Antimosquito Phenylpropenoids from the Stem and Root Barks of Uvariodendron

The phenylpropenoids O-methyleugenol, O-methylisoeugenol and 2,3dimethoxycinnamaldehyde, have been isolated as the antimosquitocidal principles of the stem and root bark extracts of Uvariodendron pycnophyllum (Diels) R.E. Fr. The extracts and compounds exhibited activity with LC50 values in the range 17-59 ppm against the Anopheles gambiae s.s Giles mosquito larvae, while the constituent phenylpropenoids showed long term mortality effects to adult An. gambiae mosquito on impregnated bednets, and mosquito repellency that was stronger than the activity of the standard repellent DEET. @ JASEM Uvariodendron (Family Annonaceae) is a small genus comprising about 16 species, which are either shrubs or trees, the genus being restricted to tropical Africa and about 6 species are confined to East Africa (Verdcourt, 1971). According to the Flora of Tropical East Africa (Verdcourt, 1971), four Uvariodendron species, namely U. kirkii, U. gorgonis, U. pycnophyllum and U. usambarense are reported to occur in Tanzania. In continuation with investigations of Tanzanian plant species for antimosquito constituents as a contribution to control malaria transmission mosquitoes (Kihampa et al., 2009), we have analyzed the root and stem barks of Uvariodendron pycnophyllum (Diels) R.E. Fr. (Mkene in Kiswahili) that in preliminary assays exhibited potent antimosquito activity. We now report the antimosquito properties of O-methyleugenol (1), Omethylisoeugenol (2), 2,3-dimethoxycinnamaldehyde (3) and stigmasterol (4) obtained from the stem and root bark extracts. Besides being a source of firewood, building poles, knife and hoe handles, beds, bows and withies, the plant species is not used in any folk medicine. MATERIALS AND METHODS Plant Materials: The leaves, stem and root barks of U. pycnophyllum were collected from Siggi Valley, 3 km from Kisiwani village along the road to Bombani in the Amani Nature Reserve, East Usambara Mountains in Muheza District, Tanga Region. The plant species was identified on site and its identity was further confirmed at the Herbarium of the Department of Botany, University of Dar es Salaam, where a voucher specimen is deposited. Extraction and Isolation: The air dried and pulverized root and stem barks were extracted sequentially with CHCl3 and MeOH, 2 x 48 h for each solvent. The extracts were stored at –18 oC until further analysed or assayed. The active compounds were isolated following a bioassay-guided isolation from the relevant extracts using the brine shrimp lethality test (BST) (Meyer et al., 1982). Fractionation of the concentrated extracts was carried out by VLC, followed by repeated column chromatography on silica gel and/or Sephadex  LH20 eluting with pet ether and then pet ether containing increasing amounts of EtOAc, and mixtures of MeOH and CHCl3 (1:1, v/v) respectively. Structural determination was achieved upon analysis of spectral data. Larvicidal Assay: The assay was carried out according to the WHO protocol (WHO, 1996). Twenty late 3rd or young 4th instar larvae were used per beaker with three beakers per concentration (the water temperature being 25 ± 1oC) and for each test three beakers containing distilled water and test larvae but without sample were used as controls. Larvae mortality and deformities was recorded after every 24 h of continuous exposure and expressed as percent mortality (WHO, 1996). The lethal concentration at which 50% of the test larvae were killed (LC50) was determined using POLO PLUS computer package. Mosquitocidal Assay: This was performed as described in the literature (Joseph et al., 2004). Mosquito Repellency Assay: The assay was conducted as reported in the literature (Innocent et al., 2008) using serial dilutions, the highest concentration being 1% (0.01 g/ml) and the other concentrations were 0.000015, 0.00015, 0.0015, 0.015 and 0.15 mg/cm 2 corresponding to 10 -5 , 10 -4 , Antimosquito Phenylpropenoids from the Stem..... CHARLES KIHAMPA; MAYUNGA H. H. NKUNYA; COSAM C. JOSEPH; STEPHEN M. MAGESA 10 -3 , 10 -2 and 10 -1 ppm solutions respectively. The screening was done sequentially starting with the lowest dose (0.001%) and ending with the highest one (1%). A test solution (0.5 ml) was dispensed on the right forearm of a volunteer from the wrist to the elbow. The rest of the hand was covered with glove to make it unattractive to the mosquitoes. Acetone (0.5 ml) was dispensed on the left forearm, to act as control. The arms were swapped regularly to eliminate any bias. The control arm was introduced into the cage immediately after releasing the 25 insects and kept there for 3 min. The mosquitoes that had landed on the untreated control arm were recorded. The treated arm was then introduced into the cage and kept there for 3 min. The number of mosquitoes that landed on the treated arm was also recorded. Each concentration was screened using a fresh batch of mosquitoes. After the bioassay of each concentration, the arms were washed with bar soap, rinsed well with tap water and then allowed to dry for 15-20 min, before application of the next dose of the test sample and the percentage protective efficacy (PE) was calculated as