Inhibitors of bacterial multidrug efflux pumps from the resin glycosides of Ipomoea murucoides

Article abstract of DOI:10.1021/np800148w

A reinvestigation of the CHCl₃-soluble extract from flowers of the Mexican medicinal arborescent morning glory, Ipomoea murucoides, through preparative-scale recycling HPLC, yielded six new pentasaccharides, murucoidins VI - XI (1-6), as well as the known pescaprein III (7), stoloniferin I (8), and murucoidins I - V (9-13). Their structures were characterized through the interpretation of their NMR spectroscopic and FABMS data. Compounds 1-6 were found to be macrolactones of three known glycosidic acids identified as simonic acids A and B, and operculinic acid A, with different fatty acids esterifying the same positions, C-2 on the second rhamnose unit and C-4 on the third rhamnose moiety. The lactonization site of the aglycone was placed at C-2 or C-3 of the second saccharide unit. The esterifying residues were composed of two short-chain fatty acids, 2-methylpropanoic and (2S)-methylbutyric acids, and two long-chain fatty acids, n-dodecanoic (lauric) acid and the new (8R)-(-)-8-hydroxydodecanoic acid. For the latter residue, its absolute configuration was determined by analysis of its Mosher ester derivatives. All members of the murucoidin series exerted a potentiation effect of norfloxacin against the NorA overexpressing Staphylococcus aureus strain SA-1199B by increasing the activity 4-fold (8 μg/mL from 32 μg/mL) at concentrations of 5-25 μg/mL. Stoloniferin I (8) enhanced norfloxacin activity 8-fold when incorporated at a concentration of 5 μg/mL. Therefore, this type of amphipathic oligosaccharide could be developed further to provide more potent inhibitors of this multidrug efflux pump.

Full text of DOI:10.1021/np800148w

J. Nat. Prod. 2008, 71, 1037–1045
1037
Inhibitors of Bacterial Multidrug Efflux Pumps from the Resin Glycosides of Ipomoea
murucoides
Lilia Che´rigo,† Rogelio Pereda-Miranda,*,† Mabel Fragoso-Serrano,† Nadia Jacobo-Herrera,† Glenn W. Kaatz,‡ and
Simon Gibbons§
Departamento de Farmacia, Facultad de Qu´ımica, UniVersidad Nacional Auto´noma de Me´xico, Ciudad UniVersitaria, Mexico City 04510 DF,
Mexico, Department of Internal Medicine, DiVision of Infectious Diseases, John D. Dingell Veterans Affairs Medical Center and Wayne State
UniVersity School of Medicine, Detroit, Michigan 48201, and Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy,
UniVersity of London, 29-39 Brunswick Square, London WC1N 1AX, U.K.
ReceiVed March 10, 2008
A reinvestigation of the CHCl3-soluble extract from flowers of the Mexican medicinal arborescent morning glory, Ipomoea
murucoides, through preparative-scale recycling HPLC, yielded six new pentasaccharides, murucoidins VI-XI (1-6),
as well as the known pescaprein III (7), stoloniferin I (8), and murucoidins I-V (9-13). Their structures were characterized
through the interpretation of their NMR spectroscopic and FABMS data. Compounds 1-6 were found to be macrolactones
of three known glycosidic acids identified as simonic acids A and B, and operculinic acid A, with different fatty acids
esterifying the same positions, C-2 on the second rhamnose unit and C-4 on the third rhamnose moiety. The lactonization
site of the aglycone was placed at C-2 or C-3 of the second saccharide unit. The esterifying residues were composed
of two short-chain fatty acids, 2-methylpropanoic and (2S)-methylbutyric acids, and two long-chain fatty acids,
n-dodecanoic (lauric) acid and the new (8R)-(-)-8-hydroxydodecanoic acid. For the latter residue, its absolute configuration
was determined by analysis of its Mosher ester derivatives. All members of the murucoidin series exerted a potentiation
effect of norfloxacin against the NorA overexpressing Staphylococcus aureus strain SA-1199B by increasing the activity
4-fold (8 µg/mL from 32 µg/mL) at concentrations of 5-25 µg/mL. Stoloniferin I (8) enhanced norfloxacin activity
8-fold when incorporated at a concentration of 5 µg/mL. Therefore, this type of amphipathic oligosaccharide could be
developed further to provide more potent inhibitors of this multidrug efflux pump.
In the New World flora there are 13 tree-like morning glory
species in the genus Ipomoea series Arborescentes, most of them
are confined to Mexico and nearby Central America.1 These species
have long been of medicinal and economical interest to the native
people, who coexist with them in the same ecosystem. In central
Mexico, six species collectively called “cazahuate”2 are a conspicu-
ous floristic element of the seasonal dry tropical forest. They have
been used since Prehispanic times3 and share several morphological
features, e.g., trees with large white flowers and funnel-shaped
corollas, and the same therapeutic application to treat itching, rashes,
and other infections by rubbing the raw flowers directly on the skin.
Traditional healers continue to use decoctions of this medicinal plant
complex4–6 considered to be of “cold-nature”7 to reduce excessive
body heat8 and relieve uncomfortable “water and cold” symptoms
believed to be produced by abrupt climatic changes resulting in
diseases considered to be “hot”, e.g., rheumatism, inflammation,
and ear pain.
development of new bacterial inhibitors to treat infections resulting
from multidrug-resistant S. aureus strains.9,10
Results and Discussion
A small portion of the resin glycoside fractions, obtained from the
CHCl3-soluble extract, was submitted to saponification and yielded a
water-soluble solid product that was resolved by HPLC into three
glycosidic derivatives of jalapinolic acid and an organic solvent-soluble
oily acidic fraction. The glycosidic acids were identified as simonic
acid A, (11S)-jalapinolic acid 11-O-R-L-rhamnopyranosyl-(1f3)-
O-[R-L-rhamnopyranosyl-(1f4)]-O-R-L-rhamnopyranosyl-(1f4)-
O-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-glucopyranoside,11 simonic acid
B, (11S)-jalapinolic acid 11-O-R-L-rhamnopyranosyl-(1f3)-
O-[R-L-rhamnopyranosyl-(1f4)]-O-R-L-rhamnopyranosyl-(1f4)-
O-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-fucopyranoside,5,11 and oper-
culinic acid A, (11S)-jalapinolic acid 11-O-ꢀ-D-glucopyranosyl-
(1f3)-O-[R-L-rhamnopyranosyl-(1f4)]-O-R-L-rhamnopyranosyl-
(1f4)-O-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-fucopyranoside,5,12previously
obtained from Ipomoea batatas,11 I. stolonifera,13 I. operculata,14
I. leptophylla,15 I. murucoides,5 and I. intrapilosa.16
The major liberated fatty acids were identified as 2-methylpro-
panoic (iba), 2-methylbutanoic (mba), and n-dodecanoic acids by
GC-MS comparison of their spectra and retention times with those
of authentic samples.5,17 The new (8R)-(-)-8-hydroxydodecanoic
acid (14) was also liberated as one of the hydrolyzed products
recovered from the organic-soluble fraction obtained from the
saponification of murucoidin X (5).
This paper presents the results of a second investigation based
on the chemical analysis of the resin glycoside mixture obtained
from the flowers of Ipomoea murucoides Roem. et Schult. Six new
acylated pentasaccharides of jalapinolic acid, murucoidins VI-XI
(1-6), as well as the known pescaprein III (7), stoloniferin I (8),
and murucoidins I-V (9-13) were separated and purified by a
recycling HPLC technique, and their structures were characterized
through the interpretation of NMR spectroscopic and FABMS data.
The antimicrobial potential of these complex macrocyclic lactones
was evaluated against a panel of Staphylococcus aureus strains
overexpressing specific efflux pumps as a starting point for the
Individual constituents of the remaining portion of these resin
glycoside fractions were separated and purified by a recycling HPLC
technique,18 using a semipreparative reversed-phase column. These
procedures led to the isolation and structural characterization of
six new compounds, for which the names murucoidins VI-XI
(1-6) have been proposed. All compounds displayed the same
fragmentation pattern produced by glycosidic cleavage in their
negative-ion FABMS, as previously described for the pescaprein19
and murucoidin5 series. Common fragment peaks reported for the
* To whom correspondence should be addressed. Tel: +52-55-5622-
5288. Fax: +52-55-5622-5329. E-mail: pereda@servidor.unam.mx.
† Universidad Nacional Auto´noma de Me´xico.
‡ John D. Dingell Veterans Affairs Medical Center and Wayne State
University.
§ University of London.
This work was taken in part from the Ph.D. thesis of L. Che´rigo.
10.1021/np800148w CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/24/2008

1038 Journal of Natural Products, 2008, Vol. 71, No. 6
Chérigo et al.
resin glycosides20,21 were observed in all mass spectra, confirming
the branched pentasaccharide core, and the resulting diagnostic
peaks also indicated the nature of the esterifying moieties. For
example, murucoidin X (5) gave a pseudomolecular [M - H]- ion
at m/z 1265, indicating a molecular formula of C63H110O25. The
ions at m/z 1181 [M - H - C5H8O]- and 1067 [M - H -
C12H22O2]- suggested the presence of a methylbutyric acid and a
monohydroxydodecanoic acid as esterifying residues. The subse-
quent losses produced by glycosidic cleavage of each sugar moiety
afforded peaks at m/z 691 [1067 - 2 × 146 (C6H10O4) - C5H8O]-,
545 [691 - 146 (C6H10O4)]-, which indicated that the lactonization
was located at the first rhamnose unit (Rha), 417 [545 + H2O -
146 (C6H10O4)]-, and 271 - [417 - 146 (C6H10O4)]-.
C), 1JCH values for fucose (159 Hz) and glucose (164 Hz) supported
the ꢀ-anomeric configuration. The R-configuration was deduced for
the rhamnose units (1JCH ) 170-172 Hz).20 All monosaccharides
were in their naturally occurring form (D-glucose, D-fucose, and
L-rhamnose), as confirmed by optical rotation measurements of the
acid-liberated individual monosaccharides.
The structure of the new acylating acid residue obtained from
the saponification of murucoidin X (5) was further supported by
its chemical transformations to its silylated methyl ester derivative
(16).17 Its mass spectrum indicated that the hydroxyl group was
attached at the C-8 position in a fatty acid chain of 12 carbons
since the R-cleavage23 on either side of the trimethylsiloxyl group
gave the diagnostic ions at m/z 245 [C9H16O3TMS]+ and 159
[C5H10OTMS]+. In compound 5, the presence of the C-8 hydroxyl
group in the aglycone was also recognized by the downfield shift
(R-effect ca. +41 ppm)24 on C-8 (δC 75) and the ꢀ-effects on C-7
and C-9 (δC 38), which resulted in shifts to higher frequency (∆δ
) +8 ppm)24 in comparison with the same resonances in the
n-dodecanoic acid (δC 29-30).25 To determine the stereochemistry
at the C-8 position, compound 15 was converted to the Mosher
esters 17 and 18 with (S)- and (R)-R-methoxy-R-trifluorometh-
1
Common features in both H and 13C NMR spectra of all six
new compounds (1-6) were noted (Tables 1 and 2). All 1H NMR
spectra showed significantly downfield shifted signals for the proton
on C-2 or C-3 of the first rhamnose unit (rha) and on H-2 and H-3
of the second and third rhamnose units (rha′ and rha′′), suggesting
esterification at these positions. The multiplets with splitting patterns
as ddd centered at δ 2.80 and 2.25 showed cross-peaks in their
COSY and TOCSY spectra, revealing the macrocyclic lactone-type
structure for all the murucoidins because these signals correspond
to the nonequivalent diastereotopic protons of the CH2-2 of the
aglycone (11S-hydroxyhexadecanoic acid, jal) when forming a
ring.20,21 The following spectroscopic features were observed: (a)
the carbonyl resonance of the lactone functionality (δ 173-174)
was assigned by the 2J coupling with each of the methylene protons
on the adjacent C-2 position of jalapinolic acid in all compounds
1-6; (b) the lactonization site at C-3 (δC 77) of the second
saccharide (rha) was established by the observed 3J coupling
between this carbonyl carbon and its downfield shifted geminal
proton (δ 5.6) in murucoidins VI (1) and IX-XI (4-6); for
compounds 2 and 3, the lactonization at C-2 (δC 70) was identified
by the 3JC-H coupling and corroborated by the significant downfield
shift for its geminal proton (δ 5.0) in comparison with the same
resonance in the rest of the compounds with the Rha C-3
macrocyclic ring closure; (c) all murucoidins showed signals for
one short-chain fatty acid residue esterifying position C-4 at Rha′′
(δC 73); H-2 of these moieties was used as a diagnostic resonance
centered at δ 2.4-2.5 (1H, tq) for the methylbutanoyl group (mba)
in murucoidins VI (1), VIII (3), X (5), and XI (6) and at δ 2.5-2.6
(1H, septet) for the methylpropanoyl group (iba) in murucoidins
VII (2) and IX (4); (d) the C-2 methylene equivalent protons were
observed as a triplet signal at δ 2.4 for the long-chain fatty acid
esterifying residues in 1, 5, and 6. In all cases it was possible by
HMBC analysis21,22 to establish the links between a specific
carbonyl ester group with their corresponding vicinal proton
resonance (2JCH) and the pyranose ring proton at the site of
esterification (3JCH). For example, the 8-hydroxydodecanoyl residue
1
ylphenylacetyl (MTPA) chloride, and their H NMR spectra were
recorded.26 The chemical shift difference (∆δ ) δS - δR) between
corresponding Me-12 protons was positive (∆δ ) +0.059 ppm),
allowing the confirmation of a C-8 R absolute configuration through
the application of the configurational model proposed by Kakisawa
and associates (Scheme 1).27 This result is consistent with the
previously reported chemical shift differences (∆δ ) +0.057 ppm)
at the terminal methyl signals that are separated by as much as
four methylene groups from the asymmetric carbon.28 It is also in
accordance with the observation that synthetic monohydroxylated
fatty acids with a dextrorotatory optical activity have an S absolute
configuration and their levoisomers have the opposite, R configu-
ration.29 The acylating 8-(R)-hydroxydodecanoic acid of murucoidin
X (5) represents the first levorotatory monohydroxylated aglycone
in the Convolvulaceae family.
Only murucoidins I (9), VI (1), and VII (2) displayed antimi-
crobial activity at the concentration tested against SA-1199B (MIC
32 µg/mL), a Staphylococcus aureus strain that overexpresses the
NorA MDR efflux pump. This may be explained by a loss in
“fitness” of this strain due to overexpression of this MDR pump
when compared to the other strains. All of the murucoidins strongly
potentiated the action of norfloxacin against this NorA overex-
pressing strain30 in experiments using a subinhibitory concentration
of these oligosaccharides (Table 3). They exerted a potentiation
effect, which increased the activity of norfloxacin by 4-fold (8 µg/
mL from 32 µg/mL) at concentrations of 5-25 µg/mL; stoloniferin
I (8) enhanced norfloxacin activity 8-fold when incorporated at a
concentration of 5 µg/mL. These strong potentiation effects were
similar to those previously observed for the amphipathic orizabins,9
which are tetrasaccharides from the Mexican scammony (Ipomoea
orizabensis). An ethidium efflux inhibition assay utilizing SA-
1199B31 demonstrated that compound 8 had modest inhibitory
activity (Figure 1) in comparison with the strong activity previously
reported for orizabin IX, which was more efficacious than reser-
pine.9 This decrease in activity could be due to solubility issues,
since the murucoidins are extremely nonpolar and at higher
concentrations the broth solution became cloudy as a result of test
compound precipitation. Compound 11 had no efflux inhibitory
activity; the basis for its profound effect on the norfloxacin MIC
in the modulation assay currently is unknown and deserves further
attention.
2
in murucoidin X (5) was identified through the observed JCH
coupling between the carbonyl resonance at δ 172.9 with the triplet-
like signal for the vicinal methylene protons (δ 2.3, 2H) and its
3
location at C-2 of Rha′ by the JCH coupling with the signal at δ
5.8. Therefore, the remaining esterified position (C-4 Rha′′ δH 4.2;
δC 73) represented the location of the additional ester linkage for
the methylbutanoyl group.
From the TOCSY experiment,22 edited 1H NMR subspectra for
each individual monosaccharide 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
1
sugar unit was deduced from 2D JCH NMR experiments. For
D-sugars in the 4C1 conformation, the R-anomeric configuration (ꢀ-
equatorial C-H bond) has a 1JCH value of 170 Hz, which is 10 Hz
higher than that for the ꢀ-anomer (R-axial C-H bond; ca. 160
Hz).22 From the anomeric signals in the 13C NMR spectra of the
glycosidic acids (i.e., simonic acids A and B and operculinic acid
The amphipathic properties of these compounds resulting from
the acylation of some of the free hydroxyl groups of the oligosac-
charide core and the lipophilic alkyl chains of their aglycones would
seem to be important in facilitating cellular uptake to its MDR pump
target. To verify this hypothesis, the modulatory activity of two

Resin Glycosides of Ipomoea murucoides
Journal of Natural Products, 2008, Vol. 71, No. 6 1039

1040 Journal of Natural Products, 2008, Vol. 71, No. 6
Chérigo et al.
Table 2. 13C NMR Data of Compounds 1-6 (125 MHz)a
carbonb
1
2
3
4
5
6
glc-1
101.4 104.5 104.6
2
3
4
5
75.3
79.8
72.1
79.8
62.8
82.0
76.5
71.9
77.9
62.8
82.0
76.5
71.9
78.0
62.8
6
fuc-1
101.6 101.6 101.5
2
3
4
5
73.4
76.7
73.6
71.3
17.2
73.4
76.7
73.6
71.3
17.2
73.5
76.5
73.5
71.2
17.2
6
rha-1
2
3
4
5
6
100.3
69.7
77.8
78.1
68.0
19.4
99.2
72.9
80.0
79.8
68.3
18.7
98.8
73.7
69.9
79.8
68.7
19.3
99.3
73.0
79.8
80.1
68.2
18.8
98.8 100.3 100.3 100.1
73.7
70.0
79.8
68.5
19.4
99.1
73.1
79.8
80.1
68.2
18.8
69.8
77.9
77.4
67.9
19.2
99.1
72.8
79.8
79.4
68.4
18.8
69.8
77.8
78.0
67.9
19.2
99.2
73.0
80.2
79.2
68.3
18.8
69.9
77.9
76.5
68.0
19.2
99.3
72.3
80.2
78.6
67.9
18.7
rha′-1
2
3
4
5
6
rha′′-1
103.7 103.8 103.8 103.9 103.7 103.4
2
3
4
5
72.6
70.2
74.8
68.1
17.9
72.7
70.2
74.9
68.5
17.8
72.8
70.2
74.8
68.7
17.9
72.6
70.2
74.8
68.2
17.9
72.6
70.2
74.8
68.2
17.9
72.3
70.2
75.2
68.1
18.0
6
rha′′′-1
104.3 104.9 104.9 104.4 104.3
2
3
4
5
72.6
72.5
73.7
70.8
18.7
72.5
72.5
73.5
68.7
18.6
72.5
72.5
73.5
68.7
18.6
72.6
72.6
73.6
70.7
18.8
72.7
72.5
73.7
70.8
18.8
6
glc-1
2
3
4
5
6
104.8
75.2
78.5
70.7
79.5
62.5
jal-1
174.9 173.4 173.3 174.8 174.9 174.6
2
11
16
33.7
79.4
14.4
34.3
82.8
14.5
34.3
82.8
14.3
33.8
79.4
14.4
33.7
79.3
14.4
34.1
79.5
14.4
mba-1
176.3 175.5 175.5 176.3 176.3 176.3
2
41.6
17.0
11.8
41.5
16.1
11.8
41.5
16.8
11.7
176.3
41.5
17.0
11.7
41.6
17.0
11.8
41.6
17.0
11.8
41.5
16.9
11.7
2-Me
3-Me
mba′-1
2
2-Me
3-Me
iba-1
2
3
3′
176.9
34.5
19.3
19.1
176.0
34.2
19.1
19.1
dodeca-1
2
12
172.9
34.4
14.3
173.5
34.5
14.3
8-hydroxydodeca-1
2
7
8
9
12
172.9
34.4
38.4
75.2
38.3
14.3
a Data in C5D5. Chemical shifts (δ) are in ppm relative to TMS.
b Abbreviations: fuc ) fucose; rha ) rhamnose; glc ) glucose; jal )
11-hydroxyhexadecanoyl;
methylpropanoyl; dodeca
8-hydroxydodecanoyl.
mba
)
)
methylbutanoyl;
dodecanoyl; 8-hydroxydodeca
iba
)
)
tetrasaccharides, tricolorins A and E (SA-1199B, MIC 8 µg/mL),
and their peracetylated derivatives (SA-1199B, MIC >256 µg/mL)

Resin Glycosides of Ipomoea murucoides
Journal of Natural Products, 2008, Vol. 71, No. 6 1041
Scheme 1a
a Key: (a) KOH, (b) CH2N2, (c) TMSiCl, pyridine, 70 °C, 15 min, (d) (R)-MTPACl or (S)-MTPACl, DMAP, pyridine, 70 °C, 5 h. *∆δ ) δ(17) - δ(18).
Table 3. Susceptibility of Staphylococcus aureus to Selected Convolvulaceous Oligosaccharides and their Cytotoxicitya
ED50 (µg/mL)
MIC (µg/mL)
SA-1199Bb
compound
KB
>20
>20
17.9
>20
15.8
12.1
15.7
>20
Hep-2
HeLa
>20
>20
16.4
12.4
>20
ATCC 25923
XU-212
EMRSA-15
Nor (-)
Nor (+)
1
2
3
4
5
6
7
8
9
10
11
12
>20
>20
14.2
10.2
>20
>20
4.7
>20
>20
>20
10.7
4.0
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
16
512
512
256
256
32
32
8c
8c
8
8
8
32
32
8c
8c
8
>512
>512
>512
>512
>512
256
>512
>512
>512
>512
>512
512
>512
>512
>512
>512
>512
64
>20
>20
>20
>20
>20
>20
512
256
32
16.0
>512
>512
>512
256
>512
>512
>512
128
256
4
>512
>512
>512
64
512
8
15.4
15.0
>20
>20
1.0
10.6
>20
8
8
>20
13
>20
>20
1.6
13.2
8c
<2d
32e
32e
orizabin IX
tricolorin A
tricolorin E
tetracycline
norfloxacin
reserpine
vinblastine
5.2
4.7
1.0
>512
8
8
64
8
>20
16
0.125
0.5
8
8
0.25
0.125
0.25
32
8f
0.003
0.007
0.008
a Abbreviations: KB ) nasopharyngeal carcinoma; Hep-2 ) laryngeal carcinoma; HeLa ) cervix carcinoma; ATCC 25923 ) standard S. aureus
strain; XU-212 ) a methicillin-resistant S. aureus strain possessing the TetK tetracycline efflux protein; EMRSA-15 ) epidemic methicillin-resistant S.
aureus strain containing the mecA gene; SA-1199B ) multidrug-resistant S. aureus strain overexpressing the NorA efflux pump. b Nor (-) ) minimum
inhibitory concentration (MIC) value determined in the susceptibility testing; Nor (+) ) MIC value determined for norfloxacin in the modulation assay
at the concentration of 25 µg/mL of the tested oligosaccharide. c MIC value for norfloxacin in the modulation assay at the concentration of 5 µg/mL of
the tested oligosaccharide. d MIC value for norfloxacin in the modulation assay at the concentration of 1 µg/mL of the tested oligosaccharide. e MIC
value for norfloxacin in the modulation assay at the subinhibitory concentration of 2 µg/mL of the tested oligosaccharide. f MIC value for norfloxacin in
the modulation assay at the concentration of 20 µg/mL of reserpine, which was used as positive control for an efflux pump inhibitor.
was tested in order to compare their effects with those obtained
for the orizabin9 and murucoidin series. The antimicrobial tricolorins
and their peracetylated derivatives exhibited no modulatory activity
at the subinhibitory concentration of 2 µg/mL for the natural
products and 25 µg/mL for the derivatives. The loss of activity for
the highly acylated compounds is a comparable situation to the
inactivity recorded for the polar analogue9 since the cellular uptake
is not facilitated, probably in this case, by the formation of complex
aggregates or micelles that could induced membrane perturbation,
provoking an imbalance in the maintenance of cellular homeosta-
sis.32 Surprisingly, the antimicrobial tricolorins A and B did not
display a potentiation effect in combination with norfloxacin. It
seems possible that compounds that are nonpolar will not interact
with the membrane efflux pumps and those that are too polar are
poorly membrane soluble. Therefore, convolvulaceous plants may
elaborate an array of amphipathic mixtures of glycolipids to confer
selective advantages against microbial infections through a com-
bination of several mechanisms of action; for example, the tricolorin
series are cytotoxic, while the murucoidin series exert their action
through inhibition of multidrug resistance pumps (Table 3), as
previously reported for the structurally related acylated disaccharides
found in the leaf exudates of Geranium species (Geraniaceae).33
The most important result from the standpoint of the murucoidins’
potential use as therapeutic agents is that by combining these plant
noncytotoxic products with commercial antibiotics, which are
substrates for these MDR pumps, the treatment of refractive
infections caused by effluxing staphylococci could be alleviated.
The use of bacterial resistance modifiers such as this type of
complex oligosaccharides as prototypes for new efflux pump
inhibitors could facilitate the reintroduction of therapeutically
ineffective antibiotics into clinical use (e.g., ciprofloxacin) and might
even contribute to the suppression of the emergence of new
multidrug-resistant bacterial strains, which are a major cause of
clinical infections.10,34 Extracts that have been standardized on these
mixtures of glycosides (e.g., orizabins) would also find utility in
the development of antiseptic creams to replace or be used in

1042 Journal of Natural Products, 2008, Vol. 71, No. 6
Chérigo et al.
compounds: murucoidin V (13, tR 6.9 min), stoloniferin I (8, tR 7.3
min), murucoidin I (9, tR 13.0 min), pescaprein III (7, tR 16.3 min),
murucoidin II (10, tR 21.9 min), murucoidin III (11, tR 24.6 min), and
murucoidin IV (12, tR 30.3 min). The eluates across the peaks with tR
values of 7.9 min (peak I), 9.6 min (peak II), 14.3 min (peak III), 17.9
min (peak IV), 18.4 min (peak V), and 22.4 min (peak VI) were
collected by the technique of heart cutting and independently reinjected
in the apparatus operating in the recycle mode18,35 to achieve total
homogeneity after 15 consecutive cycles. These techniques afforded
pure compounds 5 (29 mg) from peak II, 1 (3 mg) from peak III, 2
(3.1 mg) from peak IV, 6 (4.2 mg) from peak V, and 3 (3.0 mg) from
peak VI. An isocratic elution with CH3CN-H2O (7:3) was used for
the resolution of peak I from a complex mixture of related minor
oligosaccharide to afford pure major compound 4 (tR ) 24.4 min, 3.2
mg).
Murucoidin VI (1): white powder; mp 143-145 °C; [R]D -38 (c
0.1, MeOH); 1H and 13C NMR, see Tables 1 and 2; negative FABMS
m/z 1265 [M - H]-, 1181 [M - H - C5H8O]-, 1083 [M - H -
C12H22O]-, 853 [1083 - C6H10O4 (methylpentose) - C5H8O]-, 561
[853 - 2 × 146 (C6H10O4)]-, 433 [561 - 128]-, 271 [Jal - H]-;
HRFABMS m/z 1265.7256 [M - H]- (calcd for C63H109O25 requires
1265.7258).
Figure 1. Ethidium efflux inhibition assay against SA-1199B strain
cells. The horizontal line indicates 50% efflux inhibition.
Murucoidin VII (2): white powder; mp 141-143 °C; [R]D -39 (c
0.11, MeOH); 1H and 13C NMR, see Tables 1 and 2; negative FABMS
m/z 1153 [M - H]-, 1083 [M - H - C4H6O]-, 937 [1083 -
C6H10O4]-, 791 [937 - C6H10O4]-, 561, 433, 271; HRFABMS m/z
1153.6000 [M - H]- (calcd for C55H93O25 requires 1153.6006).
Murucoidin VIII (3): white powder; mp 150-152 °C; [R]D -20
(c 0.09, MeOH); 1H and 13C NMR, see Tables 1 and 2; negative
FABMS m/z 1167 [M - H]-, 1083 [M - H - C5H8O]-, 937 [1083 -
C6H10O4]-, 853 [937 - C5H8O]-, 561, 433, 271; HRFABMS m/z
1167.6159 [M - H]- (calcd for C56H95O25 requires 1167.6162).
Murucoidin IX (4): white powder; mp 156-158 °C; [R]D -74 (c
0.07, MeOH); 1H and 13C NMR, see Tables 1 and 2; negative FABMS
m/z 1137 [M - H]-, 1053 [M - H - C5H8O]-, 991 [M - H -
C6H10O4]-, 921 [991 - C4H6O]-, 837 [921 - C5H8O]-, 545 [837 - 2
× 146 (C6H10O4)]-, 417 [545 - 128]-, 271 [Jal - H]-; HRFABMS
m/z 1137.6051 [M - H]- (calcd for C55H93O24 requires 1137.6057).
Murucoidin X (5): white powder; mp 128-130 °C; [R]D -53 (c
0.15, MeOH); 1H and 13C NMR, see Tables 1 and 2; negative FABMS
m/z 1265 [M - H]-, 1181 [M - H - C5H8O]-, 1067 [M - H -
C12H22O2]-, 691 [1067 - 2 × 146 (C6H10O4) - C5H8O]-, 545 [691 -
146 (C6H10O4)]-, 417, 271; HRFABMS m/z 1265.7254 [M - H]- (calcd
for C63H109O25 requires 1265.7258).
combination with mupirocin and fusidic acid, which have wide
usage in containment of S. aureus and its methicillin-resistant
variants in the clinical setting.
Experimental Section
General Experimental Procedures. Melting points were determined
on a Fisher-Johns apparatus and are uncorrected. Optical rotations were
1
measured with a Perkin-Elmer 241 polarimeter. H (500 MHz) and
13C (125 MHz) NMR experiments were conducted on a Bruker DMX-
500 instrument. The NMR techniques were performed according to a
previously described methodology.18,21 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 600 E multisolvent delivery system equipped
with a Waters 410 differential refractometer detector (Waters Corpora-
tion, Milford, MA). Control of the equipment, data acquisition,
processing, and management of the chromatographic information were
performed by the Empower 2 software program (Waters). GC-MS was
performed on a Hewlett-Packard 5890-II instrument coupled to a JEOL
SX-102A spectrometer. GC conditions: HP-5MS (5%-phenyl)-meth-
ylpolysiloxane column (30 m × 0.25 mm, film thickness 0.25 µm);
He, linear velocity 30 cm/s; 50 °C isothermal for 3 min, linear
temperature gradient to 300 at 20 °C/min; final temperature hold, 10
min. MS conditions: ionization energy, 70 eV; ion source temperature,
280 °C; interface temperature, 300 °C; scan speed, 2 scans s-1; mass
range, 33-880 amu.
Murucoidin XI (6): white powder; mp 156-158 °C; [R]D -50 (c
0.32, MeOH); 1H and 13C NMR, see Tables 1 and 2; negative FABMS
m/z 1265 [M - H]-, 1181 [M - H - C5H8O]-, 1083 [M - H -
C12H22O]-, 921 [1083 - 162 (C6H10O5) - C5H8O]-, 545 [921 - 2 ×
146 (C6H10O4) - C5H8O]-, 417, 271; HRFABMS m/z 1265.7253 [M
- H]- (calcd for C63H109O25 requires 1265.7258).
Plant Material. Flowers of Ipomoea murucoides were collected at
the campus of the Universidad Auto´noma del Estado de Morelos,
Cuernavaca, Morelos, Mexico, on December 12, 2004. The voucher
specimens were archived at the Departamento de Farmacia, Facultad
de Qu´ımica, UNAM. Macroscopic anatomical features enabled the
collected material to be identified by one of the authors (R.P.-M.)
through comparison with a voucher specimen deposited at the HUMO
herbarium collection (voucher no. 1520).
Extraction and Isolation. The whole plant material (425 g) was
powdered and extracted exhaustively by maceration at room temperature
with CHCl3 to afford, after removal of the solvent, a dark brown syrup
(39 g). The crude mixture of resin glycosides was obtained after
fractionation of this extract by open column chromatography over silica
gel eluted with a gradient of MeOH in CHCl3. A total of 220 fractions
(250 mL each) were collected and combined to give a pool containing
a mixture of resin glycosides, which was subjected to fractionation by
open column chromatography over reversed-phase C18 (330 g) eluted
with MeOH to eliminate waxes and pigmented residues. This process
provided 30 secondary fractions (30 mL each). Subfractions 19-25
were combined to yield a mixture of lipophilic pentasaccharides (20
g), which were analyzed by reversed-phase C18 HPLC using an isocratic
elution with CH3CN-MeOH (9:1). For their resolution, a Symmetry
C18 column (Waters; 7 µm, 19 × 300 mm), a flow rate of 9 mL/min,
and a differential refractometer detector were used. This analysis
allowed the comparison with reference solutions of the previously
reported resin glycosides,5,19 confirming the detection of the following
Alkaline Hydrolysis of Resin Glycoside Mixture. A solution of
the crude resin glycoside mixture (100 mg) in 5% KOH-H2O (5 mL)
was refluxed at 95 °C for 3 h. The reaction mixture was acidified to
pH 4.0 and extracted with CHCl3 (30 mL). The organic layer was
washed with H2O, dried over anhydrous Na2SO4, and evaporated under
reduced pressure. The aqueous phase was extracted with n-BuOH (20
mL) and concentrated to dryness. The residue from the organic phase
was directly analyzed by GC-MS to allow the detection of three peaks.
MS for the minor constituents with <5% of total chromatogram
integration were not recorded. The major constituents were identified
as 2-methylpropanoic acid (tR 4.12 min), m/z [M]+ 88 (10), 73 (27),
60 (3), 55 (5), 45 (7), 43 (100), 41 (40), 39 (10), 29 (6), 27 (24);
2-methylbutanoic acid (tR 7.2 min), m/z [M]+ 102 (3), 87 (33), 74 (100),
57 (50), 41 (28), 39 (8); and n-dodecanoic acid (tR 17.8 min), m/z [M]+
200 (15), 183 (2), 171 (18), 157 (40), 143 (10), 129 (48), 115 (20),
101 (15), 85 (33), 73 (100), 60 (80), 57 (30), 55 (47), 43 (44), 41 (30).
Previously reported procedures18 were used for the preparation and
identification of 4-bromophenacyl (2S)-2-methylbutyrate from the resin
glycoside fraction: mp 41-42 °C; [R]D +18.2 (c 1.0, MeOH).
The residue extracted (35 mg) from the aqueous phase was subjected
to preparative HPLC on a Waters µBondapak NH2 column (7.8 × 300
mm; 10 µm). The elution was isocratic with CH3CN-H2O (4:1), using
a flow rate of 4 mL/min and a sample injection of 500 µL (35 mg/
mL). This procedure yielded simonic acid A (6.3 mg, tR ) 4.8 min),
operculinic acid C (8.2 mg, tR ) 10.9 min), and simonic acid B (17.0

Resin Glycosides of Ipomoea murucoides
Journal of Natural Products, 2008, Vol. 71, No. 6 1043
Chart 1
Alkaline Hydrolisis of Murucoidin X (5). Approximately 22 mg
of 5 was dissolved in 1 mL of MeOH and 5% KOH-H2O (4 mL).
The solution was refluxed at 95 °C for 2 h. The reaction mixture was
acidified to pH 4.0 and extracted three times with 5 mL of Et2O. The
organic layer was washed with H2O, dried over anhydrous Na2SO4,
and evaporated under reduced pressure. A small aliquot was directly
analyzed by GC-MS with two peaks detected. These were 2-methyl-
butyric acid (tR 8.0 min) and 8-hydroxydodecanoic acid (14, tR 25.0
min): m/z [M - OH - H2O]+ 181 (8), 115 (13), 97 (17), 87 (19), 83
(8), 73 (84), 69 (15), 60 (100), 57 (40), 55 (35), 45 (15), 43 (27), 41
(30). The remaining ether extract was reacted with excess diazomethane,
concentrated, and analyzed by normal-phase HPLC; for the resolution
of this reaction mixture, a normal-phase column (19 × 150 mm, 10
µm), an isocratic elution with hexane-EtOAc (4:1), a flow rate of 2
mL/min, and a differential refractometer were used. The eluates across
the peaks with tR value of 6 min (2-methylbutyric acid methyl ester,
[R]D +10 (c 0.1, CHCl3)) and 12.5 min (8-hydroxydodecanoic acid
methyl ester (15)) were collected and concentrated to dryness.
Compound 15 was dissolved in 0.25 mL of pyridine-d5 and divided
into two portions. An aliquot was derivatized with Sigma Sil-A for 15
min at 70 °C. GC-MS analysis gave one peak (16, tR 5.1 min): 274 [M
- 28]+ (1.2), 271 [M - 31]+ (2.3), 255 (7), 245 (29), 216 (10), 159
(65), 141 (26), 103 (18), 95 (37), 75 (40), 73 (100). Half of the second
aliquot was treated with 4-(dimethylamino)pyridine (3 mg, previously
heated at 70 °C for 3 h) and dry pyridine-d5 (0.75 mL) in NMR tubes.25
(R)-(+)-R-Methoxy-R-trifluoromethylphenylacetyl (MTPA) chloride
was added (20 µL). The reaction was allowed to stand at 70-75 °C
for 5 h under an atmosphere of N2. NMR spectra were then recorded
at 500 MHz by acquiring the reaction mixture. Further purification was
performed as follows. The mixture was transferred from the NMR tubes
into a vial. Saturated aqueous NaHCO3 and Et2O were added to the
mixture and stirred vigorously for 5 min. Water (5 mL) was added
and the mixture was extracted with CHCl3. The organic phases were
washed with 0.5 N HCl, dried with anhydrous Na2SO4, and concen-
trated. The crude residue was purified by normal-phase HPLC using
an isocratic elution with hexane-EtOAc (7:3) and a flow rate of 3
mL/min to give the (S)-MTPA ester (17, tR 7.5 min). NMR spectra in
CDCl3 were recorded after purification. Treatment of the remaining
compound 15 with (S)-(-)-MTPA chloride as described above yielded
the (R)-MTPA ester (18, tR 6.9 min).
Chart 2
mg, tR ) 17.0 min), which were identified by comparison of their
physical constants and NMR data with published values.
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 H2O (5 mL) and extracted with Et2O (30 mL). The aqueous
phase was neutralized with 1 N KOH, extracted with n-BuOH (30 mL),
and concentrated to give a colorless solid (5.7 mg). The residue was
dissolved in CH3CN-H2O and directly analyzed by HPLC: Waters
standard column for carbohydrate analysis (µBondapak NH2; 3.9 ×
300 mm, 10 µm), using an isocratic elution of CH3CN-H2O (17:3), a
flow rate of 1 mL/min, and a sample injection of 20 µL (sample
concentration: 5 mg/mL). Co-elution experiments with standard
carbohydrate samples allowed the identification of rhamnose (tR ) 5.9
min), fucose (tR ) 7.7 min), and glucose (tR ) 10.1 min). Each of
these eluates was individually collected, concentrated, and dissolved
in H2O. Optical activity was recorded after 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, H2O); D-fucose [R]598 +81, [R]578 +83,
[R]546 +94, [R]436 +155, [R]365 +236 (c 0.1, H2O); D-glucose [R]598
+50, [R]578 +51, [R]546 +57, [R]436 +97, [R]365 +150 (c 0.1, H2O).
The aqueous phase from the saponification of compound 5 was
extracted with n-BuOH (5 mL) and concentrated to give a colorless
solid (5 mg). The residue was methylated with CH2N2, and the physical
and spectroscopic constants (1H and 13C NMR) registered for the product
were identical in all aspects to those previously reported11 for simonic

1044 Journal of Natural Products, 2008, Vol. 71, No. 6
Chérigo et al.
acid B methyl ester: white powder; mp 113-115 °C; [R]D -82.5 (c
1.0, MeOH); HRFABMS m/z 1015.5322 [M - H]- (calcd for C47H83O23
requires 1015.5325).
(8R)-(-)-8-Hydroxydodecanoic Acid Methyl Ester (15): oil; [R]598
-15.6, [R]578 -16.3, [R]546 -16.8, [R]436 -18.8, [R]365 -20 (c 0.1,
CHCl3); 1H NMR (400 MHz, CDCl3) δH 3.67 (3H, s, OMe), 3.59 (1H,
m, H-8), 2.34 (2H, t, J ) 7.5 Hz, H-2), 1.64 (2H, m, H-3), 1.46-1.07
(14H, m, H2-4-H2-7, and H2-9-H2-11), 0.90 (3H, t, J ) 7.2, H-12);
HRESIMS m/z 229.1805 (calcd for C13H25 O3, 229.1803).
References and Notes
(1) Felger, R.; Austin, D. F. Sida 2005, 21, 1293–1303.
(2) The Mexican term “cazahuate” is derived from the Nahuatl (“cuau-
hzahuatl”) words for tree (“quauitl”) and mange (“zahuatl”). This
medicinal plant complex of arborescent morning glories is composed
of six related tree-like Ipomoea species: I. arborescens (Kunth) G.
Don, I. bracteata Cav., I. intrapilosa Rose, I. murucoides Roem. &
Schult, I. pauciflora Martens & Galeotti, and I. wolcottiana Rose.
(3) Emmart, E. W. The Badianus Manuscript (Codex Barberini, Latin
241). An Aztec Herbal of 1552; The Johns Hopkins Press: Baltimore,
1940; p 215.
Bacterial Strains and Media. Staphylococcus aureus EMRSA-15
containing the mecA gene was provided by Dr. Paul Stapleton, The
School of Pharmacy, University of London. Strain XU-212, a methi-
cillin-resistant strain possessing the TetK tetracycline efflux protein,
was provided by Dr. E. Udo.36 SA-1199B, which overexpresses the
NorA MDR efflux protein,37 and S. aureus ATCC 25923 were also
used. All strains were cultured on nutrient agar (Oxoid, Basingstoke,
UK) before determination of MIC values. Cation-adjusted Mueller-
Hinton broth (MHB; Oxoid) containing 20 and 10 mg/L of Ca2+ and
Mg2+, respectively, was used for susceptibility tests.
Susceptibility Testing. Minimum inhibitory concentration values
(MIC) were determined at least in duplicate by standard microdilution
procedures, as recommended by the National Committee for Clinical
Laboratory Standards guidelines.38 An inoculum density of 5 × 105
cfu of each of the test strains was prepared in 0.9% saline by comparison
with a McFarland standard. MHB (125 µL) was dispensed into 10 wells
of a 96-well microtiter plate (Nunc, 0.3 mL volume per well).
Glycolipids 1-13 were tested at final concentrations ranging from 1
to 512 µg/mL prepared by serial 2-fold dilutions. All test compounds
were dissolved in DMSO before dilution into MHB for use in MIC
determinations. The highest concentration of DMSO remaining after
dilution (3.125% v/v) caused no inhibition of bacterial growth. The MIC
was defined as the lowest concentration that yielded no visible growth.
Tetracycline and norfloxacin from Sigma (Poole, UK) were also tested
as positive drug controls. For the modulation assay, the murucoidins
were tested at final concentrations of 25 or 5 µg/mL, and the glycolipids
orizabin IX and tricolorins A and E at 2 µg/mL. Serial doubling
dilutions of norfloxacin in the range 1-512 µg/mL were added, and
the microtiter plates were then interpreted in the same manner as MIC
determinations. The activity of reserpine at a concentration of 20 µg/
mL was also tested as an efflux pump inhibitor for comparison purposes.
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(10×) medium supplemented with 10% fetal bovine serum. Cell lines
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Acknowledgment. This research was partially supported by Consejo
Nacional de Ciencia y Tecnolog´ıa (45861-Q) and Direccio´n General
de Asuntos del Personal Acade´mico, UNAM (IN208307). R.P.-M. was
on sabbatical leave at the Centre for Pharmacognosy and Phytotherapy
(The School of Pharmacy, UL) with the financial support of DGAPA
(UNAM). L.C. is grateful to DGAPA for a graduate scholarship. N.J.-
H. received posdoctoral scholarships from CONACyT and DGAPA.
Thanks are due to G. Duarte and M. Guzma´n (USAI, Facultad de
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