Isolation of an Antimicrobial Compound from Impatiens balsamina L. using Bioassay-Guided Fractionation Xiaolong Yang, 1 D. Kristen Summerhurst, 1 Susan F. Koval, 2 Christine Ficker, 3 Myron L. Smith 3 and Mark A. Bernards 1 * 1 Department of Plant Sciences, University of Western Ontario, London, ON, Canada 2 Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada 3 Biology Department, Carleton University, Ottawa, ON, Canada By using brine shrimp (Artemia salina) lethality test-guided fractionation, a single bioactive compound (LC 50 =26 ppm) was isolated from the 95% ethanol extract of the dried aerial parts of Impatiens balsami- na L. and subsequently identified as 2-methoxy-1,4-naphthoquinone (MNQ). The structure of MNQ was confirmed by UV, FT-IR, MS, and 1-and 2-D NMR spectroscopy. The antimicrobial activity of MNQ was evaluated using 12 bacterial and eight fungal strains. Five gram-positive and two gram-negative bac- teria as well as all eight fungi (including multi-drug resistant strains) tested were highly sensitive to MNQ. A tea prepared according to traditional methods was found to contain sufficient MNQ to account for its antimicrobial properties. Copyright # 2001 John Wiley & Sons, Ltd. Keywords: Impatiens balsamina L.; brine shrimp lethality test; Artemia salina; 2-methoxy-1,4-naphthoquinone; antibacterial activity; antifungal activity. INTRODUCTION For centuries, preparations from the aerial parts of Impatiens balsamina L. (Balsaminaceae) have been used in traditional Chinese medicine for antimicrobial, anti- rheumatic, antipruritic and antitumoural purposes as well as for the treatment of difficult labour and puerperal pain (Cai, 1996; Huang, 1998). Many compounds have been isolated from I. balsamina including phenolics (Bohm and Towers, 1962), flavonols (Clevenger, 1958; Hagen, 1966a), anthocyanin pigments (Hagen, 1966a, 1966b), quinones (Glennie and Bohm, 1965; Chapelle, 1974) and saponins (Shoji et al., 1983, 1994a, 1994b, 1994c). While the antipruritic and antianaphylactic properties of some compounds (particularly phenolics and quinones) from this plant have been studied extensively (e.g. Fukumoto et al., 1996; Oku and Ishiguro, 1999), the phytochemical bases of other traditional uses of I. balsamina extracts have received little attention (Kang and Moon, 1992). Traditionally, the dried herb is either boiled in water to make a tea used to treat systemic bacterial and fungal infections or applied directly on the skin or nails in a plaster form to treat local infections. In this paper, we report on the isolation and identification of an anti- microbial principle from I. balsamina and its quantifica- tion in a traditional tea preparation. MATERIALS AND METHODS General. Silica gel 60 (0.040–0.063 mm, 230–400 mesh, EM Sciences) was used for column chromatography. Silica gel 60F 254 was used for analytical thin-layer chromatography (TLC). TLC plates were visualized under UV light (l = 254 nm) and then dipped in molybdate solution [10 g of (NH 4 ) 6 Mo 7 O 24  4H 2 O and 10 mL H 2 SO 4 dissolved in 90 mL H 2 O and diluted with CH 3 OH (1:1)] and heated with a heating gun. NMR spectra were recorded in CDCl 3 on a Varian Mercury 400 MHz spectrometer. UV and FT-IR spectra were recorded on an Ultraspec 2000 UV/visible spectro- photometer (Pharmacia Biotech) and a BOMEM (Hart- mann & Braun) spectrometer, respectively. EIMS were recorded on a Finigan MAT MS (Model 8200). HPLC was performed on a Beckman Gold Nouveau system equipped with a photodiode array detector, using a Nucleosil C 18 reversed phase column (5 mm, 3.9  150 mm). Plant material. The dried aerial parts of I. balsamina were purchased in 1998 from a market in Zhengzhou, Henan Province, P. R. China, and authenticated by the senior Chinese herbalist, Dr Wanting Zhang, Department of Chinese Herbal Medicine, Henan Medical University, P. R. China. Extraction and fractionation of antimicrobial com- pounds. Dried, ground aerial parts of I. balsamina (40 g) were extracted with 95% ethanol (1.5 L) in a soxhlet extractor overnight. The crude extract was concentrated to aqueous in vacuo at 40 °C, and partitioned three PHYTOTHERAPY RESEARCH Phytother. Res. 15, 676–680 (2001) DOI: 10.1002/ptr.906 Copyright # 2001 John Wiley & Sons, Ltd. * Correspondence to: Dr M. A. Bernards, Department of Plant Sciences, University of Western Ontario, 1151 Richmond Street N, London, ON, Canada N6A 5B7. Email: bernards@uwo.ca Contract/grant sponsor: Natural Sciences and Engineering Research Council (NSERC) of Canada. Received 30 November 2000 Revised 25 January 2001 Accepted 5 March 2001