J. Natn. Sci. Coun. Sri Lanka 1996 24(4): 247-252 MOSQUITO-LARVICIDAL ACTIVITY OF CEYLON CITRONELLA (CTYMBOP&b@OiV NmDUS (L.) Rendle) OIL FRACTIONS S. S. RANAWEERA* and K. R. DAYANANDX * Department of Botany, University of Sri Jayewardenepura, Nugegoda. I* Natural Products Development Group, Ceylon Institute of Scientific and Industrial Research (CISIR), Colombo 7. (Received: 26 March 1996; accepted: 01 November 1996) Abstract: Fractional distillation of Ceylon citronella [Cymbopogon nardus (L.) Rendlel oil yielded 13 fractions. Monoterpene hydrocarbon fractions were highly lethai to late 3rd instar Culex quinquefmciatus larvae. The results suggest that myrcene in the monokrpene fract~ons is responsible for the larvicidal activity. Elemol andlor methyl iso-eugenol were identified as active principles responsi- ble for the larvicidal activity in later fractions. The residue after fractional distillation of oil also possessed considerable larvicidal activity. Hydrocarbon fraction and the residue after fractional distillation of Ceylon citronella oil can be used as mosquito-larvicides. Key words: Citronella oil, Culex quinquefasciatus, Cymbopogon nardzs, elemol, essential oil, fractional distillation, larvicide, monoterpenes, methyl isoeugenol, myrcene. INTRODUCTION Most of the phytochemicals with insecticidal activity are biodegradable and less harmful to mammals than synthetic insecticides.' Therefore, there is a possibility of replacing synthetic insecticides with potent bio-insecticides of plant origin. The insecticidal2 and insect repellent2s3 activities of Cymbopogon nardus (L.) Rendle ( known as "Ceylon citronella") oil against Sitroga cerealella (Oliver) have been reported. Mosquito-larvicidal activity of Cymbopogon nardus oil against six mosquito species was previously reported by Rana~eera.~ The present study describes the larvicidal activity of Cymbopogon nardus oil and its fractions against Culex quinquefasciatus mosquitoes. METHODS AND MATERIALS Source of material: Citronella oil was obtained by the steam distillation of Ceylon citronella (Cymbopogon nardus (L.) Rendlel grass obtained from a plantation at Walasmulla, Sri Lanka. Plant specimens were compared with the standard specimens deposited at the National Herbarium, Peradeniya. Fractionation of essential oil: Citronella oil (5kg) was fractionally distilled using Vacum Fractionation Unit (LT Labortechnic Normschliff) equipped with glass column packed with sulzer packing, installed at the Pilot Engineering Division of CISIR; Colombo. 248 S.S. Ranaweera and K.R. Dayanandn Mosquito bioassays: Fifty percent lethal concentration ( LC,,) values of plant extracts for 3rd instar Culex quinquefasciatus Say larvae were determined by the procedure followed by World Health Organization5 with slight modifications. For bioassays, healthy late, 3rd instar C. quinquefasciatus larvae were distributed in batches of 20 in small beakers containing 25ml water. Test dispersions (25ml) were prepared in separate beakers by adding chfferent amounts of the extract to give a series of ten final concentrations ranging from 2.0 to 20.0 mgll, when contents with larvae in small beakers were added to the latter. Tween 80 at 0.1% was used as a surfactant to disperse citronella oil. Mortality counts were taken after 24 h. The negative control contained 0.1% Tween 80 in distilled water. Bioassays were carried out at 2g°C with five replicates. LC,, values were estimated from a probifflog-concentration graph. Gas chromatographic analysis: Essential oil and its fractions were analysed by using a Varian 2700 Gas chromatograph; Column: Carbowax 20M 10% coated on Gaschrom Q ( 3m x 20mm); Injection block temp. 230°C; Detector oven temp. 230°C; Carrier gas (argon) 30mllmin; Samples (0.2pl), with programming from 100 to 230°C at 2OC/min; Recorder, Skimadsu Chromatopak 6A. Identzfzcatzon of fractionated citronella 021: Fractional steam distillation of Cymbopogon nardus oil carried out under different temperature and pressure conditions yielded 13 fractions (Table 1). Gas ckromatographic analysis of oil fractions revealed that the fractions F1-F5, contained varying amounts? of monoterpene hydrocarbons: a-pinene, carnphene, myrcene, limonene, cis-ocimine and y -terpinene. Fractions F6-F13, contained varying amounts of citronellal, linalool, B-caryophyllene, L-borneol, geranyl formate, citronellol, nerol, geraniol, geranyl butyrate, nerolidol, methyl eugenol, elemol, methyl iso-eugenol, farnesol etc. The residue (F13) contained high amounts of elemol, methyliso-eugenol and farnesol. Mosquito-larvicidal activity of oil fractions: The results indicate that fractions, F2, F3, F4 and F13 were highly lethal to mosquito larvae (Table 1). The F2 fraction had the highest larvicidal activity with a LC,, value of 1.7mgA. As a result of the fractional distillation, myrcene content in the fraction (F2) was increased to 8.73% from 0.8% in unfractionated oil. Myrcene also displayed the highest larvicidal activity (LC,,, 0.3mgA) among standard compounds tested (Table 3). Coefficients of correlation calculated between LC,, values obtained for each oil fraction and the essential oil components of fractions are presented in Table 2. The highest negative correlation (r= -0.89) was observed for myrcene (Fl-F5 fractions). Larvicidal activity of fractions, F5-F8 was observed to be low and their LC,, values ranged from 10.0 to 12.6mgA. However, the Mosquito Larvicidal Activity of Citronella Oil fractions, F9-F12 and the residue (F13) after fractional distillation displayed considerable larvicidal activity comparable to hydrocarbon fractions. When final fractions of distillation were considered high negative correlations were observed between ele~~hol (r= -0.953, methyl iso-eugenol (r= -0.95) and LC,, values observed for F9-F13 fractions. Table 1: Activity of citronella oil and its fractions against late 3rd instar Culex quinquefasciatus larvae. Oi Wraction Conditions of distillation LC,, value mgll Temperature, OC Vacuum, m.bar Flask head flask Oil 6.3 Fractions: 60-65 80-85 80-85 85-90 90-95 90-95 105-110 110-115 115-120 120-125 125-130 130-135 135-140 Residue "Unfractionated oil; "F2 fraction was tested for activity after three months of storage at 2g°C. 250 S.S. Ranaweera and K.R. Dayananda Table 2: Coefficients of correlation observed between the essential oil constituents of citronella oil fractions (FP-F5; F10-F13) and their LC,, values against late, 3rd instar Cukx quinquefasciatus larvae. Essential oil component Coefficient of correlation Fractions (Fl-F5) a-Pinene Campherbe Myrcene Limonene cis-Ocimine y -Terpinene Fractions (F10-F13) Nerolidol Methyl eugenol Elemol Methyl iso-eugenol Farnesol Table 3: Effects of standard compounds against late, 3rd instar CuZex quinquefasciatus larvae. Standard compound LC,, Value (mgA) a-Pinene Myrcene y -Terpinene Limonene Linalool Citronella1 Terpineol Nerol Mosquito Larvicidal Activity of Citronella Oil DISCUSSION High negative correlation (r= -0.89) observed between myrcene and LC,, values obtained for hydrocarbon fractions and also high larvicidal activity of the myrcene stanaard indicate that myrcene was the major active prrnciple of hydrocarbon fractions responsibile for the larvicidal activity. As y -terpmene also displayed considerable activity it can be concluded that both myrcene and y -terpinene are responsible for the larvicidal activity of monoterpene hydrocar- bon fractions. Results also showed that fractions, F5- F8 which contained mostly aeetyllisable compounds possessed very low activity when compared to other fractions. When the final fractions, F9-F13 were considered, high negative correlations observed between eiernol (r= -0.95), methyl iso-eugenol (r= -0.95) and LC,, values for fractions indicates the role of elemol and/or methyl iso- eugenol as possible active principles responsible for the iarvicidal activity of these fractions. ) Total amount of total acetylisable compounds ( "total geraniol") in citronella oil determines its market q~ality.~ Two citronella species, Cymbopogon nardus (L.) Rendle ( "Ceylon citronella") and Cymbopogon wizterianus Jowitt ("Java") are distinguished morphologically, anatomically7 and chemically.* One of the striking differences observed by Wijesekerag was the presence of monoterpene hydrocarbons amounting to more than 20% of the oil in C. nardus as against 3- 4% in C. winterianus. The total geraniol content of C. nardus oil is low ( 55-65%) when compared to C, winterianus which contains around 85%. Results showed that a three fold increase in the larvicidal activity of hydrocarbon fractions (F2-F3) resulted from fractionation of oil. Therefore, it is possible to fractionate the hydrocarbon fraction from Ceylon citronella oil and use it as a mosquito larvicide. The separation ofmonoterpene hydrocarbons from Ceylon citronella oil will also improve its perfume quality by increasing its total geraniol (acetylisables) content. In addition to the hydrocarbon fraction, it is also possible to use the residue obtained from the fractional distillation of oil as a mosquito larvicide. However, in order to use the monoterpene hydrocarbon fraction and the residue obtained from the fractionation of "Ceylon" citronella oil as mosquito-larvicides, their bioactivity and stability under field conditions should be investigated. Acknowledgements This' work was funded by a research grant from the University of Sri Jayewardenepura (USJ). We thank W.P.Thilakaratna, Department of Botany, USJ for technical assistance, the technical staff of the Naturdl Products Division, CISIR for gas chromatographic analysis of samples, B.G.D.N.K. de Silva, Department of Zoology, USJ for supplying the mosquito larvae and G.R.N. Kandambi, Department of Botany for laboratory assistance. S.S. Ranaweera and K.R. Dayananda References 1. Marini-Bettolo G.B. (1976). Modern trends in the use of natural products for controlling pests and plant diseases. In: Proceedings of Symposium on Natural Products and the Protection ofPlants, 18-23 October 1976, pp5-14, Pontificiae Academiae ~cientiarum Scripta Varia, Vatican. 2. Krisnarajah S.R. & Ganesalingam V.K. (1981). Laboratory investigations on the repellent and narcotic properties of steam distillates of local plant extracts to Sitoroga cerealella (Oliver). 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