BIOASSAY-GUIDED ISOLATION, CHARACTERISATION AND CYTOTOXICITY OF MOSQUITO REPELLENT COMPOUNDS FROM SELECTED MEDICINAL PLANTS

Abstract

Malaria, a tropical disease caused by Plasmodium parasites and spread by the bites of infected female Anopheles mosquitoes, remains a major public health issue. Mosquito repellents are used as a control measure.Existing synthetic repellents present challengesof high insecticide resistance, allergic reactions, and non-biodegradable residues. This has necessitated the search for alternative repellent compounds from natural sources.This study was designed to evaluate the repellent activity of selected ethnomedicinal plants against wild adult female Anopheles gambiae (s.l.), and characterise isolated bioactive compounds. Dry powdered plant parts of Citrus limon Linn. (seeds), Citrus paradisi Macf. (seeds), Citrus sinensisL.(seeds), Jatropha curcas L.(seeds), Dennettia tripetala G. (fruits), and Afromomum melegueta K. (fruits) were successively extracted with n-hexane (Hex), ethyl acetate (EA) and methanol by cold maceration. Phytochemical screening of the extracts was done using standard methods. The extracts were screened against A. gambiae for their repellent activity using human bait method, whileN,N-diethyl-meta-toluamide(DEET) and acetone were used as positive and negative controls, respectively. Extracts with the highest percentage repellency, calculated using standard formula, were subjected to bioassay-guided fractionation and separation using chromatographic techniques. Isolated compounds were evaluated for cytotoxicity using brine shrimp lethality assay (safety cutoff, LC50> 100µg/mL), and characterised by spectroscopic (IR, 1D and 2D NMR) and mass spectrometric analyses. Repellent activity data were analysed using one-way ANOVA at α0.05. Extraction of the plant materials yielded eighteen extracts. Phytochemical screening of the extracts revealed the presence of alkaloids, flavonoids, tannins, glycoside, saponins, and anthraquinones. The screened extracts, tested at 1.5, 2.5, and 5mg/mL, gave percentage repellency of ≤ 68.1, ≤ 74.9, and ≤ 97.5%, respectively (DEET, 100%). At 5 mg/mL, the percentage repellency of the three most active extracts (C. limon (Hex, 97.5%), D. tripetala (EA, 87.7%), and C. limon (methanol, 86.8%) were not significantly different (α˂0.05). Citrus limon (Hex) fractionation yielded 10 fractions with percentage repellency of ≤ 89.5 %, whileD. tripetala (EA) yielded eleven fractions which were pooled into four (DTH1, 55.7%; DTH2, 60.6%; DTEA, 77.2%, and DTM, 71.1%). n-Hexane-100% and Hex/EA (9:1) fractions of C. limon, DTEA and DTM fractions of D. tripetala showed the most repellency effects of 89.5%, 71.1%, 77.2% and 71.1%, respectively. Chromatographic purification of n-hexane-100%, Hex/EA (9:1) and DTEA gave seven compounds. They were identified as palmitic acid (a), 14-oxotricosanoic acid (b),n-octyl stearate(c), 15-(heptanoyloxy) pentadec-9-enoic acid (d), 6,8-dimethoxy-3-undecyl-1H- [2]benzopyran-1-one (e), 1,2,3-propanetriyl tris(5-eicosenoate) (f), and α-linoleic acid (g) with percentage repellency of 65.9, 63.6, 51.7, 77.9, 75.2, 37.0, and 57.3%, respectively. Cytotoxicity evaluation of compounds a-e gaveLC50ranging from 63.2 to 171.1 µg/mL, with compounds c (106.1 µg/mL) and e (171.1 µg/mL) being adjudged safe. Compoundd showed the highest repellent activity against A. gambiae mosquito. Extracts of Citrus limon and Dennettia tripetalacontain potentially useful mosquito repellent compounds. n-Octyl stearate and 6,8-dimethoxy-3-undecyl-1H-[2]benzopyran-1- one (both from C. limon) may serve as leads for the development of novel bio-degradable and safe mosquito repellent compounds.