Pergamon 0031-9422(95)00084-4 Phytocheraistry, Vol. 39, No. 3, pp. 609-612, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/95 $9.50 + 0.00 SESQUITERPENE LACTONE CHEMISTRY OF ZALUZANIA GRA YANA FROM ON-LINE LC-NMR MEASUREMENTS OTMAR SPRING, HOLGER BUSCHMANN,BERNHARDVOGLER,* EDWARD E. SCHILLING,~'MANFRED SPRAUL~ and MANFRED HOFFMANN~ Institut fiir Botanik and *Institut ffir Chemie, Universit/it Hohenheim, Garbenstrasse 30, 70599 Stuttgart, F.R.G.; tDepartment of Botany, University of Tennessee, Knoxville, TN 37996-1100, U.S.A; ~Bruker Analytische Messtechnik GmbH, Silberstreifen, 76287 Karlsruhe, F.R.G. (Received in revised form 5 December 1994) Key Word Index--Zaluzania #rayana; Viguiera triloba; Heliantheae; Asteraceae; sesquiterpene lac- tones; LC-NMR coupling. Abstract--On-line LC-NMR measurements were used for the first time to elucidate the sesquiterpene iactone chemistry of a Compositae species, Zaluzania grayana. Structure elucidation of three compounds, visible in HPLC analysis of the constituents of glandular trichomes, was performed on a leaf extract of less than a gram of dried plant tissue by means of direct coupling of the HPLC and NMR instruments. The structures could be assigned to the guaianolide zaluzanin C, a new modified guaianolide and to the 15-hydroxy derivative of costunolide. The taxonomic relevance of the identified compounds is discussed. INTRODUCTION Zaluzania is a small genus of nine species in 11 taxa as currently recognized by Olsen 1"11. Both the generic relationships and circumscription of Zaluzania have been controversial, in part because of its characteristic lack of a pappus. Stuessy [21 suggested its removal from the formerly broadly defined Helianthinae to be part of a newly created subtribe, Neurolaeninae, and further sug- gested possible ties to Galinsoginea. In contrast, Robin- son 1-31 placed it in a separate subtribe, Zaluzaniinae, which he placed near subtribe Rudbeckiinae. Various workers have segregated or transferred some elements of Zaluzania into other genera. These include Hybridella (Zaluzania subg. Hybridella [4, 5.1), Greenmaniella (Z. resinosa ['4.1), and Kingianthus (Z. sodiroi [1, 4, 5"1). Most recently Olsen [1.1 suggested transfer of Z. orayana to Viguiera, where it goes under the name of V. triloba. Zaluzania grayana is a perennial herb that occurs in southern Arizona (U.S.A.) and in northwestern Mexico. Olsen cited its sterile ray flowers that have showy ligules and its chromosome number of n = 17 in removing it from Zaluzania, species of which all have a base number of x = 18 and fertile ray flowers with short ligules I'1]. Within Viouiera a placement in V. ser. Pinnatilobatae was suggested, although there are also features that may relate it to V. sect. Heliomeris 1'1"]. Its sesquiterpene lactone (STL) chemistry was not considered in evaluating this transfer, although studies have reported these com- pounds from two species of Zaluzania, Z.augusta and Z.triloba 1'6, 7"], and from various species of Viguiera including one of V. ser. Pinnatilobatae 1-8-10"]. The present study shows that Z. grayana shares a highly distinctive STL chemistry with other species of Zaluzania that have been investigated and thus suggests that it should be retained within this genus. This study is notable in employing a very recently developed technique of on-line LC-NMR measurement for structure elucida- tion that is very suitable for chemotaxonomic appli- cations. RESULTS The investigation of the STL chemistry of Zaluzania grayana, collected near Cuahtemoc (Chihuahua, Mexico), was guided by HPLC analysis of glandular trichomes using the microsampling technique described recently [11]. Glands were found on leaves, ray flowers and anther appendages and contained three STLs with retention times (RR,) of 0.74, 0.78 and 1.07 relative to dimethyl- phenol (DMP) in the acetonitrile system that was used (see Experimental). The same compounds were obtained from methylene chloride extracts of plant tissue. One substance (RRt = 0.78) accounted for 87% of the peak area compared to 8 and 5% of the two minor consti- tuents. Quantitation by means of HPLC revealed the presence of ca 80 ng STLs per glandular trichome in the aforementioned ratios. The extract from 250 mg dried leaves (containing an estimated 140 #g of the major STL and between 7 and 15/~g of the minor STLs) was used for HPLC separation (RP 18, 25% MeCN in D20 ) with direct measurement of IH NMR spectra. Two different ways of measurement were employed to provide data for the structure elucida- 609