pore-forming activity of morning glory resin glycosides in model ...

Pore-Forming Activity of Morning Glory Resin Glycosides in Model Membranes Rogelio Pereda-Mirandaa,*, Ricardo Villatoro-Veraa†, Moustapha Bahb and Argelia Lorencec,* (Received May 2009; Accepted August 2009) This paper is dedicated to Professor Doctor Rachel Mata for her 60th birthday

Abstract Purgative convolvulaceous resin glycosides are a family of amphipatic polysaccharides exhibiting potent, broad-spectrum of bactericidal and cytotoxic activities. In an attempt to unravel the mechanism of action of this class of biodynamic compounds, we studied the interaction of selected members from the tricolorin series, major constituents of Ipomoea tricolor, with Spodoptera frugiperda Sf9 insect cells. A membrane potential-sensitive fluorescent dye was used to monitor the time course and dosis-response effects of the test compounds on the permeability of the plasma membrane. All evaluated tricolorins with intact macrolactone-type structure and OH groups showed the ability to increase the membrane permeability to both cations (K+ and Na+) and anions (Cl−) at a dose dependent fashion without any measurable delay, suggesting the formation of pores by these amphipatic molecules. These resin glycosides are active components of the biochemical arsenal employed by the morning glory species to kill target cells by permeabilizing their membranes to provoke an imbalance in cellular homeostasis. Key words: Resin glycosides, poreforming activity, tricolorin, Ipomoea tricolor, Convoculaceae. Resumen Las resinas glicosídicas de las convolvuláceas forman una familia de polisacáridos con una compleja estructura de naturaleza anfipática y con una amplio espectro de actividades bactericidas y citotóxicas de posible aplicación terapéutica. Con el objetivo de establecer el posible mecanismo de acción de esta clase de compuestos biodinámicos, se realizó un estudio para evaluar la interacción de algunos miembros selectos de la serie de las tricolorinas, constituyentes mayoritarios de las resinas de Ipomoea tricolor, sobre la membrana celular de la línea Sf9 del insecto Spodoptera Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, DF 04510, México. b Departamento de Química y Farmacología de Productos Naturales, Facultad de Química, Universidad Autónoma de Querétaro, Querétaro 76010, México. c Arkansas Biosciences Institute and Department of Chemistry and Physics, Arkansas State University, P.O. Box 639, State University, AR 72467, USA. *Corresponding authors. UNAM:pereda @servidor.unam.mx; ASU: Tel: +870 680 4322, Email: alorence@ astate.edu. †In memoriam a

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Rev. Latinoamer. Quím. 37/2 (2009)

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frugiperda. Se utilizó un colorante fluorescente sensible al potencial de membrana para moniterear el efecto dosis-respuesta y el tiempo de reacción de estos compuestos sobre la permeabilidad de la membrana plasmática. Todos los compuestos evaluados con una estructura macrocíclica intacta y con grupos OH libres provocaron un incremento en la permeabilidad membranal tanto para cationes (K+ and Na+) como aniones (Cl−) en una forma dependiente de la dosis y con una respuesta inmediata, lo que sugiere la formación de poros causada por estas moléculas de naturaleza anfipática. Esta clase de resinas glicosídicas constituyen componentes activos que forman parte del arsenal bioquímico empleado por los miembros de la familia de las convolvuláceas para combatir células blanco mediante cambios en la permeabilidad membranal que conducen a una alteración de la homeostasis celular.

Introduction The most conspicuous anatomical feature of species belonging to any genus of the morning-glory family (Convolvulaceae) is the occurrence of secretory cells with resinous content in foliar tissues, roots, and rhizomes. These species have the ability to synthesize an array of amphipatic glycolipids, the so-called resin glycosides, from such simple building blocks as sugars and fatty acids. These resins glycosyl derivatives of monohydroxy and dihydroxy C−14 and C−16 fatty acids and represent an important chemotaxonomic marker for this plant family and are responsible for several biological activities of importance in agriculture and medicine. These species have been worldwide recognized over the centuries as purgative agents (Pereda-Miranda and Bah, 2003). Jalapinolic acid, 11(S)-hydroxyhexadecanoic acid, is the predominant aglycon (lipophilic portion) which is always tied back to form a macrolactone ring spanning two or more units of their saccharide backbones. The chemical diversity of these oligosaccharides is further increased by the diverging possibilities of cyclization of the glycosidic acid cores into corresponding macrolactones. In addition, the multiple variations caused by acylation considerably increase their structural variety. In fact, on the whole a large number of congeners occur in the Convolvulaceae family as well as a remark-

able number in each species. Sugar units found in these metabolites are epimers of hexoses (D-glucose) and pentoses (L-rhamnose, D-fucose, and D-quinovose). Fatty acids with different chain lengths (e.g. tiglic, isobutyric, methylbutyric, decanoic, and dodecanoic) are ester-linked to the sugar cores (Pereda-Miranda and Bah, 2003). As a prototype example one may quote the tricolorin series from Ipomoea tricolor Cav. (Bah and Pereda-Miranda, 1996; 1997), a plant used in Mexican folk agriculture. Tricolorin A, the major allelochemical principle isolated from this cover crop, has been proven to be a strong inhibitor of seed germination and radical and seedling growth (Pereda-Miranda et al. 1993), as well as a potent uncoupler of photosystem II (Achnine et al., 1999). Even more interesting are recent reports on the significant cytotoxic activity of tricolorin A and congeners against human cancer cell lines (Pereda-Miranda and Bah, 2003; Chérigo et al., 2008), Mycobacterium tuberculosis (Rivero-Cruz et al., 2005), and Staphyloccocus aureus (Pereda-Miranda et al., 2006; Chérigo et al., 2008). It has been hypothesized that some of these biological activities might be due to the formation of pores induced by these amphipatic molecules (Rencurosi et al., 2004). These resin glycosides have a peculiar organization in aqueous solution in the form of micelles or large molecular aggregates similar to those formed in the crystalline

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state by tricolorin A (1) molecules, where a repartitioning of the hydrophobic and hydrophilic surfaces was observed. This packing allowed for the co-crystallization of 18 water molecules to form a dense network that creates a dividing layer between the hydrophilic faces in the unit cell. The hydrophobic surface exposed externally, in an enlongated fashion along the axis of the water channel, could interact with the lipids of a biological membrane upon insertion of tricolorin A into cell membrane. This speculative model for a transmembrane water channel could be responsible for a possible ion flux perturbation of the target cell membrane induced by non-selective pores (Rencurosi et al., 2004). Although the biological properties of resin glycosides have not yet been fully investigated, a closer look into this class of natural products seems highly promising in view of the existing data on the use of

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glycolipids as potential therapeutic agents, e.g. for treatment of severe immune disorders (Schepetkin and Quinn, 2006). The fact that many of them are isolated from plants which are essential ingredients of traditional remedies provides additional support for this notion. The wide range of antimicrobial activity displayed by these compounds is another example of synergy between related components occurring in the same medicinal crude drug extract, i.e., microbiologically inactive components disabling a resistance mechanism and potentiating the antibiotic properties of the active substances (Chérigo et al., 2008; Pereda-Miranda et al, 2006). In the present study a fluorescent cyanine dye was used to monitor the time course and dose-response effects of natural tricolorins and some derivatives on the permeability of the plasma membrane of Spodoptera frugiperda (Sf9) insect cells. The


structure-function relationship was studied by comparing the activity of the natural products and that observed for derivatives where the macrolactone structure or the amphipatic properties of the saccharide skeleton was disrupted. Material and Methods Chemicals and materials Unless otherwise stated all chemicals were purchased from Sigma Aldrich (St. Louis, MO). Natural individual glycolipds 1−3 from the CHCl3 extracts of I. tricolor were purified as previously described (Bah and Pereda-Miranda, 1996; 1997) by preparative recycling HPLC using a 600E multisolvent delivery system equipped with a 410 differential refractometer detector (Waters, Milford, MA). All solvents used were of HPLC grade. The elution was isocratic (CH3CN−H2O, 95:5; flow rate, 8 ml/min) on a µBondapak-aminopropyl column (19 × 150 mm; 10 µm; Waters) and a sample injection of 500 µl (100 mg/ml). Derivatives 4−6 were prepared according to previously described methodologies (Pereda-Miranda


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et al., 1993). Identity and purity of all tested compounds were confirmed by 1H and 13C NMR as well as by HPLC co-elution experiments with reference samples. Cell cultures Sf9 (ATCC CRL1711) cells were grown in SF900 II medium (Gibco BRL, Gaithersburg, MD) supplemented with 0.1% (v/v) Pluronic F-68 at 27oC in 250 ml Erlenmeyer flasks with a working volume of 30 ml and agitated at 100 rpm (Palomares et al., 2000). Cells were harvested during mid-logarithmic phase by centrifugation (14,000 rpm, 10 min at room temperature). Pellets were resuspended at 10-20 x 106 cells/ml with incubation buffer A (250 mM NaCl, 100 mM Tris-HCl, pH 6.8). Under these conditions, viability of cells was 90-95% as deduced from their ability to exclude tryphan blue. Cells were used within 3 h following harvesting. Fluorescence measurements Membrane potential was monitored with the fluorescent, positively charged, membrane potential-sensitive dye Dis-C 3-(5) [3,3’-dipropylthiodicarbocyanine, Molecular Probes, Eugene, OR; 1.5 µM final, 1 mM

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stock in DMSO] as described by Lorence et al. (1995). Fluorescence was recorded at the 620/670 nm excitation/emission wavelength pair using a Perkin Elmer LS50B Luminescence Spectrometer. Sf9 cells (2x106) were added to a cuvette containing 3 ml of various buffers. All determinations were made at 30oC with constant stirring. Time zero (t0) was considered to be when the Sf9 cells were added. Test compounds were supplemented (40-160 µM) after 6 minutes. Cation substitutions in the incubation buffer were made by replacing the N-methyl-D-glucamine chloride (MeGluCl) by the indicated salts (mol per mol), maintaining the osmolarity (~350 mOsm). Clwas substituted with methane sulfonate.


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Results and Discussion The purgative effect of resin glycosides is confined to the whole molecule (Pereda-Miranda et al. 2006), presumably bound to the intact complex mixture of glycoconjugates, since their glycosidic acids are inactive. In mammals, resin glycosides are known to induce peristalsis in the small intestine resulting in water elimination and numerous bowel movements within 1-2 h even after moderate dosages. It has been proposed that these compounds dissolve lecithin from the epithelial cells of the intestine resulting in its irritation (Eich, 2008) but this activity could be associated with the formation of pores by resin glycosides. To test this

Figure 1. A fluorescent membrane potential-sensitive dye was used to monitor the time course and doseresponse of the effect of tricolorins on the permeability of Sf9 in sect cell membranes. (A) Initially the dye entered the cells until it reached an equilibrium (B) After the equilibrium was reach, various doses of tricolorin A and other an alogues were tested. If these glycosidic resins had the ability to disrupthe intrinsic permeability of the membrane two scenarios were possible: 1) fluorescence increase due to exit of the dye to the incubation medium, or 2) fluorescence decrease due to entrance of the dye into the cells. By substituting the ionic composition of the incubation media we were able to establish the relative selectivity of the pores induced by tricolorins.

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Figure 2. Tricolorin A disrupts the permeability of Sf9 cell membranes at doses ≥ 80 mM. As shown results obtained in incubation buffers with identical osmolarity but differentionic composition were similar indicating that tricolorin induced pores do not discriminate between small (i.e. K+, Na+) and larger cations (i.e. methylglucarmine).

hypothesis, the effect of selected members from the tricolorin series on the membrane permeability of S. frugiperda insect cells was monitored with the fluorescent, positively charged, membrane potential-sensitive dye Dis-C3-(5) (Lorence et al., 1995). In these measurements, hyperpolarization causes dye internalization into the Sf9 cells and a fluorescence decrease, while depolarization causes the opposite effect (Figure 1). As illustrated in Figure 1 addition of 82 µM tricolorin A (1) to Sf9 cells caused an immediate drop of the fluorescence. This effect was dosis-dependant as experiments performed with lower amounts of resin (≤ 40 µM) were indistinguishable from controls (data not shown). This drop of fluorescence indicative of the formation of pores was also observed in Sf9 cell membranes treated with natural tricolorins E (2) and I (3) with an intact macrolactone-type structure (Figure 3). Tricolorins A and E showed a comparable pore forming activity, while the hyperpolarization induced by tricolorin I was higher. This larger pore-forming activity may reflect the fact that tricolorin I (3) is a dimer, facilitating its ability to form molecular ag-

gregates of higher complexity as has been suggested from the observed aggregation of tricolorin A molecules in solid state (Rencurosi., 2004). It is important to note that this in vitro activity was observed at µM concentration, doses at which physiologically relevant activities have been reported for these byodinamic oligosaccahides (Barnes et al., 2003; Cao et al., 2005; Pereda-Miranda, 1995; Pereda-Miranda and Bah, 2003). The key role of the macrolactone for the pore forming activity was evident from the data obtained with the glycosidic acid derivative 6. This saponification product of tricolorin A is highly polar and clearly incapable of membrane insertion at the highest dosis tested (160 µM). This inability to disrupt the membrane can be directly correlated with the lack of cytotoxicity previously found for all glycosidic acids derived from natural resin glycosides (Barnes et al., 2003; Pereda-Miranda and Bah, 2003). These highly polar glycosidic acids are likely incapable of forming conjugates due to the lack of alternating hydrophilic and hydrophophic surfaces needed for membrane insertion and pore formation. From these results, there seems



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Figure 3. The intact macrolactone and OH groups are requiered for the pore forming activity of tricolorins. All molecules were tested at 160 mM, as shown, peracetyl tricolorin A (5) and tricoloric acid (6) lost the ability to perturbate membrane permeability. Acetonide derivative (4) displayed a similar behavior as natural products 1 and 3. The most active compound in this assay was the dimeric structure 3.

to be a correlation between lipophilicity and pore-forming activity which resembles the antibacterial activity where the more lipophilic compounds displayed significantly more activity than their polar analogues. The lactone ring size was not crucial for activity as similar results were observed for the three tested natural products, tricolorins 1-3 which di-ffer in their macrolactone structures (Bah and Pereda-Miranda, 1996; 1997). The amphipathic properties of these compounds resulting from the acylation of some of the free hydroxyl groups of the oligosaccharide core and the lipophilic alkyl chains of their aglycones would seem to be important in facilitating cellular uptake and membrane insertion to reach its intracellular target. Interestingly, despite the presence of the macrolactone, the peracetylated compound 5, where all hydroxyl groups were protected lacked the ability to disrupt Sf9 cell membranes (Figure 3). It is possible that non-polar

compounds will lose their permeabilizing activity due to strong interactions with the cell membrane lipids and those that are polar poorly penetrate membranes. These results highlight the importance of the amphipatic character of this family of molecules for their pore forming activity. This observation is in agreement with total lost of biological activities displayed by their hydrolysis products deprived of the macrocyclic structure as well as the amphipatic properties conferred by the high degree of esterification of the oligosaccharide cores (Barnes et al., 2003; Pereda-Miranda et al., 1993). The highly lipophilic intrapilosin (Bah et al., 2007), murucoidin (Chérigo and Pereda-Miranda, 2006; Chérigo et al., 2009), and pescaprein series (Escobedo and Pereda-Miranda, 2007) were also found to be weakly cytotoxic or inactive in cytotoxicity assays, e.g., murucoidin IV exhibited marginal activity against Hep-2 laryngeal carcinoma cells: ED50 4 µg/ml (Chérigo

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et al., 2009). The most potent of all resin glycosides is ipomoeassin F that inhibits A2780 human ovarian cancer cell line with a value as low as 0.30 µg/ml. The available data with the ipomoeassin series suggest that minor variations in the peripherial oxygenation of the aglycone and acylation pattern of the oligosaccharide core modulate the biological activity of these compounds to a significant extent (Cao et al., 2005, 2007). To further confirm this observation, compound 4 was prepared. This derivative retains an intermediate polar character due to the presence of one hydroxyl group in the molecule (Pereda-Miranda et al., 1993), and as expected it displayed pore-forming activity in our assay (Figure 3). It has also been reported that the merremosides, a series of glycolipids structurally related to the tricolorins, isolated from the tuber of an Indonesian medicinal plant Merremia mammosa (Convolvulaceaea), increased the ionophoric activity towards K+, Na+, and Ca2+ in human erythrocytes (Kitagawa, 1989). The ion-transport activities were also completely lost by cleavage of the macrocyclic structure under alkaline hydrolysis. On the basis of these results and judging from the complex amphipathic structures of the tricolorins, it is possible to assume that the biological potential of this type of glycolipids relays in their poreforming properties as demonstrated by this study.

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Conclusions Convolvulaceous plants have the ability to synthesize an array of bioactive and amphipatic oligosaccharides from simple building blocks as sugars and fatty acids. The remarkable chemical diversity of this plant family, as reflected in the highly elaborated allelopathic resin glycosides, may confer the selective ecological advantages for which these species have long been used in traditional agriculture and medicinal practices. The pore-forming activity of the morning glory resin glycosides as evaluated in this study is likely to be the main mechanism of action that explains the microbial, mammalian and plant cytoxicity of this class of natural products. Acknowledgments We thank Drs. Tonatiuh Ramírez and Susana López (Instituto de Biotecnología, UNAM) for providing Sf9 cells and allowing access to fluorometer, respectively. This work was supported by Consejo Nacional de Ciencia y Tecnología Grant No. 45861-Q to RPM. AL thanks CONACYT (Grant No. J29064-B) for funding to her laboratory while working at Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos. This work was supported by DGAPA,UNAM (IN2008307-3).



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