Phytochemistry 55 (2000) 217±222 www.elsevier.com/locate/phytochem An aphid repellent glycoside from Solanum laxum S. Soule a,b,C.GuÈ ntner b,A.Va zquez b, V. ArgandonÄ a c, P. Moyna b, F. Ferreira b,* aDepartamento de QuõÂmica OrgaÂnica, Facultad de Ciencias, General Flores 2124, Montevideo, Uruguay bCaÂtedra de Farmacognosia y Productos Naturales, Facultad de QuõÂmica, CC 1157, Montevideo, Uruguay cLaboratorio de FisiologõÂa Vegetal, Facultad de Ciencias, Universidad de Chile, Santiago de Chile, Chile Received 24 January 2000; received in revised form 2 June 2000 Abstract A spirostanic saponin was isolated from the ethanolic extract of the aerial parts of Solanum laxum Steud. The compound, named luciamin, was characterised by NMR spectroscopy, mass spectrometry and chemical methods, as (22R,25S)-spirost-5-en-3 b,15a- diol 3-O-{b-d-glucopyranosyl (1!2)-b-d-glucopyranosyl-(1!4)-[a-l-rhamnopyranosyl-(1!2)]-b-d-galactopyranoside}. The com- pound was tested against the aphid Schizaphis graminum by incorporation in arti®cial diets. It showed a deterrent (toxic) activity against the insect and is the ®rst spirostane glycoside reported to have this activity. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Solanum laxum; Solanaceae; Bioassay; Glycosides; Schizaphis graminum 1. Introduction bindweed that grows in association with riverside vege- tation. Several classes of compounds, including alkaloids (ZuÂnÄ iga et al., 1985), ¯avonoids and related compounds (Dreyer and Jones, 1981), and diterpenes (Rose et al., 2. Results and discussion 1981), are toxic for and/or deter feeding of herbivores and insects. The Solanaceae family is a rich source of The material obtained after ethanolic extraction of active secondary metabolites. In particular, the genus the aerial parts of S. laxum and further evaporation, Solanum produces a great variety of steroidal saponins was partitioned between water and chloroform. During and glycoalkaloids of importance in the natural resis- this procedure, a layer formed between the water and tance of these plants against several pests (Friedman et the chloroform. It was separated and the residue after al., 1991). Solanum glycosides and glycoalkaloids aect concentration was fractionated by medium pressure the aphid Schizaphis graminum (Rondani) (Homoptera: liquid chromatography (MPLC) using ®rst reversed and Aphididae) (Soule et al., 1999). This aphid is of eco- then normal stationary phases. Two known saponins, nomic interest, since it is considered the worst cereal laxumine A and laxumine B (Ferreira et al., 1996), and a aphid pest (Anonymous, 1981), yet it does not normally third new saponin, named luciamin (1), were isolated by settle on Solanum plants. this method. In our 5-year continuous study of the structure and Luciamin (1) was obtained as a pale yellow powder, 20 activity of the glycosides produced by Uruguayan Sola- [a]D =65 (MeOH, c 0.3). The IR spectrum exhibited, num plants (Ferreira et al., 1994, 1996; GuÈ nter et al., among other signals, absorption bands indicating pre- 1997; Soule et al., 1999) we now report the isolation, sence of hydroxyl groups (3417 and 1069 cm1) (Parikh, characterisation and biological activity against Schiza- 1974). The 1H NMR spectrum and the typical steroidal phis graminum of a new spirostanic saponin named coloration produced by H2SO4 aspersion and charring luciamin isolated from Solanum laxum Steud, a native on TLC plates suggested a glycosidic structure. The sugars obtained after acid hydrolysis of luciamin * Corresponding author. Fax: +598-29241906. (Fig. 1, 1) were analysed as their alditol acetates by gas E-mail address: [email protected] (F. Ferreira). chromatography±mass spectrometry (GC±MS) using 0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00273-9 218 S. Soule et al. / Phytochemistry 55 (2000) 217±222 the successive loss of two hexose residues, the rhamnose residue and a further loss of an another hexose, respec- tively. The 1H NMR spectrum of luciamin showed, in addi- tion to the signals of the oligosaccharide residue, signals corresponding to one ole®nic proton at 5.38, two methyl groups substituted at quaternary carbons at 0.85 (s) and 1.06 (s) and three methyl groups sub- stituted at methine carbons at 0.99 (d, J=6.8 Hz), Fig. 1. Chemical structure of luciamin. 1.09 (d, J=7.2 Hz) and 1.30 (d, J=7.0 Hz). The 13C NMR spectrum showed signals for 51 car- pure reference samples (Sawardeker et al., 1965), and bons, 27 of which arose from the aglycone moiety. The the absolute con®gurations were determined by GC of spectrum displayed, among other, signals corresponding the glycosides obtained by reaction with (+)-2-butanol to two ole®nic carbons at 141.4 and 123.2, and a spir- and trimethylsilylation (Gerwig et al., 1979). d-Glucose, ostanic carbon atom at 110.7 (Agrawal et al., 1985). l-rhamnose and d-galactose in the relative proportions These data, and comparison with published data (Agra- 2:1:1 were the only sugars detected. The linkages by wal et al., 1985, 1998; Barbosa Filho et al., 1989; Ferreira which the sugars are connected were determined by et al., 1994, 1996), suggested that luciamin consisted of a methylation analysis according to Jansson et al. (Jans- Á5-spirosten aglycone, which is a structure commonly son et al., 1976). Analysis of the products by GC±MS found in Solanum plants, substituted with a tetra- and comparison with pure reference samples yielded the saccharide. acetates of 2,3,4-tri-O-methyl-l-rhamnitol, 2,3,4,6-tetra- The CID mass spectrum of the [M+H]+ ion at m/z O-methyl-d-glucitol, 3,4,6-tri-O-methyl-d-glucitol and 1063 produced by FAB ionisation of luciamin, showed 3,6-di-O-methyl-d-galactitol. These results indicated the an ion at m/z 431 corresponding to the protonated presence of terminal rhamnose and glucose groups, a 2- aglycone. This indicated the molecular weight of the substituted glucose and a 2,4-disubstituted galactose aglycone to be 430 mass units, which corresponds to a residue. molecular formula of C27H42O4, suggesting the pre- From the 1H and 13C NMR spectra of luciamin infor- sence of a Á5-spirosten skeleton substituted with two mation on the sugar residues and their anomeric con- hydroxyl groups. ®gurations were obtained. Using dierent 1D and 2D The proton spin system of the A, B and C rings were experiments (H±H COSY, relay and double relay H±H assigned by dierent H±H correlation experiments (H± COSY), most of the 1H NMR signals could be assigned H double quantum ®ltered phase sensitive COSY and 3 and the JH1,H2 values determined (Table 1). relay and double relay COSY). These data, together 1 1 The H NMR spectrum of luciamin in methanol-d4 at with the proton-carbon correlation from a H-decou- 30C showed signals corresponding to four anomeric pled HMQC experiment and comparison with the lit- protons at 5.14 (J=1.6 Hz), 4.80 (J=7.9 Hz), 4.58 erature, allowed the assignment of the carbon signals. (J=7.6 Hz) and 4.48 (J=7.6 Hz), con®rming the pre- H-3 ( 23.59) was readily identi®ed by its coupling with sence of a tetrasaccharide moiety consistent with the H-4a ( 2.45), H-4b ( 2.29), H-2 ( 1.91) and H-2b ( sugar and methylation analyses. Proton decoupled 1.60). The C-2, C-3 and C-4 signals ( 30.5, 80.3 and HMQC experiments allowed the assignment of the cor- 38.3, respectively) are similar to the corresponding responding carbon signals (Table 1). values for diosgenin 3-O-b-d-galactoside ( 30.5, 78.4 3 This information, together with the values of JH1,H2 and 39.6 respectively) (Agrawal et al., 1985). for the anomeric protons and published chemical shift The observed n.O.e. between the anomeric proton of data for each sugar residue (Jansson et al., 1989) the galactose residue ( 4.48) and H-3 ( 3.59) con®rmed allowed the assignment of the dierent spin systems to that the tetrasacharride was linked to the 3-OH of the speci®c sugar residues and the determination of their aglycone. The signals for H-1a ( 1.08) and H-1b ( respective anomeric con®gurations. The data showed 1.87) were assigned by their coupling with H-2a and H- that the rhamnose residue was an a-pyranoside whereas 2b. the other residues were b-pyranosides. The coupling between the signal for the vinylic H-6 ( The FAB mass spectrum of luciamin (1) gave a 5.38) and H-7b ( 2.36) and H-7a ( 1.75) allowed their [M+Na]+ ion at m/z 1085 and a [M+H]+ ion at m/z identi®cation. H-8 ( 1.70) was identi®ed by its coupling 1063 indicating that the molecular weight was 1062. with both H-7 and a cross peak between its signal and The high energy collision induced dissociation mass the H-6 signal in the relay H±H COSY spectrum. The spectrum (CID-MS) of the luciamin [M+H]+ ion at m/z H-9 signal at 1.07 was readily assigned by its coupling 1063 using He as the collision gas, showed among others, with H-8. Similarly, all the 1H and 13C NMR signals for fragments at m/z 901, 739, 593 and 431 corresponding to the A, B and C rings were assigned (Table 1) and were S. Soule et al. / Phytochemistry 55 (2000) 217±222 219 Table 1 The continuity of the proton spin system is broken 1 13 H and C chemical shifts of luciamin in CD3OD solutions at 30 C between rings E and F in the spirostan system due to the Aglycone Sugar residues presence of the quaternary C-22 ( 110.7).