites was also evaluated in vinblastine-resistant human breast carcinoma cells (MCF-7/Vin). The noncytotoxic compound 3 exerted the strongest potentiation effect of vinblastine susceptibility to over 2140-fold, while a moderate activity was observed for 1 (3.1-fold) and 2 (2.6-fold) at a concentration of 25 μg/mL.

Full text of DOI:10.1021/np300414d

Article
pubs.acs.org/jnp
Mammalian Multidrug Resistance Lipopentasaccharide Inhibitors
from Ipomoea alba Seeds
Sara Cruz-Morales,† Jhon Castaneda-Gomez,† Gabriela Figueroa-Gonzalez,† Alma Delia Mendoza-García,†
́
́
̃
Argelia Lorence,‡ and Rogelio Pereda-Miranda*,†
†Departamento de Farmacia, Facultad de Química, Universidad Nacional Auton
04510 DF, Mexico
́ ́
oma de Mexico, Ciudad Universitaria, Mexico City,
‡Arkansas Biosciences Institute and Department of Chemistry and Physics. Arkansas State University, P.O. Box 639, State University,
Arkansas 72467, United States
S
* Supporting Information
ABSTRACT: As part of an ongoing project to identify inhibitors of multidrug efflux pumps, three new resin glycosides,
albinosides I−III (1−3), were isolated from a CHCl3-soluble extract from the seeds of moon vine (Ipomoea alba). Their
structures were established through NMR spectroscopy and mass spectrometry as partially acylated branched pentasaccharides
derived from three new glycosidic acids, named albinosinic acids A−C (4−6). The same oligosaccharide core formed by two
D-quinovose, one D-glucose, and two L-rhamnose units was linked to either convolvulinolic or jalapinolic acid for 1 and 3,
respectively. They were partially esterified with (2R,3R)-3-hydroxy-2-methylbutanoic, acetic, or 2-methyl-2-butenoic acid.
Compound 2 has two D-quinovose and three L-rhamnose units, linked to convolvulinolic acid, and its esterifying residues were
characterized as two units of 2-methyl-2-butenoic acid. The aglycone lactonization site was located at C-2 of the terminal
rhamnose unit (Rha) for 1, at C-3 of the terminal rhamnose unit (Rha′) for 2, and at C-3 of the second saccharide unit (Glc) for
3. Reversal of multidrug resistance by this class of plant metabolites was also evaluated in vinblastine-resistant human breast
carcinoma cells (MCF-7/Vin). The noncytotoxic compound 3 exerted the strongest potentiation effect of vinblastine
susceptibility to over 2140-fold, while a moderate activity was observed for 1 (3.1-fold) and 2 (2.6-fold) at a concentration of
25 μg/mL.
Mexico and Florida. Formerly known as Calonyction aculeatum,
it is now properly assigned to the genus Ipomoea, subgenus
Quamoclit, section Calonyction.5 It is a herbaceous climbing
plant growing to a height of 5−30 m tall with twining stems,
an annual in the North and a perennial in milder regions. The
leaves are entire or three-lobed, 5−15 cm long, with a 5−
20 cm long stem. The flowers are fragrant, white, and large,
with a 8−14 cm diameter. Seeds are ovoid with a smooth hard
cover, resembling a brownish nut6 (Figure S1, Supporting
Information). In Mexico, the decoction of the leaves, bark,
and flowers of this plant has been used since colonial time to
treat paralysis and the swelling of soft tissues due to the
accumulation of water.7 In India, a decoction of the whole
orning glory resin glycosides are amphipathic non-
1
Mcytotoxic glycolipids that have been characterized as
substrates for efflux pumps that produce the multidrug-
resistant (MDR) phenotype in both Gram-positive2 and
-negative bacteria3 as well as in mammalian cancer cell lines.4
In consequence, this class of active glycolipids represents
efflux inhibitors that could be used to lower current effective
therapeutic doses of drugs, thereby decreasing toxic side
effects in refractory malignancies. In this context, the present
investigation was undertaken to gain a deeper understanding
of the chemical diversity of these MDR reversal agents
essentially by isolating major glycolipids from commercial
moon vine seeds.
Moon vine or moonflower, Ipomoea alba L. (Convolvulaceae),
is a species of night-blooming morning glories native to tropical
and subtropical regions of America, from Northern Argentina to
Received: June 13, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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plant is used to treat snake bites.8 In Africa, the leaves are
used as food and as a soap substitute for bathing.8b In North
America, this species is widely used as an ornamental plant
due to its beautiful round shaped white flowers. There are
reports of the invasiveness potential of moon vine that can
cause problems in agricultural settings.9,10
and 145. Compound 3 yielded methyl 11-hydrohexadecanoate
(m/z 287 and 173) as the aglycone.15
Calonyctin, the natural mixture of resin glycosides extracted
from the leaves of moon vine, promotes sweet potato tuber
production and increased crop yields of potatoes, yams, peanuts,
beans, and wheat.11 Two glycosides with a tetrasaccharide
core, known as calonyctins A1 and A2 (Figure S2, Supporting
Information), were previously isolated as the active
compounds.12,13 Their structural difference was the length
of the fatty acid chain that forms their aglycone moieties,
11S-tetradecanoic acid (convolvulinolic acid) for calonyctin
12
A1 and 11S-hexadecanoic acid (jalapinolic acid) for
calonyctin A2.13
This work describes the isolation, purification, and struc-
ture elucidation of three intact resin glycosides, albinosides
I−III (1−3), with a new pentasaccharide core from the CHCl3-
soluble extract of moon vine seeds. In addition, saponification
of the isolated natural compounds 1−3 yielded their
constitutive glycosidic acids (4−6), named albinosinic acids
A−C, which displayed new glycosidation sequences. Finally,
reversal of multidrug resistance by compounds 1−3 was
evaluated in vinblastine-resistant human breast carcinoma
cells.
RESULTS AND DISCUSSION
■
CHCl3-soluble extracts of moon vine seeds were subjected
to precipitation with MeOH. The resin glycoside fraction
was then submitted to preparative recycling reversed-phase
HPLC using the techniques of peak shaving and heart
cutting.14 To achieve homogeneity, each peak collected was
recycled until overlapped components were separated. This
approach allowed the purification of three major compounds,
named albinosides I−III (1−3), which represented resin
glycosides of novel structure.
GC-MS analysis of the water-soluble acid hydrolysates as
TMS derivatives led to the identification of rhamnose (Rha),
quinovose (Qui), and glucose (Glc) in the approximate ratio
2:2:1 from 1 and 3 by co-elution experiments with retention
time identification using standard samples.15 A ratio of 3:2 for
Rha/Qui was found for compound 2 (Figure S36, Supporting
Information). The monosaccharide reaction mixtures under-
went derivatization with L-cysteine to form thiazolidines, which
were identified by GC-MS as their TMS ethers (Figure S37,
Supporting Information). This sugar analysis also confirmed
the absolute configuration for all monosaccharides as the
L-series for rhamnose and the D-series for quinovose and
glucose.16
Each glycosidic acid (4−6) was acetylated and methylated
to give a residue that was purified by C18 reversed-phase
HPLC, affording compounds 4a−6a. The main approaches
used for the elucidation of these novel oligosaccharide cores
involved the use of a combination of high-resolution FABMS
and high-field NMR spectroscopy.1 Negative-ion FABMS for
4 showed a pseudomolecular [M − H]− at m/z 989.4799
(Figure S24, Supporting Information), indicating a molec-
ular formula of C44H77O24 (calcd error: δ = −0.50), a peak
was detected at m/z 973.4850 (Figure S28, Supporting
Information) for 5, which allowed the calculation of its
molecular formula as C44H77O23 (calcd error: δ = −0.51),
and a peak at m/z 1017.5112 (Figure S32, Supporting
Information) was found for 6 (C46H81O24, calcd error:
δ = −0.49). Common fragment peaks generated by glycosidic
cleavage were observed in all three spectra, confirming the
branched pentasaccharide core.17 For example, the consec-
utive elimination of sugar units for compound 4 produced
the peaks at m/z 843 [989 − C6H10O4 (methylpentose unit)]−,
Pure compounds 1−3 were each saponified to yield their cor-
responding water-soluble glycosidic acids (4−6) and an organic
solvent-soluble fraction. The released acids were identified by
GC-MS as acetic and (2R,3R)-3-hydroxy-2-methylbutanoic or
nilic (nla) acids from 1, and 2-methyl-2-butenoic (tga) acid
from 2 and 3. Fractions I−III corresponding to compounds 1−3
were hydrolyzed in acid, and their Et2O-soluble extract was
methylated and then silylated. GC-MS analysis of the aglycone
for 1 and 2 showed that the mass spectrum corresponded to
that of the trimethylsilyl derivative of methyl 11-hydroxytetrade-
canoate because of the diagnostic R-cleavage ions at m/z 287
B
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a
1
Table 1. H NMR Spectroscopic Data of Albinosides I−III (1−3)
b
b
position
1
2
3
position
1
2
3
Qui-1
4.73 d (7.7)
4.75d (4.8)
4.77 d (7.6)
4
3.68 dd (9.0, 8.9)
3.88 dq (9.2, 6.0)
1.64 d (6.1)
5.55 brs
4.13 dd (9.2, 9.2)
2
4.48 dd (9.2, 7.8)
4.26−4.30 m*
3.63 m*
3.63 m*
1.50 d (5.7)
4.39 dd (9.2, 4.8) 4.44 dd (10.0,8.0)
3.66 dd (9.2,9.2) 4.44 dd (10.0,9.0)
4.39 dd (9.2,9.2) 3.55 dd (9.0,8.4)
3.66 dq (9.2,5.6) 3.64 dq (8.0,6.0)
5
3.66 dq (9.2,6.0) 4.28 dq (9.2, 5.2)
3
6
1.32 d (6.5)
5.78 s
1.68 d (5.6)
4
Rha″-1
5
2
3
4
5
6
5.07 brs
6
1.59 d (6.0)
1.59 d (6.0)
5.94 d (7.2)
4.22 t (7.6)
4.54 dd (9.0, 3.0)
4.31 dd (9.2,9.6)
4.25 dq (9.6,6.0)
1.67 d (6.5)
3.02 dd (10.0,7.2)
2.44 dd (10.0,7.2)
3.82 brs
Glc-1
5.68 d (7.7)
2
4.04 dd (9.0, 7.8)
3.95 dd (9.1, 8.9)
3.68 dd (8.9, 8.9)
3.88 ddd (9.0, 6.0, 3.0)
4.15−4.20 m*
4.63 dd (11.7, 2.0)
4.94 brs
3
5.78 dd (9.6, 8.0)
3.83 m*
4.28 ddd (9.6,5.2, 3.0)
4.30 dd (9.6, 5.2)
4.27 dd (9.6,2.0)
5.78 s
4
Conv 2a 2.64 m*
2b
5
6a
11
3.72−3.80 m*
0.91 t (7.4)
6b
14
0.89 t (7.2)
Rha-1
5.83 s
Jal 2a
2b
3.0 dd (8.8, 7.6)
2.43 dd (8.8, 8.0)
3.83 m*
2
3
4
5
6
5.60 dd (3.2, 2.1)
3.58 dd (8.4, 2.3)
5.58 dd (8.8, 10.0)
3.63 m*
5.80 brs
5.09 brs
4.92 dd (9.0, 2.4) 4.38 dd (8.8,2.8)
4.45 dd (9.0, 9.0) 4.36 dd (8.8, 8.8)
4.86 dq (9.0,6.8) 3.69 dq (9.2,6.0)
11
16
0.86 t (6.8)
nla-2
3
2.76 dq (7.2, 7.1)
4.28 dq (7.0, 6.5)
1.32 d (7.0)
1.20 d (7.0)
1.90 s
1.40 d (5.9)
1.91 d (6.5)
5.69 s
1.32 d (6.0)
5.74 s
Rha′-1 5.77 d (1.5)
4
2
3
4
5
6
4.57 brs
4.15 dd (3.0, 1.6) 5.85 brs
5
4.69 dd (9.8, 2.8)
5.81 dd (9.8, 9.8)
5.18 dq (9.9,6.4)
1.58 d (6.5)
5.66 dd (9.0, 2.0) 4.88 dd (9.2,3.2)
3.81 dd (9.0, 9.2) 4.46 dd (9.2,9.0)
3.66 dq (9.2, 6.0) 4.85 dq (9.0,6.4)
Ace-2
tga-3
4
7.09 dq (6.0, 1.2) 7.04 dq (6.8, 0.8)
1.51 t (7.2, 6.4) 1.49 d (7.2)
1.50 d (6.5)
5.24 d (6.0)
1.92 d (6.0)
5.24 d (7.6)
5
1.85 s
1.84 s
Qui′-1 5.13 d (7.8)
2
tga′-3
4
7.10 dq (6.0, 0.8) 7.08 dq (7.2, 0.9)
3.96 dd (9.0,8.0)
4.24 dd (9.2,7.0) 4.12 dd (9.2,7.0)
4.14 dd (9.2, 9.6) 5.55 dd (9.2,9.2)
1.44 d (6.4)
1.87 s
1.41d (7.2)
1.86 s
3
4.19 dd (9.0, 8.9)
5
a
500 MHz for 1 and 400 MHz for 2 and 3 in C5D5N. Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is given in parentheses
b
(Hz). Chemical shifts marked with an asterisk (*) indicate overlapped signals. Abbreviations: qui = quinovose; rha = rhamnose; glc = glucose;
conv = 11-hydroxytetradecanoyl; Jal = 11-hydroxyhexadecanoyl; nla = niloyl; ace = acetyl; tga = tigloyl.
697 [843 − C6H10O4 (methylpentose unit)]−, 551 [697 −
C6H10O4 (methylpentose unit)]−, 389 [551 − C6H10O5 (hexose
unit)]−, and 243 [389 − C6H10O4 (methylpentose unit)]−, which
indicated 11-hydroxytetradecanoic acid, as the aglycone.15 The
difference of 28 mass units (two methylene groups) between
compounds 4 and 6 as well as the formation of the same general
fragmentation pattern by glycosidic cleavage of each sugar moiety
at m/z 1017, 871, 725, 579, 417, and 271 confirmed the same
branched pentasaccharide core in both glycosidic acids and 11-
hydroxyhexadecanoic acid as the aglycone for 6. For compound 5,
the peaks at m/z 827, 681, 535, 389, and 243 represented the loss
of five methylpentose units, and the peak at m/z 243 indicated
11-hydroxytetradecanoic acid as the aglycone.15,17
in the five monosaccharides. The interglycosidic connectivities
were established by HMBC studies.1 For example, the
following key correlations were observed for compound 4a:
H-2 (δH 4.45) of Qui with C-1 (δC 100.9) of Glc, H-2 (δH
5.16) of Glc with C-1 (δC 98.6) of Rha′, H-6 (δH 3.99) of Glc
with C-1 (δC 100.8) of Qui′, H-1 (δH 4.89) of Rha with C-2
(δC 78.1) of Qui′, and H-1 (δH 4.67) of Qui with C-11 (δC
83.1) of convolvulinolic acid. The same glycosidation
sequence was found in derivative 6a but having jalapinolic
acid as the aglycone. For compound 5a, the observed
correlations were as follows: H-2 (δH 4.14) of Qui with C-1
(δC 98.0) of Rha, H-4 (δH 5.64) of Rha with C-1 (δC 99.9) of
Qui′, H-1 (δH 5.48) of Rha′ with C-3 (δC 78.7) of Qui′, H-2
(δH 4.18) of Qui′ with C-1 (δC 99.7) of Ram″, and H-1 (δH
4.68) of Qui with C-11 (δC 83.9) of convolvulinolic acid.
Therefore, the structure of albinosinic acid A (4) was character-
ized as (11S)-convolvulinolic acid 11-O-α-L-rhamnopyranosyl-
(1→2)-O-[-α-L-rhamnopyranosyl-(1→2)-6-deoxy-β-D-glucopyra-
nosyl-(1→6)]-O-β-D-glucopyranosyl-(1→2)]-6-deoxy-β-D-gluco-
pyranoside; the structure of albinosinic acid B (5) was
characterized as (11S)-convolvulinolic acid 11-O-α-L-rhamnopyr-
anosyl-(1→2)-O-[α-L-rhamnopyranosyl-(1→3)]-O-6-deoxy-β-D-
glucopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-6-deoxy-
β-D-glucopyranoside; and the structure of albinosinic acid C (6)
was characterized as (11S)-jalapinolic acid 11-O-α-L-rhamnopyr-
anosyl-(1→2)-O-[α-L-rhamnopyranosyl-(1→2)-6-deoxy-β-D-glu-
copyranosyl-(1→6)]-O-β-D-glucopyranosyl-(1→2)]-6-deoxy-β-D-
glucopyranoside.
1
Common features in both H (Table 3; Figures S26, S30, and
S34, Supporting Information) and 13C (Table 4; Figures S27, S31,
and S35, Supporting Information) NMR spectra of derivatives 4a−
6a were noted. In the low-field region of the HSQC spectrum, five
anomeric signals were confirmed: for compound 4a: Qui-1 (δH
4.67, δC 102.8), Glc-1 (δH 5.59, δC 100.9), Rha-1 (δH 4.89, δC 99.8),
Rha′-1 (δH 5.71, δC 98.6), and Qui′-1 (δH 5.07, δC 100.8); for 5a:
Qui-1 (δH 4.68, δC 101.0), Rha-1 (δH 5.82, δC 98.0), Rha′-1 (δH
5.48, δC 98.5), Rha″-1 (δH 5.64, δC 99.7), and Qui′-1 (δH 5.02, δC
99.9); and for 6a: Qui-1 (δH 4.68, δC 101.0), Glc-1 (δH 5.61, δC
101.3), Rha-1 (δH 5.82, δC 99.7), Rha′-1 (δH 5.50, δC 97.2), and
Qui′-1 (δH 5.09, δC 101.0). Therefore, five spin systems for each
individual monosaccharide moiety were readily distinguished in
1
the H−1H COSY and TOCSY spectra. This allowed for the
assignment of chemical shift values for all C-bonded protons
C
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a
Table 2. 13C NMR Spectroscopic Data of Albinosides I−III (1−3)
b
b
position
1
2
3
position
1
2
3
Qui-1
103.5
78.8
78.9
76.6
72.9
18.2
101.2
77.9
75.4
70.0
76.5
69.6
100.2
71.7
74.2
75.6
72.2
17.4
101.1
71.9
70.1
76.1
66.5
18.1
104.4
81.3
77.7
76.7
72.5
18.4
101.1
77.4
75.7
75.5
71.2
17.1
103.2
79.6
77.6
77.8
72.8
18.9
100.5
81.2
79.6
77.4
74.2
62.5
105.2
72.6
71.3
79.3
70.3
17.5
98.9
74.3
79.4
79.5
68.6
19.7
105.1
77.5
74.8
73.2
71.2
18.9
Rha″-1
103.3
70.7
71.5
72.4
69.2
17.1
171.1
33.8
78.8
14.3
2
2
3
4
5
6
3
4
5
6
Glc-1
Conv-1
173.5
33.2
81.5
14.3
2
2
3
11
4
14
5
Jal-1
173.0
6
2
35.0
81.5
14.7
Rha-1
98.4
72.4
77.6
77.5
66.7
17.8
97.0
71.7
77.8
73.8
70.3
16.7
103.1
71.4
79.7
72.9
68.3
15.6
11
2
16
3
nla-1
174.6
48.7
68.9
20.4
13.0
170.7
25.2
4
2
5
3
6
4
Rha′-1
2
5
Ace-1
3
2
4
tga-1
166.2
136.0
12.2
168.1
137.9
14.5
5
3
6
4
Qui′-1
5
10.9
12.7
2
3
4
5
6
tga′-1
166.7
136.7
12.6
168.6
138.7
14.5
3
4
5
10.8
12.7
a
125.7 MHz for 1 and 100 MHz for 2 and 3 in C5D5N. Chemical shifts (δ) are in ppm relative to TMS. Assignments are based on 1H−1H COSY
b
and TOCSY experiments. Abbreviations: qui = quinovose; rha = rhamnose; glc = glucose; conv = 11-hydroxytetradecanoyl; Jal = 11-
hydroxyhexadecanoyl; nla = niloyl; ace = acetyl; tga = tigloyl.
Albinoside I (1) gave a pseudomolecular [M − H]− ion at m/z
1113.5320 in the negative-ion FABMS, corresponding to the
molecular formula C51H85O26 (calcd error: δ = −0.80). Peaks from
the consecutive elimination of one niloyl residue [M − H − 100
(C5H8O2)]− at m/z 1013 and one acetyl [1013 − 42 (C2H2O)]−
at m/z 971 were also observed for 1 (Figure S3, Supporting
Information). For albinoside II (2), the mass spectra revealed a
[M − H]− peak at m/z 1119.5582 (Figure S10, Supporting
Information) consistent with molecular formula C54H87O24 (calcd
error: δ = −0.44), in contrast to the [M − H]− peak detected at m/z
1163.6208 (Figure S17, Supporting Information) for 3 (C57H95O24,
calcd error: δ = −0.43). The initial loss of a tigloyl residue
[M − H − 82 (C5H6O)]− afforded a peak at m/z 1037 for 2
and at m/z 1081 for 3, in addition to the peaks at m/z 663
[1037 − C6H10O4 − C6H10O4 − 82 (C5H6O)]− and m/z 853
[1081 − C6H10O4− 82 (C5H6O)]−, which indicated the loss of
two methylpentose units and one tigloyl residue for compound 2
ad the loss of a methylpentose unit and a tigloyl residue for 3.
Other observed peaks were produced by glycosidic cleavage of
the sugar moieties, which are common to all resin glycosides,17 as
the tetrasaccharide members of the calonyctin series.12,13
Substitution patterns on each individual saccharide unit in
albinosides I−III (1−3) were studied by 1H NMR spectroscopy
(Figures S4, S11, and S18, Supporting Information). COSY and
TOCSY techniques1 made possible the assignment of chemical
shift values for all C-bonded protons in each moiety (Table 1,
D
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a
1
Table 3. H NMR Spectroscopic Data for Derivatives
4a−6a
Table 4. 13C NMR Spectroscopic Data of Derivatives 4a−6a
a
b
position
4a
5a
6a
b
position
4a
5a
6a
Qui-1
102.8
75.6
76.2
74.5
70.4
17.3
100.9
74.6
74.4
73.5
76.1
67.9
99.8
73.4
73.3
74.1
70.6
17.9
98.6
70.1
70.4
71.1
67.9
18.0
100.8
78.1
81.6
74.3
69.9
17.6
101.0
74.5
76.1
74.2
69.8
17.7
101.0
73.5
75.9
73.9
69.5
17.6
101.3
69.8
71.2
71.8
69.6
63.0
99.7
70.5
70.4
71.2
68.5
17.6
97.2
72.8
79.0
76.2
68.0
18.7
101.0
77.0
75.9
69.8
72.6
16.0
2
Qui-1
4.67 d (8.0)
4.68 d (7.5)
4.68 d (7.5)
3
2
4.45 dd (8.0, 8.0)
5.61 dd (9.0, 8.5)
5.16 dd (9.5, 9.5)
3.68 dq (9.5, 6.0)
1.42 d (6.0)
4.14 dd (9.5, 8.0)
5.54 t (9.5)
4.19 dd (10.0, 7.5)
5.54 t (9.5)
4
3
5
4
5.08 t (9.5)
5.06 t (9.5)
6
5
4.12 dq (9.5,6.5)
1.35 d (6.0)
4.08 dq (9.5, 6.5)
1.35 d (6.5)
Glc-1
6
2
Glc-1
5.59 d (8.5)
5.61 d (7.5)
3
2
5.16 dd (9.5, 9.5)
5.67 t (10.0)
5.82 t (10.5)
5.64 t (10.5)
5.83 t (10.5)
4.13 m*
4
3
5
4
5.50 dd (9.0, 9.0)
4.01 ddd (9.0,5.0,3.0)
3.99 dd (10.0, 5.0)
3.98 dd (10.0, 3.0)
4.89 brs
6
5
Rha-1
98.0
74.2
76.6
71.1
68.8
17.7
98.5
71.1
74.8
76.7
67.7
18.8
99.9
77.5
78.7
71.7
69.8
16.8
99.7
71.0
73.0
78.8
70.7
17.6
171.2
35.0
83.9
14.9
2
6a
4.33 dd (12.5, 5.0)
4.62 dd (12.0, 4.5)
5.82 brs
3
6b
4
Rha-1
5.82 s
5
2
5.16 brs
6.12 brs
6.12 dd (3.0, 1.5)
6
3
5.5 dd (8.5, 2.0)
4.97 dd (9.5, 9.5)
3.48 dq (10.0, 6.0)
1.06 d (6.0)
5.99 dd (10.0, 3.0) 5.99 dd (10.0, 3.0)
5.64 dd (10.0, 9.0) 5.63 t (9.5)
Rha′-1
2
4
5
4.36 dq (9.5, 6.5)
1.35 d (6.0)
5.48 brs
4.33 m*
3
6
1.36 d (6.5)
5.50 brs
4
Rha′-1
5.71 brs
5
2
6.08 dd (4.0, 1.6)
5.78 dd (9.0, 4.0)
5.66 dd (10.0, 10.0)
4.76 dq (10.0, 6.5)
1.61 d (6.0)
4.81 brs
5.47 dd (3.5, 1.5)
4.77 dd (9.5, 4.0)
6
3
4.35 dd (9.5, 3.0)
Qui′-1
2
4
4.55 dd (10.0, 9.5) 4.41 t (9.0)
3
5
4.54 dq (9.5, 6.5)
1.80 d (6.0)
4.53 dq (9.5, 6.0)
4
6
1.80 d (6.0)
5
Qui′-1
5.07 d (7.5)
5.02 d (7.5)
5.09 d (8.0)
6
2
4.05 t (8.5)
4.18 dd (8.0, 8.0)
4.18 dd (10.0, 8.0)
Rha″-1
2
3
4.52 t (9.0)
5.65 dd (10.0, 9.0) 5.75 t (9.5)
4
4.93 dd (9.0, 8.0)
3.98 dq (8.0, 6.0)
1.33 d (6.0)
5.04 dd (9.5, 7.5)
4.12 dq (8.0, 6.5)
1.12 d (6.5)
5.36 t (10.0)
3
5
4.1 dq (10.5, 6.5)
1.12 d (6.5)
4
6
5
Rha″-1
5.64 s
6
2
3
4
5
6
5.85 dd (2.8, 2.4)
5.65 dd (9.0, 3.0)
5.03 dd (9.5, 9.0)
3.77 dq (10.0, 6.0)
1.29 d (6.0)
Conv-1
2
171.4
34.4
83.1
14.9
11
14
Jal-1
2
174.2
34.3
81.0
14.3
51.5
Conv 2 2.34 t (7.5)
2.35 t (7.5)
11
11
3.75 m*
3.65 m*
16
14
0.96 t (7.5)
0.93 t (7.5)
OCH3
51.5
51.5
Jal 2a
11
2.36 t (7.5)
3.65 m*
a
125.7 MHz in C5D5N. Chemical shifts (δ) are in ppm relative to
TMS. Assignments are based on H−1H COSY and TOCSY experi-
ments. Abbreviations: qui = quinovose; rha = rhamnose; glc =
glucose; conv = 11-hydroxytetradecanoyl; Jal = 11-hydroxyhexadecanoyl.
1
b
16
0.90 t (7.0)
3.62 s
OCH3
3.58 m*
3.62 s
a
500 MHz in C5D5N. Chemical shifts (δ) are in ppm relative to TMS.
The spin coupling (J) is given in parentheses (Hz). Assignments
b
are based on 1H−1H COSY and TOCSY experiments. Abbreviations:
(splitting as a ddd) centered ca. δ 2.7−2.8 and 3.2−3.4 showed
cross-peaks in their COSY and TOCSY spectra, because these
signals correspond to the nonequivalent diastereotopic protons
for the methylene C-2 of the aglycone when forming a
ring.1,14,17 The carbonyl carbon corresponding to the aglycone
qui = quinovose; rha = rhamnose; glc = glucose; conv = 11-hydroxy-
tetradecanoyl; Jal = 11-hydroxyhexadecanoyl.
2
Figures S6/S7, S13/S14, and S20/S21, Supporting Informa-
tion). 13C NMR signals were assigned by HMQC studies1
(Table 2, Figures S5, S8, S12, S15, S19, and S22, Supporting
Information). These natural products proved to be individual
macrocyclic lactone-type pentasaccharides since the multiplets
was identified through the JCH correlations (HMBC) with
these protons. The site for the macrolactonization was located
3
by the observed JCH correlations as follows: for albinoside I
(1), it was placed at C-2 of the terminal rhamnose unit (Rha)
by the observed correlation of H-2 (δH 5.60) of Rha with C-1
E
dx.doi.org/10.1021/np300414d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 5. Modulation of Vinblastine Cytotoxicity in Drug-Sensitive MCF-7 and Multidrug-Resistant MCF-7/Vin by Albinosides
I−III (1−3)
c
IC50 (μg/mL)
reversal fold
a
MCF-7/Vin−
MCF-7/Vin+
MCF-7 sens
RFMCF‑7/Vin
RFMCF‑7/Vin
RFMCF‑7 sens
−
+
compound
vinblastine
1.08 0.06
0.26 0.002
0.84 0.082
<0.00064
1.37 0.23
0.44 0.03
0.54 0.061
<0.00064
0.047 0.01
0.022 0.05
0.025 0.002
<0.00064
1
2
3
4.2
1.3
3.1
2.6
2.1
1.9
>1687.5
29.2
>2140.6
4.4
>73.4
15.7
b
reserpine
0.037 0.01
0.31 0.19
0.003 0.001
a
b
Serial dilutions from 0.00064 to 10 μg/mL of vinblastine in the presence or absence of glycolipid (25 μg/mL). Reserpine = 5 μg/mL as positive
c
control. RF = IC50 vinblastine/IC50 vinblastine in the presence of glycolipid. Each value represents the mean
SD from three independent
experiments.
performed on a Bruker MicrOTOF-Q high-resolution quadruple-time-
of-flight mass spectrometer. The samples were dissolved in HPLC-
grade MeOH (0.4 mg/mL) and infused directly to the ESI source
using a syringe pump at a flow rate of 180 μL/h. The nebulizer and
drying gas was nitrogen set at 0.4 bar and 4.0 L/min, respectively, with
the drying gas temperature being 180 °C. Capillary voltage was 3.2 kV.
Mass spectra were acquired over the range 50−3500 Da. The samples
were processed using Bruker Data Analysis software. The instru-
mentation used for HPLC analysis consisted of a Waters (Millipore
Corp., Waters Chromatography Division, Milford, MA, USA) 600E
multisolvent delivery system equipped with a refractive index detector
(Waters 410). Control of the equipment, data acquisition, processing,
and management of the chromatographic information were performed
by Empower 2 software (Waters). GC-MS was performed on a
Thermo-Electron instrument coupled to a Thermo-Electron spec-
trometer. GC conditions: DB-5MS (5% phenyl)-methylpolysiloxane
column (30 m × 0.25 mm, film thickness 0.1 μm); He linear velo-
city, 30 cm/s; 50 °C isothermal for 4 min, linear gradient to 300 at
40 °C/min; final temperature hold, 20 min. MS conditions: ionization
energy, 70 eV; ion source temperature, 250 °C; interface temperature,
270 °C; mass range, 45−600 amu.
Chemicals, Cell Lines, and Cell Cultures. RPMI 1640 medium
and fetal bovine serum were obtained from Gibco (Life Technologies,
Carlsbad, CA, USA). Sulforhodamine B, reserpine, and vinblastine
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Drug-
sensitive human breast carcinoma cell line MCF-7 was obtained from
the American Type Culture Collection (ATCC HTB-22). The
resistant counterpart MCF-7/Vin was developed through continuous
exposure to vinblastine during three consecutive years as previously
reported.4 All cell lines were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum and were cultured at 37 °C
in an atmosphere of 5% CO2 in air (100% humidity). To maintain
drug resistance, MCF-7/Vin+ cells were cultured in medium con-
taining 0.192 μg/mL vinblastine. At the same time, a stock of MCF-7/
Vin cells was maintained in vinblastine-free medium.
Plant Material. Seeds of moon vine (Ipomoea alba var. calonyction;
item 01052-PK-P1) were acquired from Park Seed (Greenwood, SC,
USA) in January 2008. For authentication purposes, 20 seeds were
germinated in soil, and four seedlings were grown to maturity under
greenhouse conditions (Figure S1, Supporting Information). One
specimen was deposited at the Ethnobotanical Collection of the
National Herbarium (MEXU 1143969), Instituto de Biología, UNAM.
Extraction and Isolation. Dried and milled seeds (363.83 g) were
exhaustively extracted by maceration at room temperature with hexane
and then with CHCl3 to give, after removal of the solvents, two
extracts: an oily residue (8 g) and a dark syrup (6 g). Both extracts
were compared by TLC (silica gel 60 F254 aluminum sheets; CHCl3−
MeOH, 4:1) with a reference solution of an authentic I. orizabensis
collection,15 which confirmed the similar lipophilic resin glycoside
mixtures (Rf 0.45) in the CHCl3-soluble extract. This extract was
subjected to a precipitation process with MeOH to obtain a resin
glycoside crude mixture, which was filtered under reduced pressure,
yielding a white solid (5 g). This mixture was resolved by HPLC on a
Symmetry C18 column (Waters; 7 μm, 19 × 300 mm) with isocratic
(δC 173.5) of convolvulinolic acid; for albinoside II (2), the
macrolactonization was placed at C-3 of the terminal rhamnose
unit (Rha′) by the correlation of H-3 (δH 5.66) of Rha′ with
C-1 (δC 171.1) of convolvulinolic acid; and for albinoside III
(3), the lactonization was placed at C-3 of the second unit
saccharide (Glc) by the correlation between H-3 (δH 5.78) of
Glc with C-1 (δC 173.0) of jalapinolic acid.
HMBC experiments1 also provided evidence for the location of
the ester substituents on the saccharide core through links
between a specific carbonyl ester group with their vicinal proton
resonance (2JCH) and the pyranose ring proton at the site of
esterification (3JCH) (Figures S9, S16, and S23, Supporting
Information). The following spectroscopic features were observed:
(a) for albinoside I (1), H-4 (δH 5.58) of Rha correlated with the
C-1 (δC 174.6) signal of the niloyl group and H-4 (δH 5.81) of
Rha′ with C-1 (δC 170.7) of the acetyl group; (b) for albinoside II
(2), H-2 (δH 5.80) of Rha with C-1 (δC 166.2) of the tigloyl
group (tga) and H-4 (δH 5.55) of Qui′ with C-1 (δC 166.7) of the
tigloyl group (tga′); (c) for albinoside III (3), H-2 (δH 5.85) of
Rha′ with C-1 (δC 168.1) of the tigloyl group (tga) and H-3 (δH
5.55) of Qui′ with C-1 (δC 168.6) of the tigloyl group (tga′)
(Figures S38 and S39, Supporting Information).
Screening for cytotoxicity of compounds 1−3 utilized both
vinblastine-sensitive and vinblastine-resistant human breast
carcinoma cells (MCF-7/Vin).4 Calculated IC50 values are
shown in Table S40 (Supporting Information). The lack of
cytotoxicity of these compounds (IC50 > 20 μg/mL) allowed us
to explore their potential as MDR modulators. Results of the
modulation assay are shown in Table 5. All tested compounds
displayed modulation of vinblastine susceptibility (Figures S41,
S42, and S43, Supporting Information) as previously observed
for other morning glory resin glycosides.4 Compound 3 was
extremely potent (reversal fold, RFMCF‑7/Vin+ > 2140), as it was
previously reported for murucoidin V, a proven substrate of
glycoprotein P, the chief plasma membrane associated
translocase responsible for the MDR phenotype in mammalian
cells.4 Therefore, these results further support the potential of
this family of compounds as efflux pump inhibitors for over-
coming MDR in cancer therapy, used in combination with
already approved antineoplastic drugs.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined on a Fisher-Johns apparatus and are uncorrected. Optical
rotations were measured with a Perkin-Elmer model 341 polarimeter.
1H (500 and 400 MHz) and 13C (125.7 and 100 MHz) NMR
experiments were conducted on a Varian Inova instrument. Negative-
ion LRFABMS were recorded using a matrix of triethanolamine on a
Thermo DFS spectrometer. Negative-ion HRESIMS experiments were
F
dx.doi.org/10.1021/np300414d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
elution using MeOH−CH3CN−H2O (5:4:1), a flow rate of 4 mL/
min, sample injection of 500 μL, and concentration of 0.1 mg/μL.
Eluates across the peaks with tR values 23.1 min (peak I), 99.7 min
(peak II), and 128.2 min (peak III) were collected by the technique of
heart cutting. Each subfraction was independently injected (sample
injection, 500 μL; concentration, 0.1 mg/μL) in the apparatus
operating in the recycle mode to achieve total homogeneity between
10 and 20 consecutive cycles and employing a Symmetry C18 column
(Waters; 7 μm, 19 × 300 mm), isocratic elution with MeOH−
CH3CN−H2O (10:7:3), and a flow rate of 8 mL/min for the first
subfraction. For the remaining fractions, isocratic elution with
MeOH−CH3CN (7:3) with a flow rate of 8.5 mL/min was used.
These procedures afforded pure compounds 1 (15 mg; tR 10.8 min)
from peak I, 2 (18.1 mg; tR 8.84 min) from peak II, and 3 (18.5 mg; tR
9.88 min) from peak III.
Albinoside I (1): white powder; mp 160−165 °C; [α]589 −38.3, [α]578
−40.0, [α]546 −44.16, [α]436 −75.0, [α]365 −115.0 (c 1.0, MeOH); 1H and
13C NMR, see Tables 1 and 2 (S4 and S5, Supporting Information);
negative FABMS m/z 1113 [M − H]−, 1013 [M − H − C5H8O2
(niloyl)]−, 971 [1013 − C2H2O (acetyl)]−, 825 [971 − C6H10O4
(methylpentose unit)]−, 679 [825 − C6H10O4 (methylpentose unit)]−,
533 [679 − C6H10O4 (methylpentose unit)]−, 389 [533 + H2O −
C6H10O5 (hexose unit)]−, 243 [389 − C6H10O4 (methylpentose
unit)]−; HRFABMS m/z 1113.5320 [M − H]− (calcd for C51H85O26
requires 1113.5329).
Albinoside II (2): white powder; mp 152−155 °C; [α]589 −56.0,
[α]578 −58.0, [α]546 −65.0, [α]436 −105.0, [α]365 −146.0 (c 1.0,
MeOH); 1H and 13C NMR, see Tables 1 and 2 (S11 and S12,
Supporting Information); negative FABMS m/z 1119 [M − H]−, 1037
[M − H − C5H6O (tigloyl)]−, 891 [1037 − C6H10O4 (methylpentose
unit)]−, 745 [891 − C6H10O4 (methylpentose unit)]−, 663 [745 −
C5H6O (tigloyl)]−, 517 [663 − C6H10O4 (methylpentose unit)]−, 389
[517 + H2O − C6H10O4 (methylpentose unit)]−, 243 [389 − C6H10O4
(methylpentose unit)]−; HRFABMS m/z 1119.5582 [M − H]− (calcd
for C54H87O24 requires 1119.5587).
Albinoside III (3): white powder; mp 146−150 °C; [α]589 −21.6,
[α]578 −22.4, [α]546 −25.6, [α]436 −40.8, [α]365 −57.0 (c 1.0, MeOH);
1H and 13C NMR, see Tables 1 and 2 (S18 and S19, Supporting
Information); negative FABMS m/z 1163 [M − H]−, 1081 [M − H −
C5H6O (tigloyl)]−, 935 [1081 − C6H10O4 (methylpentose unit)]−,
853 [935 − C5H6O (tigloyl)]−, 707 [853 − C6H10O4 (methylpentose
unit)]−, 561 [707 − C6H10O4 (methylpentose unit)]−, 417 [561 +
H2O − C6H10O5 (hexose unit)]−, 271 [417 − C6H10O4 (methyl-
pentose unit)]−; HRFABMS m/z 1163.6208 [M − H]− (calcd for
C57H95O24 requires 1163.6213).
Alkaline Hydrolysis of Compounds 1−3. Individual solutions of
compounds 1 (8 mg), 2 (9 mg), and 3 (9.5 mg) in 5% KOH−H2O
(0.75 mL) were refluxed at 95 °C for 3 h. Then, the reaction mixtures
were acidified to pH 5.0 and extracted with CHCl3 (2 × 5 mL) and
Et2O (2 × 5 mL). The organic layers were combined and washed with
H2O, dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The residues were directly analyzed by GC-MS. For
compound 1, acetic acid (tR 2.81 min): m/z [M]+ 60 (65), 45 (80),
43 (100), 29 (19), 15 (25); 3-hydroxy-2-methylbutyric acid (tR 7.95
min): m/z [M]+ 118 (2.0), 115 (10), 101 (20), 84 (12), 73 (70), 60
(100). For compounds 2 and 3, tiglic acid (tR 6.95 min): m/z [M]+
100 (30), 83 (18), 79 (38), 77 (40), 73 (100), 65 (9), 55 (22).
The aqueous phases were extracted with n-BuOH (10 mL) and
concentrated to give colorless solids. Saponification of compound 1
yielded 4 (7 mg), compound 2 afforded derivative 5 (8.5 mg), and
hydrolysis of compound 3 yielded glycosidic acid 6 (8.5 mg).
Albinosinic acid A (4): white powder; mp 148−150 °C; [α]589
−27.6, [α]578 −29.2, [α]546 −33.0, [α]436 −54.6, [α]365 −83.0 (c 1.0,
MeOH), negative FABMS m/z 989 [M − H]−, 843 [989 − C6H10O4
(methylpentose unit)]−, 697 [843 − C6H10O4 (methylpentose unit)]−,
551 [697 − C6H10O4 (methylpentose unit)]−, 389 [551 − C6H10O5
(hexose unit)]−, 243 [389 − C6H10O4 (methylpentose unit)];
HRFABMS m/z 989.4799 [M − H]− (calcd for C44H77O24 requires
989.4804).
Albinosinic acid B (5): white powder; mp 146−148 °C; [α]589
−25.0, [α]578 −26.6, [α]546 −30.0, [α]436 −46.6, [α]365 −71.6 (c 1.0,
MeOH), negative FABMS m/z 973 [M − H]−, 827 [973 − C6H10O4
(methylpentose unit)]−, 681 [827 − C6H10O4 (methylpentose unit)]−,
535 [681 − C6H10O4 (methylpentose unit)]−, 389 [535 − C6H10O4
(methylpentose unit)]−, 243 [389 − C6H10O4 (methylpentose
unit)]−; HRFABMS m/z 973.4850 [M − H]− (calcd for C44H77O23
requires 973.4855).
Albinosinic acid C (6): white powder; mp 142−144 °C; [α]589
−36.4, [α]578 −37.5, [α]546 −42.1, [α]436 −69.2, [α]365 −106.0 (c 1.0,
MeOH), negative FABMS m/z 1017 [M − H]−, 871 [1017 −
C6H10O4 (methylpentose unit)]−, 725 [871 − C6H10O4 (methyl-
pentose unit)]−, 579 [725 − C6H10O4 (methylpentose unit)]−, 417
[579 − C6H10O5 (hexose unit)]−, 271 [417 − C6H10O4 (methyl-
pentose unit)]; HRFABMS m/z 1017.5112 [M − H]− (calcd for
C46H81O24 requires 1017.5117).
Derivatization of 4−6. Each individual glycosidic acid (5 mg of 4,
6.5 mg of 5, and 6.0 mg of 6) was acetylated (Ac2O−C5H5N, 2:1) and
methylated with CH2N2 to give a residue (8 mg of 4a, 9 mg of 5a, and
8.5 mg of 6a), which was subjected to preparative HPLC on a
reversed-phase C18 column (7 μm, 19 × 300 mm). The elution was
isocratic with CH3CN−H2O (95:5) using a flow rate of 8.0 mL/min.
For compound 4a, the eluate across the peak with a tR value of 11.89
min was collected by heart cutting and independently reinjected in the
apparatus operated in the recycle mode to achieve total homogeneity
after 10 consecutive cycles employing the same isocratic elution. The
same method was used for compounds 5a (tR 18.98 min) and 6a
(tR 21.14 min).
Peracetylalbinosinic Acid A Methyl Ester (4a): white powder; mp
99−101 °C; [α]589 −5.7, [α]578 −5.7, [α]546 −7.1, [α]436 −10.0, [α]365
−15.0 (c 1.0, MeOH); 1H and 13C NMR, see Tables 3 and 4 (S26 and
S27, Supporting Information); MALDIMS m/z [M + Na]+ 1531.
Peracetylalbinosinic Acid B Methyl Ester (5a): white powder; mp
89−92 °C ; [α]589 −2.5, [α]578 −2.5, [α]546 −5.0, [α]436 −10.0, [α]365
−18.7 (c 1.0, MeOH); 1H and 13C NMR, see Tables 3 and 4 (S30 and
S31, Supporting Information); MALDIMS m/z [M + Na]+ 1473.
Peracetylalbinosinic Acid C Methyl Ester (6a): white powder; mp
78−80 °C; [α]589 −4.0, [α]578 −6.0, [α]546 −8.0, [α]436 −8.0, [α]365
−12.0 (c 1.0, MeOH); 1H and 13C NMR, see Tables 3 and 4 (S34 and
S35, Supporting Information); MALDIMS m/z [M + Na]+ 1559.
Determination of Configuration of 3-Hydroxy-2-methylbu-
tyrate. Preparation and identification of 4-bromophenacyl (2R,3R)-3-
hydroxy-2-methylbutyrate were performed according to a previously
reported procedure: mp 56−59 °C; [α]D −6.0 (c 1.0 CHCl3); GC-MS
m/z 118 (2.0), 115 (10), 101 (20), 84 (12), 73 (70), 60 (100). This
transesterification procedure has been used to confirm the absolute
configuration for 3-hydroxy-2-methylbutyrate.14
Acid Hydrolysis and Sugar Analysis. Fractions I, II, and III
(15 mg of each one) in 10 mL of 4 N HCl were refluxed at 90 °C for
2 h. Then, the reaction mixtures were diluted with 5 mL of H2O and
extracted with ether (3 × 10 mL). The organic layer was evaporated to
dryness, dissolved in CHCl3 (3 mL), and treated with CH2N2. The
aqueous phase was neutralized with 1 N KOH and extracted with n-
BuOH (10 mL), then washed with H2O (2 × 5 mL) and concentrated
to give a colorless solid. The sugar units were converted into volatile
derivatives by treatment with chlorotrimethylsilane (Sigma Sil-A) and
then analyzed by GC-MS, as previously described,18 by applying the
following conditions: DB-5MS (10 m × 0.18 mm, film thickeness
0.18 μm); He, 2 mL/min; 100 °C isothermal for 3 min, linear gradient
to 300 at 20 °C/min. Retention times for TMS derivatives of common
sugars were used as standards for GC identification: L-rhamnose tR
5.25 min, D-fucose tR 5.47 min, D-quinovose tR 6.17 min, and D-glucose
tR 7.15 min. D-Quinovose, D-glucose, and L-rhamnose were detected
in fractions I and III, while in fraction II only D-quinovose and
L-rhamnose were detected.
Aditionally, thiazolidine derivatives of compounds 4−6 were pre-
pared according to previously described procedures.16 The TMS
thiazolidine derivatives were directly analyzed by GC under the same
conditions described above. TMS derivatives of D-quinovose, D-glucose,
and L-rhamnose were used as standard authentic samples: L-rhamnose,
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tR 4.58 min; D-quinovose, tR 4.64 min; and D-glucose, tR 4.53 min.
D-Quinovose, D-glucose and L-rhamnose were detected in fractions I and
III, while only D-quinovose and L-rhamnose were detected in fraction II.
Identification of Aglycones. The derivatized (CH2N2) organic
layer residues obtained from acid-catalyzed hydrolysis of fractions I
and II were individually submitted to normal-phase HPLC (ISCO,
21.2 × 250 mm, 10 μm), using isocratic elution [n-hexane−CHCl3−
Me2CO (6:3:1)] and a flow rate of 6 mL/min to give 3.0 mg (fraction I)
and 3.5 mg (fraction II) of methyl (11S)-hydroxytetradecanoate15
(convolvulinolic acid methyl ester): tR 18.6 min; mp 27−29 °C; [α]D
+1.5 (c 2, CHCl3); 13C NMR 174.4, 71.7, 51.5, 39.6, 37.5, 34.1, 29.6,
29.5, 29.3, 29.2, 29.1, 25.6, 24.9, 18.8, 14.1. An aliquot of this pure
sample (2 mg) was derivatized with Sigma Sil-A for 5 min at 70 °C.
GC-MS analysis gave one peak (tR 7 min): m/z [M]+ 330 (0.3), 315
(3.5), 287 (66.8), 145 (100), 73 (35.4). Treatment of fraction III as
described above yielded 2.0 mg of methyl (11S)-hydroxyhexadeca-
noate15 (jalapinolic acid methyl ester): tR 16.4 min; mp 42−44 °C;
[α]D +7.3 (c 2, CHCl3); 13C NMR 174.4, 72.0, 51.4, 37.5, 37.4, 34.1,
31.9, 29.6, 29.5, 29.4, 29.2, 29.1, 25.6, 25.3, 24.9, 22.6, 14.1. This
aglycone (1 mg) was derivatized by treatment with Sigma Sil-A and
subjected to GC-MS analysis (tR 12.8 min): m/z [M]+ 358 (0.3), 343
(0.5), 311 (10.5), 287 (59.7), 173 (100), 73 (46.3).
Cytotoxicity and Modulation of Multidrug-Resistance
Assays. Cytotoxicity and reversal fold of the resin glycosides 1−3
were determined by using the SRB assay.19,20 The cells were harvested
at log phase of their growth cycle, treated in triplicate with various
concentrations of the test samples (0.2−25 μg/mL), and incubated for
72 h at 37 °C in a humidified atmosphere of 5% CO2. Results are
expressed as the concentration that inhibited 50% control growth after
the incubation period (IC50). The values were estimated from a
semilog plot of the drug concentration (μg/mL) against the per-
centage of growth inhibition.20 Vinblastine was included as a positive
control. The reversal effects as modulators were further investigated
with the same method. MCF-7 and MDR MCF-7/Vin cells were
seeded into 96-well plates and treated with various concentrations of
vinblastine (0.00064−10 μg/mL) in the presence or absence of
glycolipids at 25 and 5 μg/mL for 72 h as previously described.4 The
ability of glycolipids to potentiate vinblastine cytotoxicity was mea-
sured by calculating the IC50 as described above. In these
experiments, reserpine (5 μg/mL) was used as a positive control.
The reversal fold value, as a parameter of potency, was calculated
from dividing IC50 of vinblastine alone by IC50 of vinblastine in the
presence of test compounds.
ACKNOWLEDGMENTS
■
This research was supported by Direccion
del Personal Academ
́
General de Asuntos
́
ico, UNAM (IN217310), and CONACyT
(101380-Q). S.C.-M., J.C.-G., G.F.-G., and A.D.M.-G. are
grateful to CONACyT for graduate student scholarships.
Ipomoea alba seeds were purchased with funds to A.L. from
the Arkansas Biosciences Institute, the major research
component of the Arkansas Tobacco Settlement Proceeds
Act of 2000. Thanks are due to Dr. D. Mills (BioCentre
Facility, University of Reading, UK), G. Duarte, and M.
́
Guzman (Facultad de Química, UNAM) for the recording of
mass spectra. We also wish to thank Dr. M. Fragoso-Serrano
(Facultad de Química, UNAM) for HPLC technical assistance.
The final draft of this contribution was prepared during a
sabbatical visit of R.P.-M. as a Fulbright Fellow and Visiting
Research Scholar at Lehman College, City University of New
York, NY, USA, with partial financial support from DGAPA,
UNAM.
REFERENCES
(1) Pereda-Miranda, R.; Rosas-Ramírez, D.; Castaneda-Gom
■
́
ez, J.
̃
Progress in the Chemistry of Organic Natural Products; Kinghorn, A. D.,
Falk, H., Kobayashi, J., Eds.; Springer-Verlag: New York, 2010; Vol. 92,
Chapter 2, pp 77−152.
(2) (a) Pereda-Miranda, R.; Kaatz, G. W.; Gibbons, S. J. Nat. Prod.
2006, 69, 406−409. (b) Cher
Serrano, M.; Jacobo-Herrera, N.; Kaatz, G. W.; Gibbons, S. J. Nat.
Prod. 2008, 71, 1037−1045. (c) Cherigo, L.; Pereda-Miranda, R.;
́
igo, L.; Pereda-Miranda, R.; Fragoso-
́
Gibbons, S. Phytochemistry 2009, 70, 222−227. (d) Escobedo-
Martínez, C.; Cruz-Morales, S.; Fragoso-Serrano, M.; Rahman, M.
M.; Gibbons, S.; Pereda-Miranda, R. Phytochemistry 2010, 71, 1796−
1801.
(3) Corona-Castaneda, B.; Pereda-Miranda, R. Planta Med. 2012, 78,
̃
128−131.
́
(4) Figueroa-Gonzalez, G.; Jacobo-Herrera, N.; Zentella-Dehesa, A.;
Pereda-Miranda, R. J. Nat. Prod. 2012, 75, 93−97.
(5) Several synonyms exist for this plant: Calonyction aculeatum (L.)
House, C. album (L.) House, C. pulcherrimum Parodi, Convolvulus
bona-nox (L.) Spreng, Ipomoea aculeata (L.) Kuntze, I. bona-nox (L.),
and I. noctiflora Griff.
(6) McDonald, A. Flora de Veracruz; Instituto de Ecología, A.C.
Xalapa, University of California: Riverside, CA, 1994; pp 13−18.
ASSOCIATED CONTENT
■
(7) Osadao, R. El Libro Judío o Medicina Domes
́
́
tica. Descripcion de las
Virtudes de las Yerbas Medicinales de Yucatan
Ed.; Mexico, 1834; pp 100−208.
́
, Merida; de Zapata, A. H.,
́
S
* Supporting Information
Photographs of moon vine (Ipomoea alba) (Figure S1). Struc-
́
(8) (a) Gunn, C. Brittonia 1972, 24, 150−168. (b) Burkill, I. H. A
Dictionary of the Economic Products of the Malay Peninsula; The
Government of the Straits Settlements and Federated Malay States.
The Crown Agents for the Colonies: London, 1935; Vol. 2, pp 2101−
2108.
1
tures of calonyctins A1 and A2 (Figure S2). FABMS and H,
13C, 1H−1H COSY, TOCSY, HSQC, and HMBC NMR spectra
of albinosides 1−3 (Figures S3−S23). FABMS of glycosidic
1
acids 4−6 (Figures S24, S28, S32). MALDI-MS and H and
13C NMR spectra of derivatives 4a−6a (Figures S25−S27,
S29−S31, S33−S35). GC chromatograms of TMS ethers and
thiazolidine derivatives (Figures S36, S37). HMBC correlations
for 1−3 (Figures S38, S39). Cytotoxicity and modulation assay
of vinblastine with 1, 2, and 3 (Table S40 and Figures S41−
S43). This material is available free of charge via the Internet at
http://pubs.acs.org.
(9) Space, J. C.; Flynn, T. Observations on Invasive Plant Species in
American Samoa; USDA Forest Service, Pacific Southwest Research
Station, Institute of Pacific Islands Forestry: Honolulu, HI, 2000; pp
1−50.
(10) (a) Foxcroft, L.; Richardson, D.; Wilson, J. Environ. Manage.
2008, 41, 32−51. (b) Macdonald, I. A.; Reaser, J. K.; Bright, C.;
Neville, L. E.; Howard, G. W.; Murphy, S. J. Invasive Alien Species in
Southern Africa: National Reports & Directory of Resources; Preston, G.,
Ed.; Global Invasive Species Programme: Cape Town, South Africa,
2003; pp 1−125.
(11) (a) Shen, S. H.; Shen, H. M.; Wu, J. H. J. Plant Growth Regul.
1996, 15, 27−31. (b) Shen, S. H.; Wu, J. H.; Zen, D. L. Potato Res.
1996, 39, 63−68.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +52 55 5622-5288. Fax: +52 55 5622 5329. E-mail:
pereda@servidor.unam.mx.
(12) Fang, Y.; Chai, W.; Chen, S.; He, Y.; Zhao, L.; Peng, J.; Huang,
H.; Xin, B. Carbohydr. Res. 1993, 245, 259−270.
(13) Furukawa, J.; Kobayashi, S.; Nomizu, M.; Nishi, N.; Sakairi, N.
Tetrahedron Lett. 2000, 41, 3453−3457.
Notes
The authors declare no competing financial interest.
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Journal of Natural Products
Article
(14) Pereda-Miranda, R.; Hernan
58, 3145−3154.
(15) Pereda-Miranda, R.; Fragoso-Serrano, M.; Escalante-San
E.; Hernandez-Carlos, B.; Linares, E.; Bye, R. J. Nat. Prod. 2006, 69,
́
dez-Carlos, B. Tetrahedron 2002,
́
chez,
́
1460−1466.
(16) (a) Hara, S.; Hikaru, O.; Kunihide, M. Chem. Pharm. Bull. 1987,
35, 501−506. (b) Miyase, T.; Saitch, H.; Shiokawa, K.; Ueno, A. Chem.
Pharm. Bull. 1995, 43, 466−472.
(17) (a) Pereda-Miranda, R.; Escalante-San
́
chez, E.; Escobedo-
igo, L.; Pereda-
Martínez, C. J. Nat. Prod. 2005, 68, 226−230. (b) Cher
́
Miranda, R. J. Nat. Prod. 2006, 69, 595−599. (c) Escobedo-Martínez,
C.; Pereda-Miranda, R. J. Nat. Prod. 2007, 70, 974−978. (d) Escalante-
San
(e) Bah, M.; Cher
Hammond, G. B.; Pereda-Miranda, R. J. Nat. Prod. 2007, 70, 1153−
1157. (f) Escalante-Sanchez, E.; Rosas-Ramírez, D.; Linares, E.; Bye,
́
chez, E.; Pereda-Miranda, R. J. Nat. Prod. 2007, 70, 1029−1034.
́
igo, L.; Taketa, A. T. C.; Fragoso-Serrano, M.;
́
R.; Pereda-Miranda, R. J. Agric. Food Chem. 2008, 56, 9423−9428.
(g) Castaneda-Gom
1148−1153.
́
ez, J.; Pereda-Miranda, R. J. Nat. Prod. 2011, 74,
̃
(18) Pereda-Miranda, R.; Mata, R.; Anaya, A. L.; Wickramaratne, D.
B. M.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod. 1993, 56, 571−582.
(19) Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112−1116.
(20) (a) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon,
J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J.
Natl. Cancer Inst. 1990, 82, 1107−1112. (b) Angerhofer, C. K.;
Guinaudeau, H.; Wongpanich, V.; Pezzuto, J. M.; Cordell, G. A. J. J.
Nat. Prod. 1999, 62, 59−60.
I
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