Acylsugar diversity in the resin glycosides from Ipomoea tricolor seeds as chemosensitizers in breast cancer cells

Phytochemistry Letters 32 (2019) 77–82

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Acylsugar diversity in the resin glycosides from Ipomoea tricolor seeds as chemosensitizers in breast cancer cells

T

Jhon Castañeda-Gómeza, Pedro Lavias-Hernándezb, Mabel Fragoso-Serranob, Argelia Lorencec, ⁎ Rogelio Pereda-Mirandab, a

Grupo Químico de Investigación y Desarrollo Ambiental, Programa de Licenciatura en Ciencias Naturales, Facultad de Educación, Universidad Surcolombiana, Neiva, Colombia b Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City, 04510, Mexico c Arkansas Biosciences Institute and Department of Chemistry and Physics, Arkansas State University, P.O. Box 639, State University, AR 72467, United States

ARTICLE INFO

ABSTRACT

Keywords: Resin glycoside Chemosensitizer HPLC-MS profiling Structural spectroscopy

Mexican morning glory, Ipomoea tricolor Cav., is native to America and widely cultivated as an ornamental plant. High performance liquid chromatography profiling coupled with off-line electrospray mass spectrometry detection was applied to identify novel acylsugar from the CHCl3-soluble resin glycoside from seeds. Dereplication of known tricolorins A-J included the recording of mass values for ions [M + Na]+ and [M – H]−, in addition to comparison of retention times. Recycling HPLC was used for the purification of novel tricolorins K-M. NMR analysis revealed acylation at the C-2 and C-4 positions on the third pyranose unit of the tetrasaccharides by short chain aliphatic acyl groups. Acylsugar diversity results from variations in the esterification of sugar cores. Tricolorin A at a concentration of 25 μg/ml exerted a strong potentiation of vinblastine susceptibility in multidrug-resistant human breast carcinoma cells with a reversal factor of 2164-fold.

1. Introduction Ipomoea tricolor Cav. (Convolvulaceae) is a perennial morning glory native to the tropical New World, widely cultivated and naturalized around the world as an ornamental plant because its beautiful funnel shaped flowers (Fig. S1). The seeds are used as a hallucinogen, since they contain ergoline-type alkaloids produced by a symbiotic Periglandula fungi (Steiner and Leistner, 2018), and have been used for centuries by many native Mexican societies as an entheogen (Schultes et al., 2000). In Mexico, farmers use this species as a cover crop during the fallow period in sugar cane fields. Bioactivity-guided fractionation of the active CHCl3-soluble extract from the aerial parts led to the isolation of the allelopathic resin glycoside mixture (Pereda-Miranda et al., 1993). The resolution of this complex mixture was carried out successfully through the application of HPLC using an amino propyl silica based stationary phase column for the analysis of carbohydrates. Separation used a mobile phase of water/acetonitrile with a UV detector (Bah and Pereda-Miranda, 1996). The major fraction that concentrated the biological activity allowed the isolation of five major components, tricolorins A-E (1-5), all with a macrocyclic tetrasaccharide core and 11S-hydroxyhexadecanoic (jalapinolic) acid as the aglycone (Bah and Pereda-Miranda, 1996). Tricolorin A (1), the major ⁎

acylsugar isolated from this active fraction, inhibited seed germination and radical growth (Pereda-Miranda et al., 1993), acting as a pre- and post-emergence plant growth inhibitor (Lotina-Hennsen et al., 2013) (Fig. S2). This principle was also found to be a potent inhibitor of seed respiration and uncoupler of photophosphorylation in spinach chloroplasts through inhibition of H+-uptake and adenosine 5′-triphosphate synthesis (Achnine et al., 1999). Tricolorin F and G were similarly identified as trisaccharides of jalapinolic acid (Fig. S3), which were also found forming ester type dimers, tricolorins H-J (Fig. S4) (Bah and Pereda-Miranda, 1997; Pereda-Miranda et al., 1993). The Convolvulaceae family belongs to the order Solanales and is taxonomically related to Solanaceae (Eich, 2008). Both families are characterized by the presence of a high diversity of acylsugars or resin glycosides built on distinct sugar cores (Moghe et al., 2017; PeredaMiranda et al., 2010), which seems to play important roles in the natural pest resistance, providing protection against fungi, herbivores, bacteria, and mechanical damage, e.g., synergistic antimicrobial (Luu et al., 2017) or insect protective action (Leckie et al., 2016; Liu et al., 2017), as well as in plant-plant interactions of the producing species (Lotina-Hennsen et al., 2013). These mixtures are a source of inhibitory compounds of efflux pumps (EPs) which play an important role in extruding xenobiotics outside the cells providing a protective means

Corresponding author. E-mail address: [email protected] (R. Pereda-Miranda).

https://doi.org/10.1016/j.phytol.2019.05.004 Received 2 April 2019; Received in revised form 5 May 2019; Accepted 7 May 2019 Available online 16 May 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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responsible for the multidrug resistant (MDR) phenotype in Gram-positive (Pereda-Miranda et al., 2006) and -negative bacteria (CoronaCastañeda and Pereda-Miranda, 2012; Corona-Castañeda et al., 2016), as well as in mammalian cancer cells (Castañeda-Gómez et al., 2013; Figueroa-González et al., 2012). These EP inhibitors of plant origin could have therapeutically important benefits for the introduction of new alternatives for the treatment of refractory malignances (Silva et al., 2015; Spengler et al., 2017). Hence, in this context, the purpose of the present study was to associate the efficient resolution provided by high performance liquid chromatography combined with off line electrospray mass spectrometry to dereplicate and identify novel multidrugresistance glycolipid inhibitors in seeds of the Mexican morning glory I. tricolor. 2. Results and discussion The resin glycoside extract of heavenly blue morning glory seeds was analyzed by preparative reversed-phase HPLC with refractive index detection coupled off-line to electrospray mass spectrometry to dereplicate known tricolorins that were previously isolated from the aerial parts of this species (Pereda Miranda et al., 1991) and identify novel constituents. The application of peak-shaving and heart-cutting techniques in recycling preparative HPLC (Pereda-Miranda and HernándezCarlos, 2002) allowed the resolution of ten major constituents from the total crude extract (Fig. S5), which produced a strong modulation of vinblastine cytotoxicity in the MCF-7/Vin + cells with a reversal fold value > 255-fold (25 μg/mL). Each peak was collected and analyzed by ESIMS in both positive and negative modes (Table 1), as well as, independently evaluated for cytotoxicity and modulation of multidrugresistance. For dereplication of tricolorins A-E (1-5; Figs. 1 and 2) and F-G (9 and 10; Fig. S3), retention times, coelution experiments with pure samples, and m/z values for ions [M + Na]+ and/or [M – H]− were used (Castañeda-Gómez et al., 2017). The eluates with Rt values of 13.73 min (fraction F-I V) and 20.25 min (fraction F-V II) showed nonpreviously registered m/z values which indicated the presence of new resin glycosides. Positive HRESIMS of fraction F-V II (Fig. S6) showed a cationized molecule at m/z 1031.54736 [M + Na]+ corresponding to the molecular formula C49H84O21Na (calcd error: δ = +7.4 ppm) for tricolorin K (6), while tricolorin L (7) presented a ion [M + Na]+ peak at m/z 1017.53204 (C48H82O21Na, calcd error: δ = +7.8 ppm). The difference of 14 mass units between 6 and 7, suggested the presence of a methylbutanoyl residue for 6 and isobutanoyl residue for 7. These esterifying residues were confirmed by gas chromatography technique, once they were released as organic acids from the saponification of fraction F-V II. Both macrocyclic oligosaccharides afforded the same glycosidic acid by alkaline hydrolysis (LRESIMS m/z 871 [M – H]−), previously reported as tricoloric acid A (10) (Bah and Pereda-Miranda, 1996). In MS, the observation of the same deprotonated molecule ion peak for the known compound 2 and 6 allowed to recognize both compounds as diastereoisomers with the same linear tetrasaccharide core (Fig. S7).

Fig. 1. Chemical structures of tricolorins.

Resolution of fraction F-V II by recycling HPLC (Fig. S8) allowed the separation of tricolorins K and L (6 and 7), while fraction F-I V afforded tricolorin M (8). The 1H and 13C NMR spectra of the tricolorins K and L (6 and 7, Fig. S12 and S18) with those previously recorded for tricolorins A and B (1 and 2) were almost superimposable (Table 2 and 3; Fig. S9 and S11). For compound 6, four signals for anomeric protons were confirmed by HSQC: δH 4.66 (1H, d, J =7.8 Hz; δC 103.1, Fuc-1); 5.79 (1H, d; J =7.5 Hz; δC 99.9, Glc-1); 5.57 (1H, d; δC 98.4, Rha-1), and 5.52 (1H, d;

Table 1 Dereplication of tricolorins with HPLC coupled to off-line ESI-MS. Peaka

Rt (min)

Yield (mg)

MW

Formula

[M−H]−

[M + Na]+

Tricolorin

F-I F-II F-III F-IV F-V F-VI F-VII F-VIII F-IX F-X

8.45 10.57 12.23 13.73 15.71 18.30 20.25 21.98 24.69 31.03

7.04 4.47 3.20 10.91 5.93 13.23 18.64 8.44 39.57 95.01

708 708 808 1021 1038 1038 994 1008 1022 1008 1022

C34H60O15 C34H60O15 ND C50H86O22 C50H86O21 C50H86O22 C48H82O21C49H84O21 C50H86O21 C49H84O21 C50H86O21

707 707 807 1037 1021 1037 993 1007 1007 1007 1021

731 731 831 1061 1045 1061 1017 1031 1031 1031 1045

G F ND M E C LK D B A

a

Collected fraction; ND: not determined; unidentified. 78

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Table 2 1 H NMR Spectroscopic Data for Tricolorins K–M (6-8) in C5D5N. position

6

7

8

Fuc-1 2 3 4 5 6 Glc-1 2 3 4 5 6

4.66 d, 7.8 4.73 dd, 8.7, 7.8 4.27-4.22 4.04 d, 3.1 3.83 q, 6.3 1.59 d, 6.3 5.79 d, 7.5 4.14 dd, 9.0, 7.5 5.83 dd, 9.0, 9.0 4.35 dd, 9.0, 8.5 3.51 ddd, 8.5, 3.1, 2.4 4.15 dd, 11.8, 3.1 3.94 dd, 11.8, 2.4 5.57 d 5.81 m 4.79 m 5.71 dd, 9.8, 9.8 4.95 dq, 10.0, 6.3 1.65 d, 6.3 5.52 d 4.52 dd, 1.0, 3.0 4.42 dd, 8.0, 3.0 4.27-4.22 4.27-4.22 1.73 d, 6.5 3.00 ddd 16.4, 8.5, 2.0 2.47 ddd 16.4, 7.5, 1.5 3.83 m 0.85 t, 7.0 2.34 tq, 7.0, 7.0 1.18 d, 7.0 0.94 t, 7.0 2.41 sep, 7.0 1.12 d, 7.0 0.90 d, 7.0

4.67 d, 7.8 4.73 dd, 9.4, 7.8 4.27-4.22 4.02 d, 3.1 3.82 q, 6.2 1.57 d, 6.2 5.79 d, 7.8 4.12 dd, 9.0, 7.8 5.82 dd, 9.0, 9.0 4.35 dd, 9.0, 9.0 3.52 ddd, 9.0, 3.2, 2.4 4.15 dd, 11.8, 3.2 3.94 dd, 11.8, 2.4 5.57 d, 1.5 5.80 dd, 1.5, 3.5 4.76 dd, 3.5, 9.6 5.70 dd, 9.6, 9.6 4.92 dq, 9.6, 6.0 1.64 d, 6.0 5.50 d 4.49 dd, 1.0, 3.0 4.41 dd 4.27-4.22 4.27-4.22 1.71 d, 6.2 2.99 ddd, 16.4, 8.5, 2.0 2.46 m

4.68 d, 7.9 4.72 dd, 7.9, 7.9 4.35-4.32 4.06 d, 3.5 3.91 q, 6.5 1.56 d, 6.5 5.76 d, 7.4 4.15 dd, 9.0, 7.5 5.82 dd, 9.2, 9.2 4.35-4.32 m 3.49 ddd, 9.0, 3.2 4.14 m 3.95 dd, 11.9, 2.5 5.58 s 5.81 m 4.87 dd, 9.8, 3.3 5.78 dd, 9.8, 9.8 5.09-5.06 dq, 9.8, 6.0 1.82 d, 6.0 5.58 s 4.59 dd, 1.5, 3.3 4.43 dd, 3.3, 8.5 4.27-4.20 4.27-4.20 1.72 d, 6.2 2.97

3.82 m 0.84 t, 7.0 – – – 2.56-2.48 1.11 d, 7.0 1.13 d, 7.0 2.56-2.48 1.12 d, 7.0 1.15 d, 7.0

3.80 0.82 d, 7.0 2.43 tq, 7.0, 7.0 1.19 d, 6.7 0.94 t, 7.4

Rha-1 2 3 4 5 6 Rha'-1 2 3 4 5 6 Jal-2a 2b 11 16 Mba- 2 2-Me 3-Me Iba-2 3 3’ Iba-2 3 3’ nla-2 3 2-Me 3-Me

Fig. 2. Structure of tricolorin D.

δC 104.7, Rha′-1) (Fig. S15). The HSQC spectrum registered for compound 7 was also identical to 1 and 2 with four anomeric signals centered at δH 4.67 (1H, d, J =7.8 Hz; δC 103.1, Fuc-1); 5.79 (1H, d; J =7.8 Hz; δC 99.9, Glc-1); 5.57 (1H, d, J =1.5 Hz; δC 98.6, Rha-1), 5.50 (1H, d; δC 104.6, Rha′-1) (Fig. S20). HMBC spectra were employed to confirm the glycosylation sequence through long-range heteronuclear coupling correlations (3JCH); thus, the following connectivities were observed for 6 and 7: (a) H-1 of the fucose (6: δH 4.66; 7: 4.67) and C-11 of the fatty acid (δC 80.9); (b) H-2 of fucose (δH 4.73) with C-1 of glucose (δC 99.9); (c) H-2 of glucose (6: δH 4.14; 7: 4.12) with C-1 of rhamnose (6: δC 98.4; 7: 98.6); H-3 of rhamnose (6: δH 4.79; 7: 4.76) with C-1 of the second unit of rhamnose (6: δC 104.7; 7: 104.6) (Fig. S16). HMBC spectra were also used to establish the differences between both natural products 2 (tricolorin B) and 7 (Fig. S17), in relation to the positions of esterification through 3JCH connectivities (Pereda-Miranda et al., 2010); thus, for compound 6, the 3-methylbutanoyl group (δC 175.7) was located at C-4 of the inner rhamnose (δH 5.71); this carbonyl signal was assigned by virtue of its additional 2JCH correlation with the multiplet at 2.34 ppm, while the isobutanoyl residue (δC 175.8) was located at C-2 of this rhamnose through the observed connectivity with H-2 (δH 5.81). For 7, the isobutanoyl residues (δC 176.2, 176.4) were identified at C-2 (δH 5.80) and C-4 (δH 5.70) of the first rhamnose unit (Bah and Pereda-Miranda, 1996). Tricolorin M (8) presented a cationized molecule at m/z 1061.54810 [M+Na]+ corresponding to the molecular formula C50H86O22Na (calcd error: δ =–2.1 ppm) by HRESIMS technique (Fig. 21). The mass value for the deprotonated molecule at m/z 1037.55152 ([M – H]− (calcd error: δ =–2.2 ppm) of 8 was identical to that reported for the tricolorin C (3), allowing to identify this new resin glycoside as an isomer of tricolorin C (3). Four anomeric signals were observed in the 1H, 13C and HSQC techniques for Fuc-1 (δC 103.5, δH 4.68), Glc-1 (δC 100.3, δH 5.76), Rha-1 (δC 98.7, δH 5.58), and Rha'-1 (δC 105.1, δH 5.58) (Fig. S22 and S25), which additionally indicated the presence of tricoloric acid A as the oligosaccharide core (Bah and Pereda-Miranda, 1996). Saponification of this compound (8) liberated

2.45

2.69 dq, 7.1, 7.1 4.27-4.20 1.19 d, 7.1 1.28 d, 6.2

2-methylbutanoic and 3-hydroxy-2-methylbutyric acids, which were identified by GC-MS. The glycosylation sequence for the oligosaccharide core was determined by the long-range heteronuclear coupling correlations (3JCH) in the HMBC spectra and confirmed by the comparison between compounds 1 and 3. Therefore, the following connectivities were observed: H-1 of fucose (δH 4.6) with C-11 of the fatty acid (δC 80.9); H-2 of fucose (δH 4.72) with C-1 of glucose (δC 100.3); H-2 of glucose (δH 4.15) with C-1 of rhamnose (δC 98.7); H-3 of rhamnose (δH 4.87) with C-1 of the second unit of rhamnose (δC 105.1) (Fig. S26). The structural differences that allowed determining the isomerism between 3 and 8 were located by the correlations of the carbonyl signals for the esterifying residues with the oligosaccharide proton resonances. Thus, the carbonyl groups of 3-hydroxy-2-methylbutanoyl (δC 174.9) and 3-methylbutanoyl (δC 176.2) residues correlated with H-2 of inner rhamnose (δH 5.81) and H-4 (δH 5.78), respectively. Numerous species of morning glories, like the Mexican morning glory, accumulate acylsugars with the presence of a tetrasaccharide core with a substantial diversity in the number and length of the acyl chain aliphatic acids, similar to those previously characterized resin glycosides from the moon vine, I. alba, (Castañeda-Gómez et al., 2017). The number of acylating chains on the sugar cores normally ranged from one to four with chain lengths from 2 to 12 carbons (PeredaMiranda et al., 2010). Across the Convolvulaceae, numerous species 79

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glycolipid) was very similar to the activity displayed by acylsugars isolated from other morning glories (Figure S28): murucoidin V (RFMCF7/Vin+ > 255-fold) from I. murucoides (Figueroa-González et al., 2012), jalapinosides I and II (Bautista et al., 2015, 2016), in addition to purgin II, all from I. purga (Castañeda-Gómez et al., 2013), and albinoside III from I. alba (Cruz-Morales et al., 2016), all with RFMCF-7/Vin+ > 2000fold. In terms of the relationship between the chemical structure and the observed modulatory activity, none could be deduced. Neither the size of the lactone ring nor the length of the oligosaccharide was crucial for activity. Therefore, it has been suggested that the only common property among resin glycosides, as EP substrates, is their relative amphiphilic nature. These active glycolipids have shown some of the ideal structural features recognized for efflux pumps inhibitors or substrates, various hydrophobic units, which are represented by the glycosidic acid aglycone, in addition to the esterifying residues, sometimes containing aromatic rings, and various H-bond acceptors and Hbond donor centers at the oligosaccharide core (Figure S28). Therefore, both covalent and non-covalent interactions will modulate the threedimensional EP protein structure, resulting in conformational changes, which are associated with a loss or reduction in the effluxing activity.

Table 3 13 C NMR Spectroscopic Data for Tricolorins K-L (6-8) in C5D5N. position

6

7

8

Fuc-1 2 3 4 5 6 Glc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha'-1 2 3 4 5 6 Jal-1 2 11 16 Mba- 1 2 2-Me 3-Me Iba-1 2 3 3’ Iba-1 2 3 3’ nla-1 2 3 2-Me 3-Me

103.1 74.7 76.4 73.2 71.4 17.4 99.9 80.1 79.0 70.6 76.3 61.3 98.4 72.8 76.0 73.4 67.3 18.4 104.7 72.4 72.6 73.5 69.6 18.6 172.4 34.5 80.9 14.3 175.7 41.7 17.3 11.9 175.8 34.8 18.6 18.4

103.1 74.6 76.3 73.2 71.3 17.4 99.9 80.9 79.1 70.6 76.4 61.3 98.6 72.8 76.2 73.5 67.4 18.4 104.6 72.4 72.6 73.4 69.6 19.0 172.4 34.4 80.9 14.3

103.5 75.2 76.5 73.8 71.6 17.4 100.3 80.6 79.4 69.9 76.6 61.8 98.7 72.9 76.3 73.6 67.8 18.7 105.1 72.78 72.6 73.9 69.6 18.90 172.7 34.7 80.9 14.6 176.2 41.9 17.3 12.2

176.2 34.4 19.1 18.5 176.4 34.5 19. 3 18.5

3. Conclusions The results of the present investigation extended the knowledge of the hyper-diverse acylsugar phenotype within the resin glycosides of the Convolvulaceae, meaning there is large diversity within and between closely related species. This acylsugar diversity is the results of variation in the esterification of the sugar cores, most of which are unique to an individual given species as demonstrated by the complex mixtures of resin glycosides in the Mexican morning glory. Although the purpose of this structural diversity is not clear, it has been postulated that this natural variability may generate a synergistic antimicrobial or insect protective action that may provide selective adaptive defense advantages against abiotic and biotic stress, which could be explore for the discovery and development of novel resin glycosides as potent lead modulators of efflux-pumps in cancer cells. This potential of the acylsugars as modulators of EPs in cancer cells could be used to identify effective therapeutic drug combination and lower their current doses, thereby decreasing toxic side effects of chemotherapeutic agents in refractory malignancies.

174.9 50.20 70.9 14.4 21.4

4. Experimental

incorporate at least one oligosaccharide chain and one common aliphatic acid as an esterifying residue in all their acylsugars: for example, methylbutyric acid in the Mexican morning glory, tiglic acid in the moon vine, I. alba (Castañeda-Gómez et al., 2017; Cruz-Morales et al., 2012), and long-chain fatty acid were found in multiple species, like decanoic and dodecanoic acids in the beach morning glory, I. pes-caprae (Pereda-Miranda et al., 2005) and sweet potato, I. batatas, (RosasRamírez et al., 2011; Rosas-Ramírez and Pereda-Miranda, 2013), inter alia. This structural diversity could be the result of promiscuous enzymes during the biosynthetic pathways of these bioactive secondary metabolites (Moghe et al., 2017). All tricolorins 1-11 were tested to determine their cytotoxic potential in various cancer cells using the sulforhodamine B method (Vichai and Kirtikara, 2006) (Table S1). The absence of cytotoxic activity in all tested compounds was an important criterion to carry out further trials of modulation with MDR human breast carcinoma (MCF7/Vin) cells and differentiate clearly any effect of potentiation (by inhibition of EPs) of a possible synergy between an active tested compound and the selected drug to be extruded, in this case vinblastine (Figueroa-González et al., 2012) (Table S2, Fig. S27). The effect of modulation of vinblastine cytotoxicity in the MDR MCF-7/Vin + cells by tricolorin A was 2164-fold at 25 μg/mL; this reversal fold value (RFMCF-7/Vin+ = IC50 vinblastine/IC50 vinblastine in the presence of

4.1. General experimental procedures A Fisher-Johns apparatus was employed for the determination of melting points and a Perkin-Elmer model 341 polarimeter was used for the optical rotations. 1H (500 MHz) and 13C (125.7 MHz) NMR analysis were performed on Varian Inova instrument. The dereplication technique was carried out according to a previously described methodology (Castañeda-Gómez et al., 2017). Therefore, a Waters HPLC equipment (Millipore Corp., Waters Chromatography Division, Milford, MA) composed of a 600E multisolvent delivery system equipped with a refractive index detector was used to collect each eluate on preparative scale. Control of the equipment, data acquisition, processing, and management of the chromatographic information were performed on Empower 2 software (Waters). Positive and negative-ion low resolution ESIMS data were recorded using a Waters Acquity UHPLC-H class system (Waters Co., Milford, MA, USA) equipped with an electrospray ionization mass spectrometer Waters SQD2 system. Mass spectra were acquired over the range 150–2000 m/z. The nebulizer and drying gas was nitrogen and set at 25 bar and capillary voltage was 3.7 kV. For UPLC segment, a BEH C18 (2.1 × 100 mm, 1.7 μm) (Waters Corp., Milford, MA, USA) column was used as stationary phase, CH3CN– H2O (acidified with 0.1% formic acid), starting with 15:85 then increasing linearly to 100% CH3CN within 8 min, holding for 1.5 min, and then 80

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returning to the starting conditions within 0.5 min. The samples were dissolved in 150 μl of HPLC grade MeOH (2.0 mg/ml). Positive-ion high resolution ESIMS analysis, was registered with a Thermo LTQ Orbitrap XL hybrid FT mass spectrometer (ThermoFisher, San Jose, CA, USA) where each sample (1 mg/ml; MeOH-H2O 9.5:0.5, 0.1% formic acid) was directly infused into electrospray ionization source, under the following chromatographic parameters: spray voltage 6.0 kV; capillary temperature at 350 °C; capillary voltage 35 V. HRMS of compound 8 was recorded using a quadrupole time-of-flight mass spectrometer (MS Q-TOF G6530, Agilent Technologies) equipped with an electrospray ionization ion source. The finalized operating ESI source conditions were as follows, using a m/z 50–3200 mass range: capillary voltage set at 3500 V; skimmer voltage, 65 V; fragmentor voltage, 175 V; desolvation gas (325 ; 8 l/min) and nebulizing gas (nitrogen, 50 psi).

4.4.1. Tricolorin K (6) White solid; mp 110–113 °C; [α]589 –26.3, [α]578 –28.8, [α]546 –32.5, [α]436 –51.3, [α]365 –73.8 (c 0.8, MeOH); 1H and 13C NMR see Table 2 and 3; HRESIMS m/z 1031.54736 [M + Na]+; (calcd for C49H84O21Na requires 1031.539731, calcd error: δ = +7.4 ppm). 4.4.2. Tricolorin L (7) White solid; mp 113–116 °C; [α]589 –22.2, [α]578 –27.8, [α]546 –28.9, [α]436 –41.1, [α]365 –60.0 (c 0.9, MeOH); 1H and 13C NMR see Table 2 and 3; HRESIMS m/z 1017.53204 [M + Na]+; (calcd for C48H82O21Na requires 1017.524081, calcd error: δ = +7.8 ppm). 4.4.3. Tricolorin M (8) White solid; mp 116–118 °C; [α]589 – 10.0, [α]578 –10.0, [α]546 –10.0, [α]436 –30.0, [α]365 –10.0 (c 1.0, MeOH); 1H and 13C NMR see Table 2 and 3 positive HRESIMS m/z 1061.54810 [M + Na]+; (calcd for C50H86O22Na requires 1061.55029, calcd error: δ =–2.1 ppm).

4.2. Chemicals, cell lines and cell cultures Sulforhodamine B, reserpine and vinblastine were purchased of Sigma-Aldrich (St. Louis, MO, USA) and RPMI 1640 medium and fetal bovine serum from Gibco. All drug-sensitive cell lines were acquired from the American Type Culture Collection: MCF-7 (ATCC HTB-22), CaOV3 (ATCC HTB-75), HeLa (ATCC CCL-2), and HCT15 (ATCC CCL225). The resistant counterpart MCF-7/Vin was developed and obtained as previously reported (Figueroa-González et al., 2012). All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 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 vinblastine. At the same time, a stock of MCF-7/Vin− cells was maintained in vinblastine-free medium.

4.5. Determination of configuration of 3-hydroxy-2-methylbutyrate 4-Bromophenacyl (2R,3R)-3-hydroxy-2-methylbutyrate was prepared and identified according to a previously reported procedure: mp 56–59 °C; [α]D –6.0 (c 1.0 CHCl3); GC–MS: m/z 118 (2.0), 115 (10), 101 (20), 84 (12), 73 (70), 60 (100). This transesterification procedure has been used to confirm the absolute configuration for 3-hydroxy-2-methylbutyrate (nla) (Pereda-Miranda and Hernández-Carlos, 2002). 4.6. Basic hydrolysis and identification of glycosidic acid Solutions of tricolorin A (15 mg), fraction F-VII (1.5 mg) and fraction F-IV (1.5 mg) in 5% KOH-H2O (10 ml, 2.0 ml, 2.0 ml, respectively) were refluxed at 90 °C for 2 h. The mixtures were neutralized with HCl and extracted with chloroform. The organic layers were washed with water, dried over anhydrous Na2SO4, evaporated under reduced pressure and analyzed by GC–MS: Isobutyric acid (Rt 1.1 min) m/z [M]+ 88 (20), 73 (28), 70 (55), 61 (100) was identified of fraction VII, while 3hydroxy-2-methylbutyric acid (Rt 7.95 min) was detected of fraction IV: m/z [M]+ 118 (2.0), 115 (10), 101 (20), 84 (12), 73 (70), 60 (100). 2methylbutanoic acid (Rt 2.0 min) m/z [M]+ 102 (8.0), 87 (2), 74 (12), 57 (100), was registered in all solutions. The aqueous layers were extracted with n-BuOH and concentrated to give colorless solids that were evaporated to dryness. Tricolorin A afforded the tricoloric acid A, that was previously isolated and reported (Pereda-Miranda et al., 1993): ESIMS m/z 871 [M−H]−; m.p. 104–106 °C; [α]D –61.92 (c 0.86, MeOH). Comparison of the physical constants and the mass spectrometric value of the saponification products of fractions F-I V and F-V II, permitted to dereplicate the tricoloric acid A as the oligosaccharide core of the three new tricolorins K-M. All NMR data (Fig. S9) were identical with those value previously reported (Pereda-Miranda et al., 1993).

4.3. Plant material Seeds of Heavenly blue morning glory (item # 01049-PK-P1) were acquired from Park Seed Company (3507 Cokesbury Road, Hodges, South Carolina, 29653, EE. UU.) in January 2010. 4.4. Extraction and isolation The powdered seeds (167.9 g) were macerated at room temperature with hexane (2.5 l). Then, the resulting material was treated with CHCl3 and analyzed by TLC (silica gel 60 F254 aluminum sheets) and HPLC (Symmetry C18 column, Waters; 5 μm, 4.6 × 250 mm; CH3CN–H2O (9:1), flow rate of 0.4 ml/min) with a reference solution of tricolorin A (Rf 0.60), which permitted to identify the lipophilic resin glycoside mixtures. Ten eluates were collected by preparative HPLC using the peak-shaving and heart cutting techniques (Symmetry C18 column Waters; 7 μm, 19 × 300 mm, flow rate of 8.0 ml/min, sample injection of 500 μl, and concentration of 0.1 mg/μl) and registered by electrospray mass spectrometry. High-resolution mass values for ions [M + Na]+ and/or [M – H]− were detected for peaks as follows: F-I (Rt 8.45 min; m/z 707 [M−H] –); F-I I (Rt 10.57 min; m/z 707 [M−H] –); F–III (Rt 12.23 min; m/z 807 [M−H] –, m/z 831 [M + Na]+); F-I V (Rt 13.73 min; m/z 1061 [M + Na]+); F-V (Rt 15.71 min; m/z 1061 [M + Na]+); F-VI (Rt 18.30 min; m/z 1037 [M – H] –); F-VII (Rt 20.25 min; m/z 993 [M – H]– and 1017 [M + Na]+; m/z 1007 [M – H]– and 1031 [M + Na]+); F-VIII (Rt 21.98 min; m/z 1007 [M – H]–); m/z 1031 [M + Na]+); F-IX (Rt 24.69 min; m/z 1007 [M – H]–); m/z 1031 [M + Na]+) and F-X (Rt 31.03 min; m/z 1021 [M – H]–); m/z 1043 [M + Na]+). Comparison of retention times and mass values by UPLCESIMS was the key to dereplicate the known tricolorins G (10), F (9), E (5), C (3), D (4), B (2) and A (1) from peaks F-I, F-I I, F-V, F-V I, F-V III, F-I X and F-X, respectively. Fraction F-VII was purified by recycling HPLC, affording the new resin glycosides 6 (m/z 1031 [M + Na]+) and 7 (m/z 1017 [M + Na]+), while 8 (m/z 1061 [M + Na]+) was obtained from peak F-I V.

4.7. Cytotoxicity and modulation of multidrug-resistance assays Sulforhodamine B (SRB) assay was employed to determine the cytotoxicity and reversal fold of tricolorins 1-11. The cells were harvested at log phase of their growth cycle and were treated in triplicate with various concentrations of the test samples (0.2–25 μg/ml) and incubated for 72 h at 37 °C in a humidified atmosphere of 5% CO2. Results are expressed as the concentration that 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 Vinblastine was included as a positive control drug. 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 0.000128 to 2 μg/ml in the presence or absence of glycolipids at 25 μg/ ml for 72 h as previously described. The ability of glycolipids to 81

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potentiate vinblastine cytotoxicity was measured by calculating the IC50 as described above. In the modulation experiments, reserpine (5 μg/ml) was used as a positive control drug. The reversal fold (RF) 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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Acknowledgements We acknowledge the financial support by Dirección General de Asuntos del Personal Académico, UNAM (IN215016; IN208019) and Consejo Nacional de Ciencia y Tecnología (CB220535). P.L.-H. and J.C.G. are grateful to CONACYT for graduate and postdoctoral scholarships, respectively. A.L. thanks funds provided to her laboratory by the Arkansas Biosciences Institute to support this research. This contribution was prepared during a sabbatical stay of R.P.-M. as a Visiting Research Scientist at Faculdade de Farmácia, Universidade Federal de Rio de Janeiro with partial financial support from DGAPA. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.05.004. References Achnine, L., Pereda-Miranda, R., Iglesias-Prieto, R., Moreno-Sánchez, R., Lotina-Hennsen, B., 1999. Tricolorin A, a potent natural uncoupler and inhibitor of photosystem II acceptor side of spinach chloroplasts. Physiol. Plant. 106, 246–252. Bah, M., Pereda-Miranda, R., 1996. Detailed FAB-Mass spectrometry and high-resolution NMR investigations of tricolorins A-E, individual oligosaccharides from the resins of Ipomoea tricolor (Convolvulaceae). Tetrahedron 52, 13063–13080. Bah, M., Pereda-Miranda, R., 1997. Isolation and structural characterization of new glycolipid ester type dimers from the resins of Ipomoea tricolor (Convolvulaceae). Tetrahedron 53, 9007–9022. Bautista, E., Fragoso-Serrano, M., Pereda-Miranda, R., 2015. Jalapinoside, a macrocyclic bidesmoside from the resin glycosides of Ipomoea purga, as a modulator of multidrug resistance in human cancer cells. J. Nat. Prod. 78, 168–172. Bautista, E., Fragoso-Serrano, M., Pereda-Miranda, R., 2016. Jalapinoside II, a bisdesmoside resin glycoside, and related glycosidic acids from the officinal jalap root (Ipomoea purga). Phytochem. Lett. 17, 85–93. Castañeda-Gómez, J., Figueroa-González, G., Jacobo, N., Pereda-Miranda, R., 2013. Purgin II, a resin glycoside ester-type dimer and inhibitor of multidrug efflux pumps from Ipomoea purga. J. Nat. Prod. 76, 64–71. Castañeda-Gómez, J., Rosas-Ramírez, D., Cruz-Morales, S., Fragoso-Serrano, M., PeredaMiranda, R., 2017. HPLC-MS Profiling of the multidrug-resistance modifying resin glycoside content of Ipomoea alba seeds. Rev. Bras. Farmacogn. 27, 434–439. Corona-Castañeda, B., Pereda-Miranda, R., 2012. Morning glory resin glycosides as modulators of antibiotic activity in multidrug-resistant Gram-negative bacteria. Planta Med. 78, 128–131. Corona-Castañeda, B., Rosas-Ramirez, D., Castañeda-Gómez, J., Aparicio-Cuevas, M.A., Fragoso-Serrano, M., Figueroa-Gonzales, G., Pereda-Miranda, R., 2016. Resin glycosides from Ipomoea wolcottiana as modulators of the multidrug resistance phenotype

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