Phytochemistry 95 (2013) 277–283

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Phytochemistry

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Modulators of antibiotic activity from Ipomoea murucoides

0031-9422/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.phytochem.2013.07.007

⇑ Corresponding author. Tel.: +52 (55) 5622 5288; fax: +52 (55) 5622 5329.E-mail address: pereda@unam.mx (R. Pereda-Miranda).

Berenice Corona-Castañeda a, Lilia Chérigo a, Mabel Fragoso-Serrano a, Simon Gibbons b,Rogelio Pereda-Miranda a,⇑a Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City 04510DF, Mexicob Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK

a r t i c l e i n f o

Article history:Received 7 May 2013Received in revised form 7 July 2013Available online 3 August 2013

Keywords:Ipomoea murucoidesConvolvulaceaeStructural spectroscopyGlycolipidsMultidrug-resistanceGram-positive bacteriaGram-negative bacteria

a b s t r a c t

Reinvestigation of the CHCl3-soluble extract from the flowers of Ipomoea murucoides, through prepara-tive-scale recycling HPLC, yielded three pentasaccharides of 11-hydroxyhexadecanoic acid, murucoidinsXVII–XIX, in addition to the known murucoidin III and V, all of which were characterized by NMR spec-troscopy and mass spectrometry. These compounds were found to be macrolactones of the known pen-tasaccharides simonic acid B and operculinic acid A. The acylating groups corresponded to acetic, (2S)-methyl-butyric, (E)-cinnamic and octanoic acids. The esterification sites were established at the C-2 ofthe second rhamnose and C-3 and C-4 of the third rhamnose. The aglycone lactonization was placed atC-2 or C-3 of the first rhamnose. Bioassays for modulation of antibiotic activity were performed againstmultidrug-resistant strains of Staphylococcus aureus, Escherichia coli Rosetta-gami, and two nosocomialpathogens: Salmonella enterica sv. Typhi and Shigella flexneri. The tested glycolipids did not act as cyto-toxic (IC50 > 4 lg/mL) nor as antimicrobial (MIC > 128 lg/mL) agents. However, they exerted a potentia-tion effect on clinically useful antibiotics against the tested bacteria by increasing their antibioticsusceptibility up to four-fold at concentrations of 25 lg/mL.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction activity within the cell (Savjani et al., 2009; Stavri et al., 2007). Pre-

The genus Ipomoea of the morning glory family (Convolvulaceae)is distinguished by its worldwide distribution in tropical regions aswell as for its curative attributes that include laxative properties,which are derived from the presence of high yields of resin glyco-sides in the whole plant (Eich, 2008; Pereda-Miranda et al., 2010).From a chemical point of view, resin glycosides are mixtures of gly-colipids of high molecular mass that are amphipathic in nature duethe presence of a hydrophilic portion (the oligosaccharide core) anda hydrophobic aglycone (monohydroxylated fatty acids of fourteenand sixteen carbon atoms) forming the macrolactone (Pereda-Mir-anda et al., 2010). Important and diverse biological activities of ther-apeutic interest have been attributed to morning glory resinglycosides (Pereda-Miranda et al., 2010). One of which is their anti-bacterial activity against resistant nosocomial strains of Staphylo-coccus aureus (Pereda-Miranda et al., 2006).

The main mechanism in the development of bacterial resistanceis due to the presence of trans-membrane proteins that confer thephenotype of multidrug-resistance (MDR) to antibiotics, for exam-ple the Nor-A and Tet-K pumps. These proteins function as trans-port systems that are activated to expel antibiotics, preventingthe drug from reaching the concentration required to exert their

vious studies have described the enhancement of antibiotic sus-ceptibility using microbiologically inactive resin glycosides asinhibitors of efflux pumps in S. aureus (Chérigo et al., 2008, 2009;Escobedo-Martínez et al., 2010; Pereda-Miranda et al., 2006). Thesecompounds caused a decrease (at the concentration of 25 lg/mL)of the minimum inhibitory concentration value (MIC; four times,32–8 lg/mL) of therapeutic antibiotics. Experiments to test theinhibition of extracellular transport of ethidium bromide, a fluores-cent agent, have shown that these glycolipids are substrates forMDR efflux pumps (Chérigo et al., 2008; Pereda-Miranda et al.,2006) as well as, in some instances, more potent and effectiveinhibitors of efflux pumps than reserpine, an alkaloid used as a po-sitive efflux pump inhibitor control (Gibbons, 2008; Li and Nikaido,2009). These metabolites were also tested for resistance-modula-tory activity against Escherichia coli Rosetta-gami and two nosoco-mial pathogens, Salmonella enterica sv. Typhi and Shigella flexneri.These compounds exerted a potentiation effect of clinically usefulantibiotics against the tested Gram-negative bacteria by increasingantibiotic susceptibility up to 64-fold at concentrations of 25 lg/mL (Corona-Castañeda and Pereda-Miranda, 2012).

These findings constitute the starting point for the conceptualapproach that guided the present investigation, which is aimedat evaluating new oligosaccharides from I. murucoides as potentialmodulators of the multidrug-resistant (MDR) phenotype in bothGram-positive and -negative bacteria, where active efflux has

278 B. Corona-Castañeda et al. / Phytochemistry 95 (2013) 277–283

proven to be one of the most successful detoxification mecha-nisms. Expanding the knowledge of resin glycoside structuraldiversity opens the potential for possible therapeutic applicationsagainst nosocomial pathogens.

2. Results and discussion

A chemical reinvestigation of I. murucoides flowers allowed theisolation of three novel pentasaccharides (1–3) from their

Table 11H (500 MHz) NMR spectroscopic data of compounds 1–3 (pyridine-d5).a

Hydrogenb 1

Fuc 1 4.82 d (7.5)2 4.50 dd (7.5, 9.5)3 4.20 dd (9.5, 2.5)4 3.90⁄

5 3.81 dq (1.0, 6.5)6 1.52 d (6.5)

Rha 1 6.32 d (1.0)2 5.20 dd (1.0, 2.5)3 5.66 dd (3.0, 10.0)4 4.67 t (9.5)5 4.99 dq (9.5, 6.0)6 1.59 d (6.0)

Rha0 1 5.57 d (1.5)2 6.03 dd (1.5, 3.5)3 4.63 dd (3.5, 9.0)4 4.26 t (9.5)5 4.39 dq (9.5, 6.0)6 1.62 d (6.0)

Rha00 1 6.20 d (1.5)2 5.14 dd (1.5, 3.0)3 5.88 dd (3.0, 9.5)4 6.02 t (9.5)5 4.45 dq (9.5, 6.5)6 1.43 d (6.5)

Rha0 0 0 123456

Glc 1 5.09 d (7.5)2 3.87 dd (7.5, 8.8)3 4.12 dd (8.5, 9.0)4 4.14 dd (8.5, 9.0)5 3.84 ddd (2.5, 5.5, 8.0)6a 4.35 dd (11.0, 5.0)6b 4.46 dd (12.5, 3.0)

Jal 2a 2.26 ddd (2.8, 7.0, 15.0)2b 2.58 dd (2.8, 15.0)11 3.90⁄

16 0.96 t (7.0)

Mba 2 2.45 tq (7.0, 7.0)2-Me 1.13 d (7.0)3-Me 0.84 t (7.5)

Mba0 2 2.45 tq (7.0, 7.0)2-Me 1.11 d (7.0)3-Me 0.81 t (7.5)

Cna 2 6.56 d (16.0)3 7.84 d (16.0)2’ 7.32 m3’ 7.44 m4’ 7.32 m

Ac 2

Octa 28

a Chemical shifts (d) are in ppm relative to TMS. The spin coupling (J) is given in parenSpin-coupled patterns are designated as follows: s = singlet, d = doublet, t = triplet, q = q

b Abbreviations: Fuc, fucose; Rha, rhamnose; Glc, glucose, Jal, 11-hydroxyhexadecanoy

CHCl3-soluble extracts as well as the previously known murucoi-dins III and V (Chérigo and Pereda-Miranda, 2006). The structuresfor the new murucoidins XVII–XIX (1–3) were established by one-dimensional (1H and 13C NMR) (Tables 1 and 2) and two-dimen-sional (NMR: COSY, TOCSY, HSQC and HMBC) spectroscopic andspectrometric (FAB-MS negative mode) techniques (Pereda-Miran-da et al., 2010). The novel compounds were found to be macrolac-tones of the known pentasaccharides simonic acid B andoperculinic acid A. Evidences for the configuration of constitutive

2 3

4.80 d (8.0) 4.73 d (7.5)4.51 dd (8.0, 9.5) 4.16 dd (7.5, 9.5)4.16 dd (9.5, 3.0) 4.07 dd (9.5, 4.0)3.91 sa 3.99 d (3.5, 1.0)3.81 dq (1.0, 6.5) 3.78 dq (1.0, 6.5)1.52 d (6.5) 1.51 d (6.0)

6.32 d (1.5) 5.48 d (1.5)5.29 dd (1.5, 3.0) 5.95 dd (2.0, 3.5)5.59 dd (3.0, 9.5) 5.02 dd (3.0, 9.5)4.62 t (9.5) 4.23 sa4.99 dd (9.5, 6.0) 4.43 dq (9.5, 6.0)1.57 d (6.0) 1.62 d (6.0)

5.64 d (1.5) 6.12 d (2.0)5.81 dd (1.5, 3.0) 5.97 dd (2.0, 3.0)4.49 dd (3.0, 9.5) 4.61 dd (3.0, 9.5)4.21 dd (9.5, 9.5) 4.26 dd (9.5, 9.5)4.31 dq (9.5, 6.5) 4.35 dq (9.5, 6.0)1.59 d (6.5) 1.65 d (6.5)

5.89 sa 5.89 d (1.5)4.64 dd (1.5, 3.5) 4.72 dd (1.5, 3.0)4.41 sa 4.49 dd (3.0, 9.5)5.76 t (9.5) 5.81 t (9.5)4.33 dq (9.5, 6.0) 4.33 dq (9.5, 6.5)1.39 d (6.0) 1.38 d (6.5)

5.55 d (1.5) 5.64 d (1.5)4.76 sa 4.85 dd (1.5, 2.5)4.38 dd (3.5, 9.5) 4.40 dd (2.5, 9.0)4.25 dd (9.5, 9.5) 4.27 dd (8.0, 9.0)4.28 dq (9.5, 6.5) 4.25 dq (9.5, 4.5)1.69 d (6.5) 1.56 d (5.5)

2.24 ddd (2.5, 6.5, 15.0) 2.23 ddd (3.5, 9.0, 12.5)2.92 ddd (2.5, 6.5, 15.0) 2.40 ddd (3.5, 9.0, 12.5)3.87 m 3.86 m0.84 t (7.0) 0.88 t (7.0)

2.50 tq (7.0) 2.37 tq (6.5, 7.0)1.20 d (7.0) 1.07 d (7.0)0.94 t (7.5) 0.84 t (7.5)

2.05 s

2.34 t (7.0)0.95 t (7.0)

theses (Hz). Chemical shifts marked with an asterisk (⁄) indicate overlapped signals.uartet, m = multiplet, brs = broad singlet.l; Mba, methylbutanoyl, Cna, cinamoyl, Ac, acetyl, Octa, octanoyl.

Table 213C (125 MHz) NMR spectroscopic data of compounds 1–3 (pyridine-d5).a

Carbonb 1 2 3

Fuc 1 101.5 101.6 104.32 73.6 73.4 80.33 76.5 76.6 73.34 73.6 73.5 73.15 71.2 71.2 70.86 17.2 17.2 17.4

Rha 1 100.0 100.2 98.82 70.0 69.7 73.93 78.0 77.8 69.94 76.0 77.9 79.95 68.0 67.9 68.66 19.2 19.2 19.5

Rha0 1 99.2 99.2 99.22 72.3 73.0 73.03 80.1 80.2 79.64 79.1 79.2 80.05 68.1 68.3 68.16 18.8 18.7 18.8

Rha00 1 103.4 103.6 103.72 69.9 72.6 72.93 72.8 70.2 70.24 71.7 74.8 75.35 68.2 68.1 68.56 17.9 17.9 17.8

Rha0 0 0 1 104.3 104.92 72.5 72.53 72.6 72.64 73.7 73.55 70.8 70.56 18.8 18.6

Glc 1 105.12 75.23 78.24 70.85 77.76 62.5

Jal 1 174.4 174.9 173.12a 34.5 33.7 34.311 79.5 79.3 82.316 14.5 14.4 14.3

Mba 1 175.9 176.32 41.3 41.62-Me 16.9 17.03-Me 11.8 11.8

Mba0 1 176.12 41.52-Me 16.73-Me 11.6

Cna 1 166.12 118.63 145.21’ 134.72’ 129.23’ 128.54’ 130.7

Ac 1 170.62

Octa 1 172.92 34.48 14.2

a Chemical shifts (d) are in ppm relative to TMS.b Abbreviations: Fuc, fucose; Rha, rhamnose; Glc, glucose, Jal, 11-hydroxyhexa-

decanoyl; Mba, methylbutanoyl, Cna, cinamoyl, Ac, acetyl, Octa, octanoyl.

B. Corona-Castañeda et al. / Phytochemistry 95 (2013) 277–283 279

monosaccharides, the anomeric linkages, the sequence of glycosi-dation, as well as the absolute configuration for the aglycones werepublished when these oligosaccharides were first elucidated. Also,they are representative cores commonly found in the morningglory resin glycosides, e.g., the Mexican medicinal arborescent

members of the genus Ipomoea, I. murucoides (Chérigo andPereda-Miranda, 2006) and I. intrapilosa (Bah et al., 2007), amongothers (Pereda-Miranda et al., 2010).

Negative-ion FAB mass spectrometry was particularly useful indetermining the exact mass and molecular formulas for 1–3. Themass spectra showed a quasimolecular ion [M�H]� at m/z 1297(C65H101O26) for compound 1, at m/z 1193 ([M�H]�, C59H101O24)for compound 2, and at m/z 1109 ([M�H]�, C53H89O24) for 3. Inall cases, the characteristic fragment ions common to all branchedpentasaccharide resin glycosides were observed for the glycosidiccleavage of each sugar moiety (Chérigo et al., 2008; Escalante-Sán-chez et al., 2008; Pereda-Miranda et al., 2005). For example, diag-nostic fragments were observed for the aglycone (jalapinoic acidresidue: m/z 271), the aglycone and a methyl pentose residue (m/z 417), the aglycone and two methyl pentoses (m/z 545), and theaglycone and three methyl pentoses (m/z 691) (Bah and Pereda-Miranda, 1996; Pereda-Miranda et al., 2010; Castañeda-Gómezet al., 2013). For compound 1, the observed ester eliminations atm/z 1213 ([M�H]�–C5H8O [Mba, 84 amu]), 1167 ([M�H]�–C9H6O[Cna, 130 amu]), and 1083 ([M�H]�–C5H8O–C9H6O) allowed forthe identification of a triacylated simmonic acid B macrocyclicderivative, while a diacylated derivative of this glycosidic acidwas identified for 3 through fragment anions at m/z 1067([M�H]�–C2H2O [Ac, 42 amu]) and 837 ([M�H]�–C2H2O–C5H8O–C6H10O4). Finally, the fragments at m/z 1067 ([M�H]�–C8H14O),963 ([M�H]�–C5H8O–C6H10O4), and 837 ([M�H]�–C5H8O–C8H14O–C6H10O4) also identified a diacylated operculinic acid Aderivative for 2 (Castañeda-Gómez et al., 2013).

Murucoidin XVII (1) was identified as the intramolecular1,300 ester of (11S)-hydroxyhexadecanoic acid of O-b-D-glucopyran-osyl-(1?3)-O-[4-O-(2S)-2-methylbutanoyl,3-O-cinnamoyl]-a-L-rhamnopyranosyl-(1?4)]-O-a-L-rhamnopyranosyl-(1?4)-O-a-L-rhamnopyranosyl-(1?2)-O-b-D-fucopyranoside. In the 1H NMRspectrum, five anomeric protons were identified through theirchemical shifts and coupling constant as doublets (Table 1): 4.82(7.5 Hz, Fuc), 6.32 (1.0 Hz, Rha), 5.57 (1.5 Hz, Rha0), 6.20 (1.5 Hz,Rha00), and 5.09 ppm (7.5 Hz, Glc). Characteristic signals for theacylating substituents were registered: (a) (2S)-methylbutanoic,triplet-like signal at 2.45 ppm (Mba, H-2); (b) cinnamic acid, twodoublets at 6.56 (Cna, H-2) and 7.84 (H-3) ppm and two multipletsat 7.32 (H-20) and 7.44 (H-30) ppm. The 13C NMR spectrum pro-vided the following information (Table 2): five anomeric carbonsbetween 99.2 and 105.1 ppm, a signal at 62.5 ppm for the hydroxy-methylene group of glucose, and four signals corresponding to thecarbonyl groups for the three acylating substituents (Mba174.4 ppm; Mba0 176.1 ppm; Cna 166.1 ppm) and the macrocycliclactone (174.4 ppm).

Murucoidin XVIII (2) was identified as the intramolecular1,200 ester of (11S)-hydroxyhexadecanoic acid of O-a-L-rhamno-pyranosyl-(1?3)-O-[[4-O-(2S)-2-methylbutanoyl]-a-L-rhamno-pyranosyl-(1?4)]-O-[2-O-octanoyl]-a-L-rhamnopyranosyl-(1?4)-O-a-L-rhamnopyranosyl-(1?2)-O-b-D-fucopyranoside. For 2 inthe 1H NMR spectrum (Table 1), the anomeric signals were ob-served at 4.80 (8.0 Hz, Fuc), 6.32 (1.5 Hz, Rha), 5.64 (1.5 Hz, Rha0),5.89(1.0 Hz, Rha00), and 5.55 ppm (1.5 Hz, Rha0 0 0). The characteristicsignals for the ester substituents were: Mba (dH-2 2.50) and octa-noic acid (two triplets, dH-2 2.34 and dH-8 0.95). The 13C NMR spec-trum showed: five anomeric carbons (dC 99.2–104.3); and threesignals for the carbonyl groups: two for the acylating acids (OctadC 172.9 and Mba dC 176.3); and the macrolactone (dC 174.9) Fig. 1.

The murucoidin XIX (3) was identified as the intramolecular1,200 ester of (11S)-hydroxyhexadecanoic acid of O-a-L-rhamno-pyranosyl-(1?3)-O-[[4-O-acetyl]-a-L-rhamnopyranosyl-(1?4)]-O-[2-O-(2S)-2-methylbutanoyl]-a-L-rhamnopyranosyl-(1?4)-O-a-L-rhamnopyranosyl-(1?2)-O-b-D-fucopyranoside. The 1H NMRspectra of this compound showed five anomeric protons: dH 4.73

Compound

R1 R2 R3 R4

1 (2S)-methylbutanoyl = Mba cinamoyl= Cna β-D-glucopyranosyl= Glc Mba

2 Mba H α-L-rhamnopyranosyl = Rha n-octanoyl

Fig. 1. Chemical structure of murucoidins XVII and XVIII (1 and 2).

Fig. 2. Chemical structure of murucoidin XIX (3).

280 B. Corona-Castañeda et al. / Phytochemistry 95 (2013) 277–283

(7.6 Hz; Fuc), 5.48 (1.5 Hz, Rha), 6.12 (2.0 Hz; Rha0), 5.89 (1.5 Hz,Rha00), and 5.64 (1.5 Hz, Rha0 0 0); 2-methylbutanoic acid (tq, dH-2

2.37) and acetic acid (singlet, dH 2.05). The 13C NMR spectrumdisplayed five signals for the anomeric carbons (dC 98.8–104.9)and three carbonyl signals at dC 170.6 (Ac), 172.9 (Octa) and175.5 (macrolactone) Fig. 2.

Table 3Susceptibility of Staphylococcus aureus to murucoidins XVII–XIX (1–3) and their cytotoxici

Compound IC50 (lg/mL) MIC (lg/m

KB Hep-2 HeLa ATCC 2592

1 15.7 6.1 >20 >1282 >20 >20 >20 >1283 10.8 >20 19.0 >128Vinblastine 0.005 0.006 0.001Colchicine 0.002 0.003 0.001NorfloxacinReserpine

a Abbreviations: KB, nasopharyngeal carcinoma; Hep-2, laryngeal carcinoma; HeLa, ceicillin-resistant S. aureus strain containing the mecA gene; XU-212, a methicillin-resimultidrug-resistant S. aureus strain over-expressing the NorA efflux pump.

b Nor (�) = minimum inhibitory concentration (MIC) value determined in the susceptibat the concentration of 25 lg/mL of the tested oligosaccharide.

c MIC value for norfloxacin in the modulation assay at the concentration of 20 lg/mL

The acylation sites and glycosidation sequence were assignedby the 2,3JCH correlations observed in the HMBC spectrum for allof the compounds (Duus et al., 2000; Pereda-Miranda et al.,2010).The position for macrolactonization was placed at C-2 forcompound 3 and at C-3 for compounds 1 and 2 of the second sac-charide (Rha), as confirmed by the observed correlation for protonsH-2 (dH-2 5.95 for compound 3) or H-3 (dH-3 5.66 for compound 1and dH-3 5.59 for 2). Similarly, the esterification sites were locatedat C-2 of the second unit of rhamnose (Rha0) at dH-2 6.03, 5.81, and5.97 for compounds 1–3, respectively, and H-4 of the third unit ofrhamnose (Rha00) at dH-4 6.02, 5.76, and 5.81 for all compounds.Additionally, compound 1 displayed another position of acylationat C-3 of Rha00 at dH-3 5.88.

The tested glycolipids were neither cytotoxic (KB: IC50 > 4 lg/mL) nor antibacterial (Gram-positive: MIC P 128 lg/mL; Gram-negative: MIC P 512 lg/mL) (Table 3). However, all of them exhib-ited a strong antibiotic modulatory activity at a concentration of25 lg/mL (Table 4). This effect potentiated norfloxacin susceptibil-ity against SA-1199B by four-fold (32–8 lg/mL) for compounds 1and 2, and two-fold (32–16 lg/mL) for compound 3 (Table 3).The modulation of antibiotic activity on MDR Gram-negative

ty.a

L)c

3 XU-212 EMRSA-15 SA-1199Bb

Nor (�) Nor (+)

>128 >128 128 8>128 >128 128 8>128 >128 128 16

324

rvix carcinoma; ATCC 25923, standard S. aureus strain; EMRSA-15, epidemic meth-stant S. aureus strain possessing the TetK tetracycline efflux protein; SA-1199B,

ility testing; Nor (+) = MIC value determined for norfloxacin in the modulation assay

of reserpine which was used as positive control for an efflux pump inhibitor.

Table 4Susceptibility of Escherichia coli, Salmonellaenterica, and Shigella flexneri to murucoidins XVII–XIX (1–3).

Compound MIC (lg/mL)

Escherichia coli Rosetta gami Salmonella enterica sv. Typhi Shigella flexneri

(�) Tet (+) Kan (+) Cam (+) (�) Tet (+) Kan (+) Cam (+) (�) Tet (+) Kan (+) Cam (+)

Antibiotics 128 32 256 1024 1024 512 512 512 2561 >512 64 8 128 >512 512 512 256 512 256 128 2562 >512 64 8 128 >512 512 512 256 512 256 128 2563 >512 64 8 128 >512 512 512 256 512 128 128 256

Reserpine >512 64 8 128 >512 512 512 256 >512 256 64 128

Abbreviations: (�), minimum inhibitory concentration (MIC) value determined in the susceptibility testing; Tet (+), MIC value determined for tetracycline; Kan(+), MIC valuedetermined for kanamycin; Cam (+), MIC value determined for chloramphenicol in the modulation assay at the concentration of 25 lg/mL of the tested oligosaccharide.

B. Corona-Castañeda et al. / Phytochemistry 95 (2013) 277–283 281

strains was similar to that observed for reserpine, a positive con-trol, in combination with tetracycline, kanamycin, and chloram-phenicol. In most of the cases observed, a reduction in MIC wastwo-fold (Table 4). The highest reduction was observed for com-pound 3 in combination with tetracycline and kanamycin againstS. flexneri (512–128 lg/mL). The complexity of the machineryand mechanisms of multidrug efflux in Gram-negative pathogenscauses the major difference observed in potency for modulatoryactivity of the tested glycolipids against both Gram-positive and-negative bacteria, the latter of which are enveloped within a pro-tective double-layer of lipid membranes. Noxious compounds areexpelled across these membranes by active transport, providingthe pathogens with a permeability barrier to hydrophilic com-pounds like antibiotics (Stavri et al., 2007). The more complexstructure of multidrug efflux pumps in Gram-negative strains in-cludes an inner membrane transporter, a periplasmic fusion pro-tein, as well as an outer membrane channel (Zgurskaya, 2009).This tripartite efflux pump system therefore confers MDR bacteriawith the capacity to occupy lethal ecological niches by avoiding thecytotoxic effects of antibiotics and drastically limits the clinical useof these drugs (Corona-Castañeda and Pereda-Miranda, 2012).

3. Concluding remarks

The contemporary recognition of the emergence and prolifera-tion of methicillin-resistant strains of S. aureus is an epidemiolog-ical challenge as few antibiotics are effective agents in thetreatment of nosocomial infections caused by these pathogens.Multidrug-resistant strains of S. aureus are often unresponsive tomany types of clinically useful antibiotics which can include van-comycin, oxazolidinone, b-lactam and the streptogram in antibi-otic categories (Freixas et al., 2012; Shibata et al., 2005). Theliterature describes a few effective plant-derived efflux pumpinhibitors (EPIs) against Gram-positive bacteria (Stavri et al.,2007; Savoia, 2012), but information about EPIs against Gram-neg-ative nosocomial pathogens is much more scarce. From the resultsobtained by researching into the potential of resin glycosides asEPIs in microbial pathogens (Chérigo et al., 2008, 2009; Escobe-do-Martínez et al., 2010; Pereda-Miranda et al., 2006) and mam-malian cells (Castañeda-Gómez et al., 2013; Cruz-Morales et al.,2012; Figueroa-González et al., 2012), there is a clear indicationof the possibility that these metabolites could be used as synergis-tic agents in combinatorial therapies, increasing the strength andusefulness of antibiotics that are not effective in the treatment ofrefractive infections caused by MDR strains.

4. Experimental

4.1. General experimental procedures

Melting points were determined on a Fisher–Johns apparatusand are uncorrected. Optical rotations were measured with a

Perkin–Elmer 241 polarimeter. 1H (500 MHz) and 13C (125 MHz)NMR experiments were conducted on a Bruker DMX-500 instru-ment. The NMR techniques were performed according to a previ-ously described methodology (Bah and Pereda-Miranda, 1996).Negative-ion low and high-resolution FABMS were recorded usinga matrix of triethanolamine on a JEOL SX-102A spectrometer. Theinstrumentation used for HPLC analysis consisted of a Waters600 E multisolvent delivery system equipped with a Waters 410differential refractometer detector (Waters Corporation, Milford,MA). Control of the equipment, data acquisition, processing, andmanagement of the chromatographic information were performedby the Empower 2 software program (Waters).

4.2. Plant material

Flowers of Ipomoea murucoides were collected at Tepostlán,Morelos, Mexico, on April 10, 2010. The plant material was identi-fied by Dr. Robert Bye and one of the authors (R.P.-M.) throughcomparison with an authentic plant sample (RP-05) archived atthe Departamento de Farmacia, Facultad de Química, UNAM (Chér-igo and Pereda-Miranda, 2006). A voucher specimen (Robert Bye35906) was deposited in the Ethnobotanical Collection of the Na-tional Herbarium (MEXUE), Instituto de Biología, UNAM.

4.3. Extraction and isolation of compounds 1–3

The whole plant material (500 g) was powdered and extractedexhaustively by maceration at room temperature with CHCl3 to af-ford, after removal of the solvent, a dark brown syrup (35.8 g). Thecrude mixture of resin glycosides was obtained after fractionationof this extract by open column chromatography over silica geleluted with a gradient of MeOH in CHCl3. A total of 220 fractions(250 mL each) were collected and combined to give a pool contain-ing a mixture of resin glycosides, which was subjected to fraction-ation by open column chromatography over reversed-phase C18

(330 g) eluted with MeOH to eliminate waxes and pigmented res-idues. This process provided 30 secondary fractions (30 mL each).Subfractions 18–25 were combined to yield a mixture of lipophilicpentasaccharides (20 g), which were analyzed by reversed-phaseC18 HPLC using an isocratic elution with CH3CN–H2O (95:5). Fortheir resolution, a Symmetry C18 column (Waters; 7 lm,19 � 300 mm), a flow rate of 9 mL/min, and a differential refrac-tometer detector were used. This analysis allowed the comparisonwith reference solutions of the previously reported resin glycosides(Chérigo et al., 2008), confirming the detection of the followingcompounds: murucoidin V (tR 6.9 min), and murucoidin III (tR

24.6 min). Peaks with tR values of 9.2 min (peak II), and 16.5 min(peak IV) were collected by the technique of heart cutting andindependently reinjected in the apparatus operating in the recyclemode (Pereda-Miranda and Hernández-Carlos, 2002) to achieve to-tal homogeneity after 15 consecutive cycles. These techniqueyielded pure compounds: compound 1 (25 mg) from peak II, left

282 B. Corona-Castañeda et al. / Phytochemistry 95 (2013) 277–283

region; compound 2 (20 mg) from peak II, right region; and com-pound 3 (15 mg) from peak IV. Saponification of all compoundsyielded (S)-(+)-a-methylbutyric acid: [a]D +10 (c 1.0, CHCl3), whilecompounds 1–3 also afforded cinnamic, octanoic and acetic acids,respectively, all of which were purified by HPLC according to pre-viously described methologies (Pereda-Miranda and Hernández-Carlos, 2002; Pereda-Miranda et al., 2005).

4.4. Compound characterization

4.4.1. Murucoidin XVII (1)White powder; mp 133–135 �C; [a]D �98 (c 0.2, MeOH); 1H and

13C NMR, see Tables 1 and 2; negative-ion FABMS m/z 1297[M�H]� (C65H101O26), 1167 [1297�130 (C9H6O, cna)]�, 1083[1167�84 (C5H8O, mba)]�, 937 [1083�146 (C6H10O4, methylpen-tose)]�, 545 [937�162 (C6H10O5, deoxyhexose)�146(C6H10O4,methylpentose)�84 (C5H8O, mba]�, 417 [545+18 (lactone hydroly-sis)�146 (C6H10O4, methylpentose)]�, 271 [417�146 (C6H10O4,methylpentose); jalapinolic acid�H]�; HRFABMS m/z 1297.6576[M�H]� (calcd for C65H101O26 1297.6581).

4.4.2. Murucoidin XVIII (2)White powder; mp 123–126 �C; [a]D �88 (c 0.2, MeOH); 1H and

13C NMR, see Tables 1 and 2; negative-ion FABMS m/z 1193[M�H]� (C59H101O24), 1067 [1193�126 (C8H14O, octa)]�, 963[1193�84 (C5H8O, mba)�146(C6H10O4, methylpentose)]�, 837[963�126 (C8H14O, octa)]�, 545 [837�146(C6H10O4, methylpen-tose)�146(C6H10O4, methylpentose)]�, 417 [545+18 (lactonehydrolysis)�146 (C6H10O4, methylpentose)]�, 271 [417�146(C6H10O4, methylpentose); jalapinolic acid�H]� ; HRFABMS m/z1193.6678 [M�H]� (calcd for C59H101O24 1193.6683).

4.4.3. Murucoidin XIX (3)White powder; mp 150–153 �C; [a]D �50 (c 0.12, MeOH); 1H

and 13C NMR, see Tables 1 and 2; negative FABMS m/z 1109[M�H]� (C53H89O24), 1067 [1109�43 (C2H3O, ac)]�, 921[1067�146 (C6H10O4, methylpentose)]�, 837 [921�84 (C5H8O,mba)]�, 545 [837�146 (C6H10O4, methylpentose)�146 (C6H10O4,methylpentose)]�, 417 [545+18 (lactone hydrolysis)�146(C6H10O4, methylpentose)]�, 271 [417�146 (C6H10O4, methylpen-tose); jalapinolic acid�H]� ; HRFABMS m/z 1109.5738 [M�H]�

(calcd for C53H89O24 1109.5744).

4.5. Biological assays

4.5.1. Bacterial strains and mediaS. aureus EMRSA-15 containing the mecA gene was provided by

Dr. Paul Stapleton, from the UCL School of Pharmacy. Strain XU-212, a methicillin-resistant strain possessing the TetK tetracyclineefflux protein, was provided by Dr. E. Udo (Gibbons and Udo, 2000).SA-1199B, which over-expresses the NorA MDR efflux protein(Chérigo et al., 2009) was the generous gift of Professor GlennKaatz of Wayne State University. Standard strain S. aureus ATCC25923 was obtained from the ATCC. All strains were cultured onnutrient agar (Oxoid, Basingstoke, UK) before determination ofMIC values. Cation-adjusted Mueller–Hinton broth (MHB; Oxoid)containing 20 and 10 mg/L of Ca2+ and Mg2+, respectively was usedfor susceptibility tests. E. coli Rosetta-gami was provided by Dr.Federico del Río Portilla, Departamento de Bioquímica, Institutode Química, UNAM, and was cultured on Luria–Bertani nutrientagar (Sigma) before determination of MIC values. Luria–Bertanibroth (LB) was used for susceptibility tests. Nosocomial strains ofS. flexneri and S. enterica sv. Typhi were provided by Dr. FernandoCalzada Bermejo, Unidad de Investigación Médica en Farmacologíay Productos Naturales, Centro Médico Nacional Siglo XXI, InstitutoMexicano del Seguro Social, and were cultured on nutrient agar

(Mueller–Hinton; Sigma) before determination of MIC values.Mueller–Hinton (MHB) broth was used for susceptibility tests.

4.5.2. CompoundsTetracycline (purity > 98%), kanamycin (purity > 99%), chloram-

phenicol (purity > 98%), and reserpine (purity > 98%) were ob-tained from Sigma. Glycolipids 1–3 were purified as previouslydescribed by preparative recycling HPLC using a Waters 600 E mul-ti-solvent delivery system equipped with a Waters 410 refractiveindex detector (Waters) as described above, and their purity wasassessed by HPLC, NMR, and FAB-MS as >99%.

4.5.3. Susceptibility testingMIC values were determined at least in duplicate by standard

microdilution procedures. An inoculum density of 1.5 � 108 CFUof each of the test strains was prepared in 0.9% saline by compar-ison with a McFarland standard. MHB or LB (125 lL) was dispensedinto 10 wells of a 96-well microtiter plate (Nunc, 0.3 mL volumeper well). All test compounds were dissolved in DMSO before dilu-tion into MHB or LB for use in MIC determinations. The highestconcentration of DMSO remaining after dilution (3.125% v/v)caused no inhibition of bacterial growth. Then, compounds 1–3or appropriate antibiotic (125 lL) were individually dispensed intowell 1 and serially diluted across the plate (512–1 lg/mL), leavingwell 11 empty for the growth control. The final volume was dis-pensed into well 12, which being free of inoculum served as thesterile control. The inoculum (125 lL) was added into wells 1–11and the plate was incubated at 37 �C for 24 h. The MIC was definedas the lowest concentration, which yielded no visible growth. Tet-racycline, kanamycin, and chloramphenicol were also tested as po-sitive drug controls. A methanolic solution (5 mg/mL) of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT;Sigma) was used to detect bacterial growth by a color change fromyellow to dark blue. For the modulation assay, all glycolipids weretested at a final concentration of 25 lg/mL. Two-fold serial dilu-tions of antibiotics ranging from 512 to 1 lg/mL were added, andthe microtiter plates were then interpreted, after inoculum addi-tion and incubation, in the same manner as for MIC determina-tions. The activity of reserpine at a concentration of 20 lg/mLwas also tested as an efflux pump inhibitor (positive control). Allsamples were tested in duplicate (Corona-Castañeda and Pereda-Miranda, 2012).

4.5.4. Cytotoxicity assayNasopharyngeal (KB), cervix (HeLa), and laryngeal carcinoma

(Hep-2) cell lines were maintained in RMPI 1640 (10�) mediumsupplemented with 10% fetal bovine serum. Cell lines were cul-tured at 37 �C in an atmosphere of 5% CO2 in air (100% humidity).The cells at log phase of their growth cycle were treated in tripli-cate with various concentrations of the test samples (0.16–20 lg/mL) and incubated for 72 h at 37 �C in a humidified atmosphereof 5% CO2. The cell concentration was determined by the NCI sulfo-rhodamine method. Results were expressed as the dose that inhib-its 50% control growth after the incubation period (IC50). Thevalues were estimated from a semi log plot of the drug concentra-tion (lg/mL) against the percentage of viable cells. Vinblastine andcolchicine were included as positive drug controls (Vichai and Kir-tikara, 2006; Figueroa-González et al., 2012).

Acknowledgments

This research was supported by Dirección General de Asuntosdel Personal Académico, UNAM (IN212813) and Consejo Nacionalde Ciencia y Tecnología (101380-Q). B.C.-C. and L.C. received grad-uate scholarships from CONACyT. Thanks are due to GeorginaDuarte, and Margarita Guzmán (‘‘Unidad de Servicios de Apoyo a

B. Corona-Castañeda et al. / Phytochemistry 95 (2013) 277–283 283

la Investigación’’, Facultad de Química, UNAM) for the recording ofmass spectra.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytochem.2013.07.007.

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