Phytochemistry Reviews 1: 409–411, 2002. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 409 Investigation of biologically active natural products using online LC-bioassay, LC-NMR, and LC-MS techniques W. Kraus ∗ , Luu Hoang Ngoc, J. Conrad, I. Klaiber, S. Reeb & B. Vogler Department of Chemistry, University of Hohenheim, Garbenstrasse 30, D-70593 Stuttgart, Germany; ∗ Author for correspondence (Tel: +49-711-459-2174; Fax: +49-711-459-2319; E-mail: kraus130@uni-hohenheim.de) Key words: Jasminum subtriplinerve, LC-bioassay, LC-MS, LC-NMR, tannins, Terminalia macroptera, terpene glycosides Plant extracts are widely used as pesticides and in folk medicine in tropical and subtropical areas. In order to study possible applications of extracts or compounds derived from extracts, methods to screen for biological activities, and separation techniques to isolate the act- ive principles have to be established. We found the combination of separation methods with bioassays and spectroscopic techniques such as NMR and MS to be a very attractive tool (Roos, 1997; Roos et al., 1998a, b; Vogler et al., 1998). We report on HPLC-MS and HPLC-NMR on- line and offline analyses of the extracts, isolation of bioactive compounds via bioassay guided separa- tion techniques including online HPLC-bioassays, and structure determination by MS and NMR of the bioact- ive principles obtained from Jasminum subtriplinerve and Terminalia macroptera. Terminalia macroptera (Combretaceae) The plant material (bark) was collected in Cameroon and successively extracted with petrol ether, ethyl acetate and methanol. Bioassay coupled chroma- tography of the ethyl acetate extract gave 23- galloylarjunolic acid (Conrad et al., 1998) and a new hydrolyzable tannin, isoterchebulin (1) (Conrad et al., 2001a), along with a number of known triter- penes and 3,3 ′ ,4 ′ -tri-O-methylellagic acid. From the methanol extract by solvent partition (BuOH/water, CHCl 3 /MeOH/water) followed by bioassay coupled chromatography on Sephadex (MeOH) we isolated 23-galloylarjunolic acid 28-O-β -D-glucopyranosyl ester (Conrad et al., 1998), vanillic acid 4-O-β -D- (6 ′ -O-galloyl)-glucopyranoside (Conrad, 2000; Con- rad et al., 2001b) 4,6-O-isoterchebuloyl-D-glucose (2) (Conrad, 2000; Conrad et al., 2001a) and four known triterpene glucosides (Conrad, 2000). All structures were elucidated by extensive 1D and 2D NMR studies, MS, and chemical transform- ations. For isoterchebulin (1) the position of the linkage between rings C and D could not be local- ized by NMR alone. Therefore the compound was permethylated to give the hexadecamethyl ether (3) from which nonamethylisoterchebulic acid dimethyl ester (4) and dimethyl-(S)-hexamethoxydiphenoate (5) were obtained by methanolysis with sodium methox- ide in methanol. The structure of 4 was determined on the basis of 1 H-, 13 C-, HSQC, HMQC, ROESY, NOE- difference, and NOESY experiments. Compounds 1 and 2 were tested for biological activity using Bacillus subtilis, Pseudomonas fluor- escens, Cladosporium cucumerinum, Caenorhabditis