Molecular docking studies of quercetin and its analogues against human inducible nitric oxide synthase.pdf
RESEARCH Open Access Alkaloids from single skins of the Argentinian toad Melanophryniscus rubriventris (ANURA, BUFONIDAE): An unexpected variability in alkaloid profiles and a profusion of new structures H Martin Garraffo1*, Nirina R Andriamaharavo1, Marcos Vaira2,3, María F Quiroga4, Cecilia Heit5 and Thomas F Spande1 Abstract GC-MS analysis of single-skins of ten Melanophryniscus rubriventris toads (five collections of two toads each) captured during their breeding season in NW Argentina has revealed a total of 127 alkaloids of which 56 had not been previously detected in any frog or toad. Included among these new alkaloids are 23 new diastereomers of previously reported alkaloids. What is particularly distinguishing about the alkaloid profiles of these ten collections is the occurrence of many of the alkaloids, whether known or new to us, in only one of the ten skins sampled, despite two skins being obtained from each breeding site of the five populations. Many of the alkaloids are of classes known to have structures with branched-chains (e.g. pumiliotoxins and tricyclic structures) that are considered to derive from dietary mites. A large number of previously reported and new alkaloids are also of unclassified structures. Only a very few 3,5-disubstituted-indolizidine or -pyrrolizidine alkaloids are observed that have a straight-chain carbon skeleton and are likely derived from ant prey. The possible relationship of these collections made during the toad’s brief breeding episodes to sequestration of dietary arthropods and individual alkaloid profiles is discussed. Keywords: Alkaloids, Gas chromatography–mass spectrometry, Melanophryniscus Toads, Reproduction, Sequestration Introduction Nine of the 25 currently known species (Frost, 2011) of South American bufonid toads of the genus Melanophry- niscus have been surveyed for lipophilic alkaloids in skin glands. These alkaloids are considered to provide a defense against predation, particularly during the mainly diurnal breeding episodes of the toads (Santos and Grant 2010a). The toads examined for alkaloids are M. atroluteus (Mebs et al. 2007), M. cupreuscapularis (Daly et al. 2008a), M. devincenzii (Mebs et al. 2007), M. klappenbachi (Daly et al. 2008a), M. moreirae (Daly et al. 1984), M. montevidensis (Garraffo et al. 1993a; Mebs et al. 2005), M. rubriventris (Daly et al. 2007), and M. stelzneri (Garraffo et al. 1993a; Daly et al. 2007). The Brazilian species, M. simplex has recently been investigated and contains both known and new alkaloids (Grant et al. 2012). A tenth spe- cies, M. cambaraensis from southern Brazil, is reported to have “defensive chemicals” in skin, not proven, however, to be alkaloids (Colombo and Grant, cited in Santos and Grant 2010a). Eighty-one skins of a Uruguayan Melanophryniscus toad, M. montevidensis from six southeastern sites were individually examined and had widely varying amounts of pumiliotoxin (PTX) 251D ranging from none, or scarcely detectible, to as much as 1 mg per toad. Traces of six other PTX alkaloids (Mebs et al. 2005) were also seen as well as traces of indolizidines that were not characterized. From two northern Uruguayan toads, M. atroluteus and M. devincenzii, PTX 251D was the major alkaloid identi- fied along with five known trace PTX alkaloids and * Correspondence: garraffo@helix.nih.gov 1Laboratory of Bioorganic Chemistry, NIDDK, NIH, DHHS, Bethesda, MD 20892, USA Full list of author information is available at the end of the article a SpringerOpen Journal © 2012 Garraffo et al; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Garraffo et al. SpringerPlus 2012, 1:51 http://www.springerplus.com/content/1/1/51 another trace PTX congener (Mebs et al. 2007). Skins of the remaining six Melanophryniscus species that are known to have alkaloids, are reported to have many more alkaloids, ranging from 10–25 alkaloids (Daly et al. 2007) to as many as 36 (Daly et al. 2008a), all with 251D and other alkaloids of the PTX class, several di- and tri- substituted indolizidines, disubstituted pyrrolizidines, di- substituted quinolizidines and tricyclic alkaloids. Some classes, considered from their straight-chain structures to derive from ants, were significant in a 1989 collection of M. stelzneri from Córdoba, Argentina, (Garraffo et al. 1993a) but were scarce in a collection from the same site made ten years later (Daly et al. 2007). Combined skin collections from four populations of M. rubriventris likewise reflected temporal variation (Daly et al. 2007). Melanophryniscus rubriventris, the subject of the current study, is a species of aposematic toads with a snout vent length (SVL) of 32–43 mm and restricted to the NW Argentinian provinces of Salta and Jujuy and temperate interandean valleys of south- ern Bolivia (Frost 2011). Although the species was first for- mally described as a toad with a black background and bright orange coloration covering the scapular region and partially the head, dorsum and flanks, with a uniform red ventral coloration (Vellard 1947), subsequent studies on population color variations in Argentina showed that they differed significantly in dorsal and ventral coloration pat- terns (Vaira 2002). Northern and central populations (Salta and central east of Jujuy province) have individuals with bright uniform dorsum, differing mainly in the extent of black patches, whereas the southern population (southeast of Jujuy province) has toads that predominately showed a more cryptic olive to black dorsal pattern. Concomitant individuals from northern and central populations have a mostly uniform yellow to red belly whereas the southern population has toads with well-demarcated yellow, red and black speckled bellies (see Figure 1). Earlier work on four M. rubriventris multi-skin collections made in 2002 from two sites in Salta province and two in Jujuy province (see Figure 2) indicated all contained various representatives of the PTX class but only two populations (Canto del Monte, Salta and Tiraxi, Jujuy) contained pumi- liotoxin 251D, normally found in Melanophryniscus (bufo- nid) toads and common also to some dendrobatid, Mantella (mantellid) and Pseudophryne (myobatrachid) frogs. The Tiraxi two-skin collection had alkaloid 267A, the only occur- rence of an allopumiliotoxin among the four collections in this earlier study. Extreme variability was also seen in the following izidine (Iz) classes detected from the 2002 collec- tions. All four collections had 195G (5-propyl-6,8-dimethy- lindolizidine) or other 5,6,8-trisubstituted indolizidines and only two collections of the four had 5,8-disubstiuted indolizi- dines, however none of these shared common structures. Likewise, all collections had several izidines (Iz) or tricyclic structures but none of these alkaloids were shared. The present study analyzing single skins of M. rubriventris, focuses on alkaloid profiles of a single species of toad from undisturbed contiguous and non-contiguous sites and whether any conclusions can be drawn as to the variety of arthropod prey consumed during their explosive breeding bouts that might allow a comparison in alkaloid compos- ition with earlier collections taken during non-breeding periods. We have deviated considerably from the data formats we used earlier in other journals in that we have included much more original data such as GC retention times, high reso- lution EIMS, CIMS, total-ion mass chromatograms and complete EIMS mass spectra (not just major peaks). We think this is important in appreciating the unexpected complexity and variation in the toad skin alkaloids of this species and the unique time and place of these collec- tions. Almost half have never been seen before in any Old or New World frog or toad. The new alkaloids are not the result of improved analytical sensitivity or more concen- trated samples as we have used basically the same protocols and instruments as in our earlier work on single-skins of mantellid frogs (Daly et al., 2008b). The corrected GC retention times are necessary to con- firm that a particular alkaloid is either new or a new dia- stereomer of a known alkaloid. In an effort to assist other investigators, this data (see Additional file 1 Tables S1-S10) will also update our earlier review that included approxi- mately 800 poison frog alkaloids then known, with their corrected GC retention times (Daly et al. 2005). The total ion current (TIC) chromatograms (Additional file 3 Figure S1-S10) are critically illustrative here because of the alkal- oid variability observed and also because of the wide range in alkaloid amounts. Because so many alkaloids are new, we cannot, as is usual, simply refer to characterization cita- tions in previously published papers on poison frog or toad alkaloids. So we chose to enter the whole of our mass spec- tral data here in hopes of stimulating further work and as an explanation for the departure of these toads from expected analytical results, where only half a dozen or so new alkaloids would have been expected. With the current state of the art of electronic publishing, there is no good reason to depict the mass spectra in any other format but the graphical one, which is the best to compare with new data to be obtained in the future. Methods and materials Two toads were collected at each of five sites: three sites in Salta province of NW Argentina and two sites in the contiguous province of Jujuy (see Figure 2). Both pro- vinces border Bolivia (see inset, Figure 2). The two speci- mens from each site were captured within an area of roughly 4 m x 4 m at the dates indicated in the footnotes to Additional file 1 Tables S1-S11. Table S11 provides GPS coordinates and elevations of the sites along with the size Garraffo et al. SpringerPlus 2012, 1:51 Page 2 of 15 http://www.springerplus.com/content/1/1/51 (M. F. Quiroga and M. Vaira unpub. data). This state- ment is supported by the following two experiments: 1) A laboratory test of stomach passage time was per- formed in a similarly sized frog, Eleutherodactylus coqui, ranging in snout-to-vent length from 25 to 50 mm. The results indicate that prey items pass out of a frog stomach in 10–14 h after being ingested (Woolbright & Stewart, 1987). So, given that Melanophryniscus rubriventris is active early in the morning (around 7 am) we can expect that in these toads, foraging during the daylight, the stom- ach would be empty by midnight (see next). 2) During 2007/2008 some of us (MFQ and MV) col- lected 71 M. rubriventris toads and analyzed the stomach contents by the stomach-flushing method (Leger and Sullivan, 1979; Solếet al. 2005) with approximately equal number of toads taken at 3 pm, 7 pm and 1 am the next day, defined as afternoon, evening and night, respectively. By flushing, the stomach eliminates all the contents (full stomach) in one bolus or almost nothing when the sto- machs are empty. The proportions of full stomachs over all the frogs taken in each period, afternoon, evening and night, were 66%, 48% and 9%, respectively. Therefore, it seems clear to state that toads feed mainly during the morning until afternoon hours, although we found a few specimens still active at night (around 1 to 2 am). The major problem with inferences from stomach contents is that such results represent the last meal or two of the toad, while the observed skin alkaloids are accumulated over a much longer time, likely an adult’s lifetime. Also the softer bodied prey items will be digested more rapidly than the hard-bodied mites and ants. Nevertheless, stomach content analyses do provide presumptive evidence of the dietary preferences of the poison frogs (Clark et al. 2005; Woodhead et al. 2007; Saporito et al. 2012) and toads so far examined. Of more limited application has been the analysis of toad feces using electron microscopy techniques to identify un- digested body parts of prey (Mebs et al. 2005). A sex-link with alkaloids in M. rubriventris toads? Of the ten skins examined, nine were from males and only one from a female (#8, Tablada site). While earlier studies of the dendrobatid frog Dendrobates (Oophaga) pumilio (Saporito et al. 2008) indicated some significant differences with sex in alkaloid profiles in both amounts and components, the present study, reveals no obvious differences in variety of alkaloids when all the toad males are considered. The M. stelzneri collection of 2001 reported in 2007 (Daly et al. 2007) examined two com- bined male and two combined female skins and the results neither in amounts (male, 9 major/minor alka- loids; female 8 major/minor alkaloids) nor variety (male 23 alkaloids total; female, 22 alkaloids total) resembled the results found in studies with mantellid (Daly et al. 2008b) or dendrobatid frogs (Saporito et al. 2010), where the slightly larger and presumably wider ranging females had skin alkaloids in both larger amounts and greater variety. The lack of a sex link seen with currently inves- tigated toad alkaloid profiles, although from very limited samples (in fact, only one female toad), suggests that the male and female Melanophriniscus toads may not separ- ate significantly during foraging. Conclusion The present ten collections were made during the lengthy, hot, spring-summer breeding season of these toads (November to February) where toads were cap- tured as pairs in small (ca. 4 m x 4 m) areas with ponds (Additional file 2: Figure S27). The Salta and Jujuy regions where these toads are found are of a typical sub- tropical humid montane/woodland forest that is mature and well-structured. Moisture and occasional fog arise from ascending air currents and promote a lush epi- phytic growth of bromeliads, ferns, mosses and liver- worts. Temporary ponds and streams where breeding occurs arise from runoff from higher elevations during the rainy season (October to April) when average yearly rainfall is over 2 m, but are short-lived due to the steep slopes and porous soils (Vaira, 2005). Several breeding periods occur during the rainy season. The fact that specimens collected from the same site have more alkaloids shared than toads from different sites, suggests that during the approximately three-day breeding period, arthropods are being consumed at or near the site, but overlaying this fresh input from a lim- ited area will be a larger background of previous alkaloid sequestration that likely represents foraging from a more extensive area in the forest as supported by some pre- liminary observations of ours. The varying profiles of contents of stomach analyses also implies this. At the moment, it is not clear how extensive is the territorial range of the toads that just happen to be collected at the same time in the same small pond. Proximity of the col- lected toads in the present study, in any case, is mislead- ing since foraging during intermittent breeding, both close to and more remote from such ponds, has been observed by us. Work is underway to determine more exactly the territorial range of toads on land or adjacent swamp areas before and during their typical three-day breeding bouts in the ponds. Of the 48 previously reported alkaloids (i.e. those hav- ing codes followed by no asterisk in Table 1, Additional file 1 Tables S1-S10) observed in the present survey of the skin alkaloids from M. rubriventris toads, most were seen before in more than one of the other anuran fam- ilies. The Venn diagram of Additional file 5 Figure S28 indicates that 22 of the 48 alkaloids were seen earlier in skin extracts of dendrobatid and/or mantellid poison Garraffo et al. SpringerPlus 2012, 1:51 Page 13 of 15 http://www.springerplus.com/content/1/1/51 frogs, while only 9 alkaloids were previously reported from bufonids alone. Thus, it appears that we are begin- ning to find more similarity in alkaloid structures be- tween these poison toads of South America and the poison frog families of both hemispheres. This impli- cates common dietary prey and the common evolution of an alkaloid uptake system between toad and frog. The six previously reported bufonid alkaloids shared with dendrobatids include one 5,8-disubstituted indolizidine, one izidine and one tricyclic, all likely alkaloids with skeletal branch points and of probable mite origin. Inter- estingly, there are currently no known mantellid alka- loids shared only with bufonids. There are, however, 11 alkaloids shared among bufonids, mantellids and den- drobatids. Only two (223H, 223AB) are known to have structures with a linear carbon-chain; the rest have branch points. Very recent work (Grant et al. 2012) on eleven M. simplex skins from three sites in Brazil indicates the occurrence of many new alkaloids or diastereomers of known dendrobatid alkaloids as we have observed with the Argentinian M. rubriventris. Fourteen of the total of 47 alkaloids or new diastereomers they report are shared with our collections but the overall pattern they observe has many more alkaloids (eleven) likely seques- tered from ants. Pumiliotoxins and various izidines dom- inate their collections but they also include nine alkaloids of unclassified structures, two that we also report. Santos and Grant have recently studied in detail the reproduction of a Brazilian Melanophryniscus species and concluded that the observed strongly diurnal reproduction preference likely preceded the development of sequestra- tion of toxic alkaloids in these and other bufonids and in poison frogs in general (Santos and Grant 2010). What is not clear at the moment is at what stage in evolution, did coloration begin to play a role to advertise toxicity. Additional files Additional file 1: Table S1-S10. The Rt (obs) of the known alkaloids in the present study was corrected to compare with the corresponding retention times of our most recent summary (Daly et al.2005). A simple plot with observed vs. corrected retention times of the known alkaloids, selected from several of the tables below, generates a straight line; the corrected retention times for the unknowns are obtained using the observed values and interpolating within this line. Both values, Rt (obs.) and Rt (corr.), were fairly reproducible among the tables S1-S10 for any repeated alkaloid. Table S11: Collection information on skins of Melanophyriscus rubriventris. Table S12: GC-eims ion current intensities. Table S13: Effect of proximity between 2002 and 2008 collections-Salta province. Table S14: Effect of proximity between 2002 and 2008 collections-Jujuy province. Additional file 2: Figure S27. Typical ponds where collections were made: a) Cedral de Baritú, Salta; b) Los Paños, Jujuy. The blue parallelogram indicates the average collection area. Additional fle 3: Figures S1-S10. Total mass spectral ion current chromatograms for the alkaloid extracts of toad skin samples #1-10. Additional file 4: Figures S11-S26. Mass spectra of all the alkaloids (127) found in this study, 8 spectra per page (total of 16 pages) in increasing order of MW, with the exception of alkaloid 195N added at the end. Additional file 5: Figure S28. Venn diagram showing occurrence and overlaps of previously known anuran skin alkaloids (N = 48) detected in the Argentinian toad Melanophryniscus rubriventris of this study with those previously seen in bufonid toads, dendrobatid or mantellid frogs. See Supplemental Information of Daly et al. 2005 for alkaloids and sources. The asterisk in the dendrobatid/mantellid alkaloid overlap indicates two occurrences also in myobatrachid frogs of Australia. Competing interest The authors declare that they have no competing interests. Authors’ contribution MV and MFQ collected specimens; NRA prepared skin extracts; CH undertook some preliminary analyses; NRA and HMG performed detailed GC-MS analyses; TFS organized most of the data; MV, HMG and TFS drafted the manuscript. All authors read and approved the final manuscript. Acknowledgments HMG, NRA and TFS wish to thank NIDDK, NIH, HHS for support. Part of the project was supported by a research fellowship from Universidad Nacional de Jujuy to MFQ, and was supported also by a Secter-UNJu grant #D-037. Permits for sample collection of the species were provided by the Delegación Técnica de Parques Nacionales, Regional Noroeste and Dirección Provincial de Políticas Ambientales y Recursos Naturales, Jujuy. Author details 1Laboratory of Bioorganic Chemistry, NIDDK, NIH, DHHS, Bethesda, MD 20892, USA. 2CIBA, Facultad de Ingeniería, Universidad Nacional de Jujuy, Gorriti 237, Jujuy 4600, Argentina. 3Instituto de Bio y Geociencias del NOA, Universidad Nacional de Salta, Mendoza 2, Salta, Argentina. 4Facultad de Ciencias Agrarias, Universidad Nacional de Jujuy, Alberdi 47, Jujuy, Argentina. 5Laboratorio de Análisis de Residuos y Trazas (LAnaRT), Zorrilla de San Martín, 2170, Jujuy, Argentina. 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Title: Alkaloids from single skins of the Argentinian toad Melanophryniscus rubriventris (ANURA, BUFONIDAE): An unexpected variability in alkaloid profiles and a profusion of new structures ​
Authors: H Martin Garraffo, Nirina R Andriamaharavo, Marcos Vaira, María F Quiroga, Cecilia Heit, Thomas F Spande ​
Publisher: SpringerPlus
Date: November 23, 2012 ​

Abstract: True, or traction, esophageal diverticulum (TED) is seen in the middle one third of the thoracic esophagus in a peribronchial location, occurs secondary to mediastinal inflammatory lesions such as tuberculosis or histoplasmosis. ​ The resultant desmoplastic reaction in the paraesophageal tissue causes full thickness pinching on the esophageal wall, producing a conical, broad-mouthed true diverticulum. ​ They often project to the right side because subcarinal lymph nodes in this area are closely associated with the right anterior wall of the esophagus. ​ TED usually presents with symptoms such as dysphagia, postural regurgitation, belching, retrosternal pain, heartburn, and epigastric pain. ​ As in patients with pharyngoesophageal (Zenker’s) diverticula, pulmonary symptoms are often present but underestimated in TED patients. ​ These symptoms range from mild nocturnal cough to life-threatening massive aspiration. ​ In this particular report we describe a rare case of TED presenting as a symptomatic upper gastrointestinal bleeding. ​ Diagnostic evaluation of TED includes chest X-ray, barium esophagogram and manometry. ​ A significant proportion of lower esophageal diverticula are associated with motility disorders. ​ Management of TED include treating the underlying cause sometimes a surgical resection of diverticulum along with esophageal myotomy is necessitated in symptomatic patients. ​GC-MS analysis of single-skins of ten Melanophryniscus rubriventris toads (five collections of two toads each) captured during their breeding season in NW Argentina has revealed a total of 127 alkaloids of which 56 had not been previously detected in any frog or toad. ​ Included among these new alkaloids are 23 new diastereomers of previously reported alkaloids. ​ What is particularly distinguishing about the alkaloid profiles of these ten collections is the occurrence of many of the alkaloids, whether known or new to us, in only one of the ten skins sampled, despite two skins being obtained from each breeding site of the five populations. ​ Many of the alkaloids are of classes known to have structures with branched-chains (e.g. ​ pumiliotoxins and tricyclic structures) that are considered to derive from dietary mites. ​ A large number of previously reported and new alkaloids are also of unclassified structures. ​ Only a very few 3,5-disubstituted-indolizidine or -pyrrolizidine alkaloids are observed that have a straight-chain carbon skeleton and are likely derived from ant prey. ​ The possible relationship of these collections made during the toad’s brief breeding episodes to sequestration of dietary arthropods and individual alkaloid profiles is discussed. ​
 