618 F. CALZADA ET AL. Copyright © 2003 John Wiley & Sons, Ltd. Phytother. Res. 17, 618–619 (2003) Copyright © 2003 John Wiley & Sons, Ltd. Received 9 October 2001 Revised 14 March 2002 Accepted 18 March 2002 PHYTOTHERAPY RESEARCH Phytother. Res. 17, 618–619 (2003) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1210 Antiamoebic Activity of Benzyl glucosinolate from Lepidium virginicum Fernando Calzada, 1 * Elizabeth Barbosa, 1 † Roberto Cedillo-Rivera 2 1 Unidad de Investigación Médica en Farmacología de Productos Naturales 2 Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, IMSS, México D. F., México In a continuation of our search for potential antiprotozoal agents from plants, we found that a crude extract from the roots of Lepidium virginicum exhibited antiprotozoal activity against Entamoeba histolytica trophozoites (IC 50 of 100.1 μg/mL). Bioassay-guided fractionation resulted in the isolation of one known glucosinolate responsible for such activity. This compound was identified as benzyl glucosinolate. It showed in vitro activity against Entamoeba histolytica strain HM1-IMSS (IC 50 of 20.4 μg/mL). The results support the anecdotal reports for the traditional use of L. virginicum roots in the control of diarrhoea and dysentery in the highlands of Chiapas, Mexico. Copyright © 2003 John Wiley & Sons, Ltd. Keywords: antiamoebic activity; Entamoeba histolytica; glucosinolate; benzyl glucosinolate; Lepidium virginicum. MATERIALS AND METHODS Plant material. Roots of Lepidium virginicum L. (Brassicaceae) were collected in Tláhuac Federal Dis- trict, Mexico, and a voucher specimen was deposited in IMSSM Herbarium under the code number 12608. Ground roots (116.6 g) were exhaustively extracted by maceration with methanol (1 L) and the extract evapo- rated to dryness. The yield of methanol extract was 11.6 g. Isolation of benzyl glucosinolate from L. virginicum. The methanol extract (9 g) was suspended in 10% ethanol-water (100 mL) and partitioned with CHCl 3 (100 mL × 3). The ethanol-water-soluble fraction (7.4 g) was then subjected to column chromatography (CC) on Sephadex LH-20 (100 g, Pharmacia), using water and ethanol gradient to obtain 6.5 g of sucrose and 320 mg of benzyl glucosinolate as major active com- pound. The CHCl 3 soluble fraction (1.54 g) was sub- jected to CC over silica gel (15 g, 70–230 mesh, Merck, Germany) with CHCl 3 to give 96 mg of β-sitosterol. The identity of sucrose and β-sitosterol were established by direct comparison with authentic material available in our laboratory (TLC and mixed melting points). Benzyl glucosinolate was identified by reported 1 H and 13 C-NMR data (Olsen and Sorensen, 1980; Cox et al., 1984). Benzyl glucosinolate: [α] D + 16.50° (c 0.3, MeOH); IR (KBr) ν max 3581, 3226, 2929, 2856, 1641, 1595, 1504, 1463, 1350, 1250, 1180, 1165, 1155, 1087, 835 cm -1 , FABMS m/z [M+H] + 432 (1), 307 (25), 154 (100), 136 (68); 1 H-NMR and 13 C-NMR (Olsen and Sorensen, 1980; Cox et al., 1984). Antiprotozoal assay. Entamoeba histolytica strain HM1: IMSS was used in all experiments. Strain was grown Enteric protozoan infections are a health problem in developing countries and are an important cause of morbidity and mortality. Symptomatic patients typi- cally present abdominal pain and tenderness, diarrhoea, and bloody stools. Although a number of different pro- tozoa may cause these symptoms, Entamoeba histolytica is the most commonly reported. The drug of choice used to treat amoebic dysentery is metronidazole, which has been associated with side effects such as headache, nausea, dry mouth, a metallic taste and neurotoxicity. It has also been shown to be mutagenic in a microbio- logical system and carcinogenic to rodents (Conde- Bonfil and De la Mora-Zerpa, 1992; Wright et al., 1994; Cedillo-Rivera et al., 1997; Kapoor et al., 1999). Thus, seeking new therapeutic agents relevant to treat amoe- bic dysentery, the medicinal plants are an important alternative to find new antiamoebic compounds. L. virginicum L. (Brassicaceae) is an erect annual herb, which grows in pine-oak and savanna forests in ecological zones of the highlands of Chiapas, Mexico. The Maya Indians call this herb pich’ tuluk’ and em- ploy the roots decoction of this plant as a treatment for diarrhoea and dysentery and as a remedy for abdom- inal pain (Berlin and Berlin, 1995). In this paper, we report the isolation of the benzyl glucosinolate, as the major antiamoebic compound from the roots of L. virginicum. * Correspondence to: Dr F. Calzada, Unidad de Investigación Médica en Farmacología de Productos Naturales, Hospital de Pediatría, 2° piso, Centro Médico Nacional Siglo XXI, IMSS, Av. Cuauhtémoc 330, Col. Doctores, CP 06725, Mexico, D. F. Tel: (+525) 627-6941. Fax: (+525) 761-0952. E-mail: fercalber1@hotmail.com † This work was the B. S. thesis of E. Barbosa. Contract/grant sponsor: National Council of Science and Technology (CONACYT). Contract/grant number: 28328N.