New Mosquito Larvicidal Tetranortriterpenoids from Turraea wakefieldii and Turraea floribunda MARY W. NDUNG’U, ² BAKARI KAONEKA, ² AHMED HASSANALI, ² WILBER LWANDE, ² ANTONY M. HOOPER, ‡ FRANCIS TAYMAN, § OLIVER ZERBE, # AND BALDWYN TORTO* ,² International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya; Biological Chemistry Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom; Department of Chemistry, University of Cape Coast, Cape Coast, Ghana; and Department of Pharmacy, Swiss Federal Institute of Technology, Winterthurestrasse 190, CH-8057 Zurich, Switzerland The crude methanol extracts of the root barks of Turraea wakefieldii and Turraea floribunda were found to show mosquito larvicidal activity against third-instar larvae of Anopheles gambiae sensu stricto. Four new limonoids comprising a vilasininoid 1 and three havanensinoids 2-4 were isolated from the chloroform fractions of the methanol extracts of T. wakefieldii and T. floribunda, respectively. The structures of the compounds were elucidated by NMR spectroscopy. Compounds 1, 2, and 4 had LD 50 values of 7.1, 4.0, and 3.6 ppm, respectively, and were more potent than azadirachtin, which had an LD 50 value of 57.1 ppm when tested against larvae of A. gambiae. KEYWORDS: Turraea wakefieldii; T. floribunda; Anopheles gambiae; limonoids; mosquito larvicidal activity INTRODUCTION Plants in the family Meliaceae are characterized by the presence of limonoids (tetranortriterpenoids) that exhibit a wide range of anti-insect effects (1-4). In eastern Africa, a number of Meliaceae plants are used in traditional medicine, including Turraea floribunda (Hochstetter), a small tree or shrub, which is used as an emetic and a purgative (5). Turraea wakefieldii Oliv., a plant native to Kenya, is not known for any traditional uses (6). The root barks of these plants contain limonoids (7-10), some of which are mosquito larvicides (10). We now report on the structures of four new limonoids that were isolated from the root barks of T. wakefieldii and T. floribunda and their activities against larvae of Anopheles gambiae sensu stricto. MATERIALS AND METHODS Plant Material. The root barks of T. wakefieldii and T. floribunda were collected from Shimba Hills National Park, Kwale, southern coast of Kenya, and were identified by S. G. Mathenge of the Botany Department, University of Nairobi. Voucher specimens WM 3/99 and 89/401 of T. wakefieldii and T. floribunda, respectively, have been deposited in the herbarium of that department. Insects. Larvae of A. gambiae used for bioassays were obtained from a colony maintained at the International Centre of Insect Physiology and Ecology (ICIPE) Insect Mass Rearing Unit. This strain of A. gambiae was originally obtained from Njage village, 70 km from Ifakara, southeastern Tanzania. The colony has been reared under laboratory conditions since April 1996. Larvae were allowed to emerge from eggs in plastic containers filled with distilled water and were transferred to larger pans at densities of 200-300 at second-instar stage. Larvae were fed on Tetramin fish food, and the water temperature was maintained between 28 and 30 °C throughout larval development. Mosquito Larvicidal Assay. The effects of different doses of meth- anol and chloroform extracts and isolated limonoids on larval mor- tality after 24 h were determined and LD50 values computed. Aza- dirachtin, a potent anti-insect naturally occurring limonoid, was tested as a positive control (1). The standard World Health Organization (WHO) larvicidal assay procedure was used (10, 12). Briefly, 1 mL of standard w/v concentrate of each test material in acetone was made up to 20 mL with distilled water in a 100 mL beaker in three replicates. Control solutions with 1 mL of acetone were similarly prepared. Twenty late third-instar larvae each were transferred into the test and control solutions, and larval mortality was recorded after 24 h. Probit analysis was used to estimate LD 50 values of extracts and limonoids (SAS version 8.0). Data Analysis. A two-way analysis of variance was carried out to evaluate the combined effects of the extracts, limonoids, and dose on larval mortality, respectively. In addition to single factors, that is, extract or limonoid and dose, the effect of interaction between the test material (extract, limonoid) and the doses was evaluated. Mean mortalities induced by each dose for each extract and limonoid were compared by Student-Newman-Keuls (SNK) test (R) 0.05). Extraction and Isolation from T. wakefieldii. A limonoid-bearing fraction (12 mg), obtained from previous work on the plant (10), was analyzed by semipreparative LC on a 250 mm × 10 mm i.d. ultrasphere * Address correspondence to this author at the USDA-ARS/CMAVE, 1700 S.W. 23rd Dr., Gainesville, FL 32608 [telephone (352) 374-5765; fax 352-374-5707; e-mail btorto@gainesville.usda.ufl.edu]. ² International Centre of Insect Physiology and Ecology. ‡ Rothamsted Research. § University of Cape Coast. # Swiss Federal Institute of Technology. J. Agric. Food Chem. 2004, 52, 5027-5031 5027 10.1021/jf049474y CCC: $27.50 © 2004 American Chemical Society Published on Web 07/14/2004