A Novel Acylated Flavonol Glycoside Isolated from Brunfelsia grandiflora ssp. grandiflora. Structure Elucidation by Gradient Accelerated NMR Spectroscopy at 14T Gerhard Brunner, 1² Ulrich Burger, 1 Pierre Castioni, 2 Ilias Kapetanidis 2 and Philippe Christen 2 * 1 Department of Organic Chemistry, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland 2 Laboratory of Pharmaceutical Analytical Chemistry, University of Geneva, 20, Boulevard d’Yvoy, CH-1211 Geneva 4, Switzerland Kaempferol-3-O-[6 Glc -O-feruloyl]-b-D-glucopyranosyl-(1!2)-O-[a-L-rhamnopyranosyl-(1!6)]-b-D-galac- topyranoside (1) was isolated from the aerial parts of Brunfelsia grandiflora ssp. grandiflora. The structure of this novel flavonol glycoside was established by fast atom bombardment-mass spectrometry and by means of NMR spectroscopy at 14T, using two-dimensional NMR experiments with pulsed-field gradients for coherence selection. The presence of the constituent aglycones and sugar units of 1 was confirmed independently by acid hydrolysis. Copyright # 2000 John Wiley & Sons, Ltd. Keywords: gradient accelerated NMR spectroscopy; South American Solanaceae; Brunfelsia grandiflora. INTRODUCTION As part of our continuing research on the South American Solanaceae, the phytochemical investigation of Brunfelsia grandiflora ssp. grandiflora has been undertaken. This species is one of the most important native remedies employed against rheumatism, arthritis, fevers and snake bites in the upper Amazon region (Plowman, 1977). This fact led us to examine, beside the alkaloids and the volatile constituents (Castioni, 1996; Castioni and Kapetanidis, 1996), the flavonoid content of the aerial parts of the plant. From a pharmacological point of view, some flavonoids possess interesting properties, such as the reported beneficial activity on the permeability and resistance of capillaries (Bruneton, 1983). Moreover, flavonoids have been shown to be endowed with anti-spasmodic, diuretic, hepatoprotective, anti-inflammatory, anti-free-radical, and even anti-bac- terial properties (Bruneton, 1983; Spilkova and Hubik, 1988, 1992). Thus, flavonoids are likely to play a major role in the anti-inflammatory activity of B. grandiflora spp. grandiflora. In this paper, the complete structural elucidation of a novel acylated flavonol triglycoside (1) by means of 2D NMR spectroscopy at 14T is described. EXPERIMENTAL Plant material. Seeds of Brunfelsia grandiflora ssp. grandiflora were provided by the Fairchild Tropical Garden (Miami, FL, USA) and grown at the Station Fe ´de ´rale de Recherches Agronomiques, (Centre des Fouge `res, Conthey, VS, Switzerland) at which site a voucher specimen is available. Aerial parts of the plants were collected and dried at 40°C. General procedures. Fractions were monitored by TLC on cellulose-coated aluminium sheets (Merck, Darm- stadt, Germany) using aqueous acetic acid (15%) or t- butanol:water:acetic acid (3:1:1) (Markham, 1982). Compounds were detected under UV light (254 and 366 nm) before and after spraying with Neu’s reagent (1% solution of 2-aminoethyl diphenylborate in metha- nol) followed by 5% PEG-4000 solution in ethanol (Markham, 1982). Sugar analysis was performed on silica gel (Si60 F 254 )-coated aluminium sheets (Merck) using propanol:water (85:15). Chromatograms were sprayed with an ethanolic solution of phthalic acid (3%) and aminohippuric acid (0.3%), heated at 110°C for 10 min and observed under UV light (366 nm). UV–vis spectra were recorded on a Zeiss (Oberkochen, Germany) DMR 21 spectrophotometer, following the procedure described by Markham (1982). Acid hydrolysis was performed by refluxing 2 mg of the compound in 5 mL of 2 M hydrochloric acid:methanol (1:1) for 2 h, evaporating the mixture to dryness, dissolving the residue in 1–2 mL water, and extracting the solution with ethyl acetate. Aglycones and sugars were identified by TLC by comparison with authentic samples. FAB-MS were recorded in the negative ion PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 11, 29–33 (2000) CCC 0958–0344/2000/010029–05 $17.50 Copyright # 2000 John Wiley & Sons, Ltd. * Correspondence to: P. Christen, Laboratory of Pharmaceutical Analytical Chemistry, University of Geneva, 20, Boulevard d’Yvoy, CH-1211 Geneva 4, Switzerland. E-mail: philippe.christen@pharm.unige.ch ²Givaudan Roure Research Ltd, CH-8600 Du¨bendorf, Switzerland. Contract/grant sponsor: Swiss National Science Foundation; Contract/grant number: 20-45806.95. Received 14 August 1998 Revised 30 December 1998 Accepted 15 January 1999