Jalapinoside, a macrocyclic bisdesmoside from the resin glycosides of ipomea purga, as a modulator of multidrug resistance in human cancer cells

Article abstract of DOI:10.1021/np500762w

The first macrocyclic bisdesmoside resin glycoside, jalapinoside (4), was purified by preparative-scale recycling HPLC from the MeOH-soluble extracts of Ipomoea purga roots, the officinal jalap. Purgic acid C (3), a new glycosidic acid of ipurolic acid, was identified as 3-O-β-d-quinovopyranoside, 11-O-β-d-quinovopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→3)-O-[β-d-fucopyranosyl-(1→4)]-O-α-l-rhamnopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→2)-O-β-d-quinovopyranoside (3S,11S)-dihydroxytetradecanoic acid. The acylating residues of this core were acetic, (+)-(2S)-methylbutanoic, and dodecanoic acids. The site of lactonization was defined as C-3 of the second saccharide moiety. Reversal of multidrug resistance by this noncytotoxic compound was evaluated in vinblastine-resistant human breast carcinoma cells.

Full text of DOI:10.1021/np500762w

Note
pubs.acs.org/jnp
Jalapinoside, a Macrocyclic Bisdesmoside from the Resin Glycosides
of Ipomea purga, as a Modulator of Multidrug Resistance in Human
Cancer Cells
Elihu Bautista, Mabel Fragoso-Serrano, and Rogelio Pereda-Miranda*
́
́ ́
Departamento de Farmacia, Facultad de Química, Universidad Nacional Autonoma de Mexico, Mexico City, 04510 DF, Mexico
S
* Supporting Information
ABSTRACT: The first macrocyclic bisdesmoside resin glycoside, jalapinoside (4), was purified by preparative-scale recycling
HPLC from the MeOH-soluble extracts of Ipomoea purga roots, the officinal jalap. Purgic acid C (3), a new glycosidic acid of
ipurolic acid, was identified as 3-O-β-D-quinovopyranoside, 11-O-β-D-quinovopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→3)-O-
[β-D-fucopyranosyl-(1→4)]-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)-O-β-D-quinovopyranoside (3S,11S)-
dihydroxytetradecanoic acid. The acylating residues of this core were acetic, (+)-(2S)-methylbutanoic, and dodecanoic acids.
The site of lactonization was defined as C-3 of the second saccharide moiety. Reversal of multidrug resistance by this
noncytotoxic compound was evaluated in vinblastine-resistant human breast carcinoma cells.
soluble content.2 According to Mannich and Schumann,8
esin glycosides are complex mixtures of glycolipids found
Rin members of the morning glory family (Convolvula- convolvulin resin glycosides are composed as large, high
ceae).1 Several biological effects of the crude extracts of the
molecular weight polymers of oligosaccharides glycosidically
linked to a hydroxylated fatty acid. Previously, the isolation of
purgins I−III,9 partially acylated ester-type dimers of branched
pentasaccharides from the jalap root, permitted the character-
ization of these compounds as individual macrolactones of
distinctive glycosidic acids. Therefore, the hypothesis of a high
molecular weight polymeric structure for this class of
compounds had to be revisited.
The structural complexity of the highly polar alcohol-soluble
resin glycosides has seriously hampered the isolation of the
individual glycolipids, limiting chemical studies to the character-
ization of their degradation products, i.e., esterifying organic
acids and major glycosidic acids.2,10 Saponification of the jalap
root MeOH-soluble contents afforded purgic acids A and B (1
and 2) as their major glycosidic acids.2 Compound 1 was
identified as (11S)-hydroxytetradecanoic acid 11-O-β-D-quino-
vopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→3)-O-[β-D-fuco-
pyranosyl-(1→4)]-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glu-
copyranosyl-(1→2)-O-β-D-quinovopyranoside. Compound 2
purgative Mexican jalaps (e.g., Ipomea purga, I. orizabensis, and
I. stans) have been attributed to their resin glycosides.1,2 These
resin glycosides represent noncytotoxic inhibitors of multidrug
efflux pumps in Gram-positive3 and -negative4 bacteria as well
as in mammalian cancer cells.5
Phytochemical reports on the jalap root (“officinal jalap”, I.
purga), a drug used since pre-Columbian times in Mexico,2
were published as early as the second half of the 19th century.6
Most of the early and even recent botanical and chemical
descriptions are confusing because of poor plant material
identification. HPLC and 13C NMR spectroscopic profiles of
the glycosidic acids obtained through saponification of the resin
glycoside contents were used as analytical tools for
authentication and quality control of the Mexican jalaps.2,7
The ratio of ether-soluble (“jalapin”) to alcohol-soluble
(“convolvulin”) resin glycoside portions can also distinguish
between authentic and false crude drugs; for example, the
officinal jalap root yields the highest amount of MeOH-soluble
resin glycosides (15−20% dried weight), in comparison to the
yields from the Mexican scammony, or false jalap (I.
orizabensis): 10−18% ether-soluble resins and <1% alcohol-
Received: September 29, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np500762w
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Journal of Natural Products
Note
was characterized as the (11S)-hydroxyhexadecanoic acid
homologue of 1 (Figure 1).2 In this context, the present
readily detectable ions resulting from glycosidic cleavage2 and
eliminations of the esterifying groups10,12 with observed ions at
m/z 1543 [1689 (M + H) − 146 (methylpentose unit)]+, 1459
[1689 (M + H) − 146 − 84 (methylbutyrate)]+, 1297 [1459 −
162 (hexose unit)]+, 739 [1297 − 146 − 182 − 146 − 84]+
(Figures 2 and S1, Supporting Information). Purgic acid C (3)
Figure 1. Chemical structures of purgic acids A−C (1−3).
investigation describes the structural elucidation of an intact
partially acylated macrocyclic bisdesmoside resin glycoside,
having ipurolic acid [(3S,11S)-dihydroxytetradecanoic acid] as
the aglycone, while having the same glycosidation sequence in
the oligosaccharide core as purgic acids A and B (1 and 2). The
second sugar linkage of an additional quinovose moiety was
established at C-3 of the aglycone. This new glycosidic acid
obtained from the saponification of the isolated resin glycoside
was named purgic acid C (3). Purification of the highly polar
bisdesmoside, jalapinoside (4), found as a minor constituent in
the H2O-insoluble resin glycosides from the “officinal jalap”
MeOH-soluble extract, was performed by preparative-scale
HPLC through the application of column overload, peak
shaving, heart cutting, and recycling techniques.11 Finally,
reversal of multidrug resistance (MDR) by compound 4 was
evaluated in vinblastine-resistant human breast carcinoma
cells.5
Alkaline hydrolysis of 4 produced a nonpolar fraction, which
was analyzed by GC-MS to establish acetic, methylbutyric, and
dodecanoic acids as the acylating residues. The water-soluble
polar fraction extracted with n-BuOH afforded purgic acid C
(3), which was hydrolyzed in acid, and their Et2O-soluble
extract was methylated. The product corresponded to methyl
(3S,11S)-dihydroxytetradecanoate, which was identified by
comparison of optical rotation and MS data with published
values.12a The monosaccharide mixture was derivatized with L-
cysteine to form thiazolidines,13 identified by GC-MS as their
TMS ethers. This sugar analysis also confirmed the absolute
configuration of all the monosaccharides as the L-series for
rhamnose and the D-series for fucose, quinovose, and glucose.
The MS analysis of compound 4 was conducted by
HRESIMS and MALDI-TOFMS in the positive ion detection
mode. HRESIMS identified the quasi-molecular ion at m/z
1689.8448 [M + H]+ (C80H137O37). LC-ESIMS/MS provided
Figure 2. Positive ESIMS fragmentation for compound 4.
showed a quasi-molecular ion of low abundance at m/z 1313
[M − H]−, corresponding to the molecular formula C56H97O34,
and an intense ion at m/z 1167 [M − H − 146]−,
corresponding to the elimination of one methylpentose unit,
which, in turn, produced the fragmentation pattern of a
hexasaccharide having the same glycosidation sequence as
purgic acid A2 (Figure 1) but with a difference of 16 amu based
upon a negative ion FAB with diagnostic ions at m/z 1021
[1167 − 146]−, 859 [1021 − 162]−, 713 [859 − 146]−, 567
[713 − 146]−, and 405 [567 − 162]−, indicating the presence
of ipurolic acid as the aglycone.12
Structure elucidation of jalapinoside (4) was accomplished
by comparison of its 1H and 13C NMR data (Figures S2 and S3,
Supporting Information) with reported data for the purgic acids
A (1) and B (2) since all of them possess the same
hexasaccharide unit (Figure 1).2 The main differences were in
the esterifying residues and macrolactonization in 4, as well as
the presence of ipurolic acid as the aglycone, containing an
additional quinovose glycosidically linked at C-3 to constitute a
macrocyclic resin glycoside bisdesmoside. In order to identify
each constitutive monosaccharide moiety, COSY (Figure 3)
and TOCSY experiments (Figures S4 and S5, Supporting
Information) were used to assign chemical shift values, after
1
identifying and differentiating each of the H NMR signals
B
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Journal of Natural Products
Note
1
Table 1. H (700 MHz) and 13C (175 MHz) NMR Data of
Compound 4 in Pyridine-d5 (δ in ppm, J in Hertz)
position
δH
δC
position
δH
δC
Qui-1
4.90 d (7.6)
102.9
80.4
78.6
Fuc-1
5.78 br d (7.6)
101.8
73.6
74.2
a
a
2
3
4.27−4.22
4.75 dd (8.8,
6.3)
2
3
4.26−4.23
5.99 d (7.8)
a
4
5
3.52 dd (9.0,
8.8)
76.8
75.5
4
5
6
4.29−4.26
72.0
77.7
3.95 dd (9.0,
6.3)
3.68 m
6
1.55 d (6.3)
5.86 d (7.8)
17.2
101.6
75.9
1.67 d (5.2)
17.0
103.0
75.7
Glc-1
Qui″-1 4.82 d (7.8)
2
a
a
2
3
4.28−4.26
4.03−3.97
5.98 dd (7.6,
7.6)
74.2
3
4.15 dd (9.0,
9.0)
78.5
a
a
4
5
4.30−4.26
69.3
62.8
4
5
4.50−4.47
74.8
77.2
4.41 dd (8.7,
8.5)
3.79 br d (9.2,
6.0)
Figure 3. Key H−1H COSY and HMBC correlations of jalapinoside
1
(4).
6a
5.69 dd (10.3,
3.5)
74.9
6
1.44 d (6.0)
14.6
173.3
43.8
6b
5.66 dd (10.3,
6.8)
Ipur-1
2a
1
(Table 1). 2D H−13C HSQC NMR experiments (Figure S6,
Rha-1
6.47 d (1.3)
96.8
74.2
74.0
80.1
67.6
2.71 dd (14.7,
4.2)
Supporting Information) were used for the assignments of the
13C NMR signals (Table 1 and Figure 3). In the low-field
region of the NMR spectra, seven anomeric signals were
confirmed at δH 4.90 (1H, d, J = 7.6 Hz; δC 102.9, Qui-1); 5.86
(1H, d, J = 7.8 Hz; δC 101.6, Glc-1); 6.47 (1H, d, J = 1.3 Hz; δC
96.8, Rha-1); 6.42 (1H, d, J = 7.8 Hz; δC 101.6, Glc′-1); 5.15
(1H, d, J = 7.8 Hz; δC 106.0, Qui′-1); 5.78 (1H, d, J = 7.6 Hz;
δC 101.8, Fuc-1); and 4.82 (1H, d, J = 7.8 Hz; δC 103.0, Qui″-
1). Therefore, seven separate spin systems for sugar skeletons
were readily distinguished in the 1H−1H COSY (Figure 3) and
TOCSY spectra. The glycosylation sequence and the positions
of esterification, lactonization, and ester-type linkage were
established by the observed long-range correlations (3JCH) in
the HMBC spectrum (Figures 3 and S7, Supporting
Information). The following key correlations were observed:
connectivities between H-1 (δH 4.90, Qui-1) and C-11 (δC
81.2, Ipur-11); H-1 (δH 4.82, Qui″-1) and C-3 (δC 68.6, Ipur-
3); H-1 (δH 5.86, Glc-1) and C-2 (δC 80.4, Qui-2); H-2 (δH
4.28−4.26, Glc-2) and C-1 (δC 96.8, Rha-1); H-1 (δH 6.42,
Glc′-1) and C-3 (δC 74.0, Rha-3); H-1 (δH 5.15, Qui′-1) and C-
2 (δC 86.3, Glc′-2); and H-4 (δH 4.61, Rha-4) and C-1 (δC
101.8, Fuc-1). The locations of the acyl residues were
established by the following HMBC correlations: H2-6 (δH
5.69 and 5.66, Glc-6) and the carbonyl of an acetate group (δC
168.5); H-2 (δH 5.91, Rha-2) and C-1 of a 2-methylbutyrate
residue (δC 176.6); H-3 (δH 5.35, Qui′-3) and C-1 of a second
methylbutyrate moiety (δC 176.2); and H-3 (δH 5.99, Fuc-3)
with the carbonyl of a dodecanoate group (δC 173.1).
Therefore, the structure of jalapinoside was elucidated as
(3S,11S)-dihydroxytetradecanoic acid 11-[3-O-(2S-methylbu-
tyryl)]-O-6-deoxy-α-L-mannopyranosyl-(1→2)-O-β-D-glucopyr-
anosyl-(1→3)-O-[6-deoxy-β-D-galactopyranosyl-(1→4)]-O-α-
L-[2-O-(2S-methylbutyryl)]-6-deoxy-β-D-glucopyranosyl-(1→
2)-O-β-D-[6-O-acetyl]-glucopyranosyl-(1→2)-O-6-deoxy-α-L-
mannopyranosyloxy]-3-O-[6-deoxy-α-L-mannopyranosyloxy]-
1,3″-lactone (4).
2
3
4
5
6
5.91 dd (3.5,
1.3)
2b
3
2.72 dd (14.2,
3.8)
a
5.50 dd (9.3,
3.5)
4.42−4.38
68.6
81.2
12.8
4.61 dd (9.5,
9.3)
11
14
3.90 m
5.26 dq (9.5,
6.2)
1.01 t (7.4)
1.96 d (6.2)
19.3
101.6
86.3
Ac-1
168.5
12.4
Glc′-1 6.42 d (7.8)
2
2
2.04 s
a
4.05−3.99
Mba-1
2
176.6
41.6
3
4.53 dd (9.0,
9.0)
72.7
2.63 t (7.3)
4
5
3.84 d (9.5, 9.0)
72.8
78.1
2-Me
3-Me
1.24 d (7.3)
0.98 t (7.4)
16.9
14.7
4.34 dd (9.0,
6.5)
a
6a
6b
4.56−4.52
4.06−3.99
63.1
Mba′-1
2
176.2
42.0
a
2.53 dq (7.2,
3.6)
Qui′-1 5.15 d (7.8)
2
106.0
77.6
76.7
2-Me
1.22 d (7.2)
0.97 t (7.0)
17.1
11.9
a
4.10−4.06
3-Me
3
5.35 dd (9.5,
9.5)
Dodeca-1
173.1
a
4
5
4.25−4.21
75.5
77.1
2
2.72 t (4.0)
43.4
3.74 br t (8.8,
6.2)
6
1.71 d (6.2)
16.9
a
Overlapped signal.
resin glycosides.12b Both compounds, as well as purgic acid C
(3), are bidesmosides of distinctive glycosidic acids having an
oligosaccharide core linked at C-11 of the aglycone (ipurolic
acid) in addition to one monosaccharide moiety at C-3.
Consequently, jalapinoside (4) represents the first intact
macrocyclic resin glycoside isolated from a convolvulin fraction.
Compound 4 was not cytotoxic (IC50 > 10 μg/mL) when
tested against colon (HCT-15), cervix (HeLa), and breast
(MCF-7 and MDA-MB-231) carcinoma cell lines (Tables 2 and
S1, Supporting Information).15 Thus, its potential as an MDR
modulator was explored. Results of the modulation assay are
shown in Table 2, and this screening utilized both vinblastine-
sensitive and vinblastine-resistant human breast carcinoma cells
Prior to this investigation, only two examples of glycosidic
acid bisdesmosides had been described, i.e., quamoclinic acids
G and H, which were isolated from the seeds of Quamoclict
pennata Bojer. These were purified from the H2O-soluble
fraction produced by alkaline hydrolysis of the ether-insoluble
C
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Journal of Natural Products
Note
Table 2. Modulation of Vinblastine Cytotoxicity in Drug-Sensitive MCF-7 and Multidrug-Resistant MCF-7/Vin by Jalapinoside
(4)
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
1.02 0.18
<0.00064
1.22 0.14
<0.00064
0.047 0.01
<0.00064
4
>1593.8
27.6
>1906.3
3.9
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.
(MCF-7/Vin; Figure S8, Supporting Information).5a Reversal
fold (RFMCF‑7/Vin+ >1906) indicated that jalapinoside (4) is
extremely potent at 25 μg/mL, as previously observed for other
morning glory resin glycosides, e.g., murucoidin V (RF
́
de Ecologia, Veracruz, Mex
́
ico (XAL ID-365843); Departamento
́
Farmacia, Facultad de Quimica, UNAM (J. Castaneda and R. Pereda
RP-06); and the National Herbarium, Instituto de Biologia,
Universidad Nacional Autonoma de Mexico (MEXU 426765).
̃
́
́
́
Extraction and Isolation. The dried, powdered roots (3.8 kg)
were extracted exhaustively by maceration with MeOH to obtain a
dried extract (202.8 g), which was suspended (22.8 g) in deionized
water (3 × 100 mL) and subjected to sonication for 30 min to afford
H2O-soluble and -insoluble fractions (9.4 and 13.3 g, respectively).
The H2O-insoluble fraction (12.0 g) was submitted to HPLC on a
Symetry C18 column (Waters, 7 μm, 19 × 150 mm) with an isocratic
elution of CH3CN−H2O (3:1), a flow of 8.2 mL/min, and a sample
concentration of 200 mg/mL, injecting 500 μL each time. The peak
with a tR value of 10.1 min was collected by the techniques of heart
cutting and peak shaving and was purified by recycling11 over 10 times
at the same column, using a flow of 4.1 mL/min to afford compound 4
(6.2 mg).
255)5a and purgin II (RFMCF‑7/Vin+ >2140),10 which
b
MCF‑7/Vin+
are proven substrates of glycoprotein P. These results further
support the potential of this family of compounds as inhibitors
of multidrug efflux pumps in mammalian cancer cells, where
glycoprotein-P is the chief plasma membrane-associated
translocase responsible for the MDR phenotype,5a but also
highlight their importance for overcoming MDR in cancer
therapy.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points (uncor-
rected) were determined on a Fisher-Johns apparatus. Optical
rotations were measured on a Perkin-Elmer 341 polarimeter. 1H
(700 MHz) and 13C (175 MHz) NMR experiments were performed
on a Bruker Avance III HD NMR spectrometer. Chemical shifts were
referenced to TMS, and J values are given in Hz. ESIMS, HRESIMS,
and MALDI-TOFMS were recorded on Bruker Daltonics Esquire
6000, Thermo LTQ Orbitrap XL, and Bruker MicrO-TOF-Q mass
spectrometers, respectively. Waters HPLC equipment was composed
of a 600E multisolvent delivery system with a refractive index detector
(Waters 410). Control of the equipment, data acquisition, and
processing of the chromatographic information was performed by the
Empower 2 software (Waters). HPLC-ESIMS was carried out on an
Agilent 1200 Rapid Resolution liquid chromatograph coupled to a
Bruker Daltonics Esquire 6000 mass spectrometer. GC-MS was
performed on a Thermo-Electron instrument coupled to a Thermo-
Electron spectrometer. GC conditions: DB-5MS (5% phenyl)-
methylpolysiloxane column (30 m × 0.25 mm, film thickness 0.18
μm); He, linear velocity 30 cm/s; 2 mL/min; 50 °C isothermal for 4
min, linear gradient to 300 °C at 40 °C/min; final temperature hold,
20 min. MS conditions: ionization energy, 70 eV; ion source
temperature, 250 °C; interface temperature, 270 °C; scan speed, 2
scans s−1; mass range, 45−600 amu. TLC was carried out on precoated
Macherey−Nagel silica gel/UV254 plates of 0.25 thickness, and spots
were visualized by spraying with 3% CeSO4 in 2 N H2SO4 followed by
heating.
Jalapinoside (4): colorless solid; mp 163−165 °C; [α]589 −33,
1
[α]578 −35, [α]546 −39, [α]436 −63, [α]365 −99 (c 0.1, MeOH); H
and 13C NMR, see Table 1; positive ESIMS m/z 1689 [M + H]+;
MALDI-TOFMS m/z 1689 [M + H]+; HRESIMS m/z 1689.8448 [M
+ H]+ (calcd for C80H137O37 requires 1689.8760).
Alkaline Hydrolysis of Jalapinoside. A solution of compound 1
(4 mg) in 5% KOH−H2O (0.75 mL) was subjected to reflux at 95 °C
for 3 h. The mixture was acidified to pH 5.0 and extracted with CHCl3
(3 × 5 mL), the organic layer was washed with H2O and dried with
Na2SO4, and the solvent was evaporated under reduced pressure. The
aqueous phase was extracted with n-BuOH (3 × 10 mL) and
concentrated to dryness. The organic phase was directly analyzed by
CG-MS2 to display three peaks, which were identified as acetic acid (tR
3 min) [m/z [M]+ 60 (65), 45 (80), 43 (100), 29 (19), 15 (25)]; 2-
methylbutanoic acid (tR 7 min) [m/z [M]+ 102 (2), 87 (25), 74 (100),
57 (64), 41 (30)]; and n-dodecanoic acid (tR 18 min) [m/z [M]+ 200
(16), 171 (9), 157 (22), 143 (5), 129 (34), 115 (18), 101 (12), 85
(28), 73 (100), 60 (95), 43 (62)]. The residue from the n-BuOH
fraction was directly analyzed by HPLC-ESIMS on a Synergi Polar
column (Phenomenex, 7 μm, 2 × 150 mm) with a gradient elution of
aqueous formic acid 0.1% and methanol, 6:4 (0 min) to 1:9 (40 min),
a flow of 0.2 mL/min, and a sample injection of 500 μL (1 mg/mL).
The peak with a tR value of 21.5 min corresponded to purgic acid C
(3) (1.7 mg).
Purgic acid C (3): colorless solid; negative ion FABMS m/z 1313
[M − H]−, 1167 [1313 − 146]−, 1021 [1167 − 146]−, 859 [1021 −
162]−, 713 [859 − 146]−, 567 [713 − 146]−, 405 [567 − 162]−.
Acid Hydrolysis and Sugar Analysis. Glycosidic acid 3 (1.5 mg)
in 4 N HCl (2 mL) was refluxed at 90 °C for 2 h. The reaction mixture
was diluted with H2O (5 mL) and extracted with ether (5 mL). The
organic layer was evaporated to dryness, dissolved in CHCl3 (2 mL),
and treated with CH2N2. The aqueous phase was neutralized with 1 N
KOH, extracted with n-BuOH (5 mL), washed with H2O (5 mL), and
concentrated to give a colorless solid. Thiazolidine derivatives for the
sugar constituents were prepared according to previously described
procedures13 and analyzed by GC-MS after conversion into volatile
derivatives by treatment with chlorotrimethylsilane (Sigma Sil-A)14 by
applying the following conditions: DB-5MS (10 m × 0.18 mm, film
thickness 0.18 μm); He, 30 cm/s, 2 mL/min; 100 °C isothermal for 3
Chemicals, Cell Lines, and Cell Cultures. Colon (HCT-15),
cervix (HeLa), and breast (MCF-7 and MDA-MB-231) carcinoma cell
lines were obtained from the American Type Culture Collection. The
resistant MCF-7/Vin was developed through continuous exposure to
vinblastine during five consecutive years as previously reported.5a 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 containing 0.192 μg/mL
of vinblastine. At the same time, a stock of MCF-7/Vin− cells was
maintained in vinblastine-free medium.
Plant Material. The roots of I. purga (Wender) Hayne were
collected in Coxmatla, Municipio de Xico, Veracruz, Mexico, in
November 2010 and identified by botanist Alberto Linajes. Three
voucher specimens were deposited at the following herbaria: Instituto
D
DOI: 10.1021/np500762w
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
min, linear gradient to 300 at 20 °C/min. Retention times for TMS
derivatives of common sugars were used as standards for GC
identification: D-fucose, tR 4.53; L-rhamnose, tR 4.58 min; D-quinovose,
tR 4.64 min; and D-glucose, tR 6.53 min. The derivatized (CH2N2)
organic layer residue obtained from acid-catalyzed hydrolysis was
submitted to normal-phase HPLC (ISCO, 21.2 m × 250 mm, 10 μm)
using an isocratic elution of hexanes−CHCl3−acetone (6:3:1)2 and a
flow rate of 6 mL/min to give 0.3 mg of methyl (3S,11S)-
dihydroxytetradecanoate (ipurolic acid methyl ester): tR 20.8 min;
colorless needles; mp 68−70 °C; [α]D +13 (c 0.02, MeOH); HPLC-
ESIMS m/z 273 [M − H]−, 255 [273 − H2O]−, 231 [M −
CH3(CH2)2]−, 103 [CH(OH)CH2COOCH3]−, 73 [CH3(CH2)2CH-
(OH)]−; identified by comparison of melting point and optical
rotation with published values.12a
The preparation and identification of 4-bromophenylacyl (2S)-2-
methylbutyrate were performed according to reported procedures:11
mp 40−41 °C; [α]D +16 (c 0.03, MeOH); GC-MS (tR 4.75 min) m/z
[M + 2]+ 272 (6.8), [M]+ 270 (7.3), 254 (3.8), 252 (3.8), 186 (2.1),
172 (8.6), 171 (100), 70 (9.7), 169 (88.7), 90 (13.9), 89 (23.4), 85
(11.5), 63 (5.3) 57 (19), 51 (2.3), 50 (2.9), 41 (8.5), 39 (9.4). This
transesterification procedure has been used to confirm the absolute
configuration for 2-methylbutyric acid.16
Cytotoxicity and Modulation of Multidrug-Resistance
Assays. The cytotoxicity and MDR reversal of compound 4 were
determined by using the SRB assay.15 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. The assay
was carried out under static conditions, and the results are expressed as
the concentration that inhibits 50% control growth after the
incubation period (IC50). The values were estimated from a semilog
plot of the drug concentration (μg/mL) against the percentage of
growth inhibition.15 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
(0.000 64−10 μg/mL) in the presence or absence of glycolipids
(dissolved in a mixture of water−DMSO, 9:1) at 25 and 5 μg/mL for
72 h as previously described.5a The ability of glycolipids to potentiate
vinblastine cytotoxicity was measured by calculating the IC50 as
described above. In these experiments, reserpine (5 μg/mL) was used
as a positive control drug. 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.
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̃
ASSOCIATED CONTENT
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74, 1148−1153. (b) Castaneda-Gom
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* Supporting Information
1D and 2D NMR spectra as well as the ESIMS/MS data of
compound 4 are available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author
*Tel: +52-55-5622-5288. Fax: +52-55-5622-5329. E-mail:
pereda@unam.mx.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We acknowledge the financial support by Direccion
́
General de
ico, UNAM (IN212813), and
́
(16) Bah, M.; Cherigo, L.; Taketa, A. T. C.; Fragoso-Serrano, M.;
Asuntos del Personal Academ
́
Hammond, G. B.; Pereda-Miranda, R. J. Nat. Prod. 2007, 70, 1153−
́
Consejo Nacional de Ciencia y Tecnologia (220535). E.B. is
1157.
grateful to DGAPA for a postdoctoral scholarship. Thanks are
́
due to G. Duarte (Facultad de Quimica, UNAM) and C.
Mar
mass spectra.
́
́
quez (Instituto de Quimica, UNAM) for the recording of
E
DOI: 10.1021/np500762w
J. Nat. Prod. XXXX, XXX, XXX−XXX