Letters in Applied Microbiology 1994, 18, 206-208 Antimicrobial activity of xanthatin from Xanthium spinosum L. E. Ginesta-Peris, F.J. Garcia-Breijo and E. Primo-Yufera Department of Biotechnology, Institute of Chemical Technology, CSIC, Polytechnic University, Valencia, Spain DST/41 : accepted 27 October 1993 E. GINESTA-PERIS, F.J. GARCIA-BREIJO AND E. PRIMO-YUFERA. 1994. Dichloromethane extracts from Xanthium spinosum L. were fractionated and the fractions tested for their bactericidal and fungicidal activity. From the active fraction, a compound was isolated and identified as xanthatin (I). Xanthatin was active against Colletotrichum gloesporoides, Trichothecium roseum, Bacillus cereus and Staphylococcus aureus. INTRODUCTION In an exploratory study of mediterranean flora, with the goal of isolating bioactive compounds, we analysed Xanth- ium spinosum L. (Asteraceae) (Cunat et al. 1990). Extracts and fractions from X. spinosum showed bactericidal and fungicidal activity. In this paper, we report the isolation, identification and antibacterial activity of the active com- pound. MATERIALS AND METHODS Plant material Xanthium spinosum was collected in Valencia and air-dried in shade. A voucher specimen was deposited in the Depart- ment of Botany (Valencia University). Fractionation, purification and identification of the active compound Air-dried X. spinosum leaves (3 kg) were extracted in soxhlet with dichloromethane for 5 h and the solvent evaporated in a rota vapor. The extract was chromato- graphed on a 60 mesh silica gel column, eluted with a gra- dient of hexane-dichloromethane-acetone, and fractions assayed. The fraction eluted with dichloromethane showed antimicrobial activity and was further refractionated with a silica gel Sep-Pak column (Waters, 10 /-lm), using dichloro- methane, ethyl and acetone as eluents. The anti- microbial activity was found in the fraction eluted with dichloromethane. From this fraction, the pure major compound was iso- lated by semi preparative HPLC (column: 8 x 30 cm, 10 Correspondence to: E. Primo- Yujera, Departamento de Biotecnologia, Universidad Polilecnica de Valencia, Camino de Vera 14, E-46022-Valencia, Spain. /-lm particle /-l-Porasil; eluents: dichloromethane: ethyl acetate, 95 : 5; flow: 2 ml min - 1; detector: Full Range u.v. Diode Array H.P. 85-B). The structure was determined by infrared, mass spec- trometry and 1H -n uclear magnetic resonance. Xanthatin. mp: 114--1 15°C; [an° = -19°C (C = 1'0, CHCI 3 ); IR I'rnax cm- 1 (nujol): 1775, 1670, 1590; ElMS m/z: 246 (M+, 8), 93 (13), 43 (100), 41 (23); lH-NMR (60 MHz, CDCI 3 ): c5 1·18 (d, 3H), 1·85-2·18 (m, 3H), 2·29 (s, 3H), 2·55 (m, 2H), 3·16 (m, I H ), 4· 5 (m, 1H), 5· 52 (m, IH), 6·1-6·5 (m, 3H), 7·1 (d, 11-1 ); Anal. Found: C, 73-15; H, 6·95; Calcd. for C 15 H 1S 0 3 : C, 73·17; H, 7·31%. Microbial strains and growth conditions The bacterial cultures used were: Escherichia coli and Sal- monella typhi, Bacillus cereus and Staphylococcus aureus. The bacterial strains were grown on Nutrient Broth Difco at 37°C and suspended in saline at A6oo0.D. 0·5 concen- tration. The fungal cultures used were: Colletotrichum gloespo- roides, Fusarium oxysporum, Botrytis cinerea, Penicillium ita- licum, Aspergillus jlavus and Trichothecium roseum. The fungi were grown on potato dextrose agar for 6 d at 28°C. Bactericidal activity The inhibition of the bacterial growth, around a filter paper disk (diam. 0·5 cm) impregnated with the pre-established amounts of the assay compound, was measured. The medium was Mueller Hinton Agar (15 ml) in Petri dishes 9 cm in diameter with I ml per dish of the bacterial suspen- sion. The compounds to be tested were dissolved in acetone (1-10 /-lg /-ll- 1). Ten /-ll of the dissolu tion were added to the paper disk, and the solvent evaporated. Parallel control ________ ________