Indian J Med Res 131, March 2010, pp 434-439

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Mosquitoes constitute a major public health problem as vectors of serious human diseases like malaria, filariasis, Japanese encephalitis, dengue fever, chikungunya and yellow fever1 cause substantial

Adulticidal activity of essential oil of Lantana camara leaves against mosquitoes

V.K. Dua, A.C. Pandey & A.P. Dash*

National Institute of Malaria Research (ICMR), Field Unit, Haridwar &*National Institute of Malaria Research New Delhi, India

Received August 1, 2008

Background & objectives: Development of insect resistance to synthetic pesticides, high operational cost and environmental pollution have created the need for developing alternative approaches to control vector-borne diseases. In the present study we have investigated the insecticidal activity of essential oil isolated from the leaves of Lantana camara against mosquito vectors.Methods: Essential oil was isolated from the leaves of L. camara using hydro-distillation method. Bioassay test was carried out by WHO method for determination of adulticidal activity against mosquitoes. Different compounds were identified by gas chromatography-mass spectrometry analysis.Results: LD50 values of the oil were 0.06, 0.05, 0.05, 0.05 and 0.06 mg/cm2 while LD90 values were 0.10, 0.10, 0.09, 0.09 and 0.10 mg/cm2 against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluvialitis and An. stephensi respectively. KDT50 of the oil were 20, 18, 15, 12, and 14 min and KDT90 values were 35, 28 25, 18, 23 min against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluviatilis and An. stephensi, respectively on 0.208 mg/cm2 impregnated paper. Studies on persistence of essential oil of L. camara on impregnated paper revealed that it has more adulticidal activity for longer period at low storage temperature. Gas chromatographic-mass spectrometric analysis of essential oil showed 45 peaks. Caryophyllene (16.37%), eucalyptol (10.75%), α-humelene (8.22%) and germacrene (7.41%) were present in major amounts and contributed 42.75 per cent of the total constituents.Interpretation &conclusion: Essential oil from the leaves of L. camara possesses adulticidal activity against different mosquito species that could be utilized for development of oil-based insecticide as supplementary to synthetic insecticides.

Key words Adulticidal activity - essential oil - gas chromatography-mass spectrometry analysis - mosquitoes

mortality and morbidity among people living in tropical and sub tropical zones. Synthetic pesticides have been extensively used for mosquito control by either killing, preventing adult mosquitoes to bite human beings or

by killing mosquito larvae at the breeding sites of the vectors2. Development of insect resistance to synthetic pesticides such as malathion, DDT, deltamethrin and even bio-pesticides such as Bacillus thuringiensis3,4, high operational cost and environmental pollution have created the need for developing alternative approaches to control vector-borne disease5. Plants products are emerging as a potential source of mosquito control and among them essential oils have special interest due to their insecticidal properties6,7.

Lantana camara Linn. (Verbenaceae) is a hardy, evergreen, straggling shrub with characteristic odour, it grows up to 3 m height, with or without minute prickles on the branches. It is a perennial shrub found growing up to 2000 m altitude in tropical, sub tropical and temperate parts of the world. The plant is spread widely over Himachal Pradesh, Uttarakhand, Uttar Pradesh and north-eastern States of India8,9. All parts of this plant have been used traditionally for several ailments throughout the world. The leaves of this plant were used as an antitumeral, antibacterial, and antihypertensive agent10, roots for the treatment of malaria, rheumatism, and skin rashes11. Several tri- terpenoids, flavonoids, alkaloids, and glycosides isolated from this plant are known to exert diverse biological activities9. Extract from the leaves of L. camara possessed larvicidal activity12 while extract from flowers of the plant showed repellent activity against mosquitoes13,14.

In the present study we have investigated the adulticidal activity of the oil extracted from the leaves of L. camara against Aedes aegypti Linnaeus, Culex, quinquefasciatus Say, Anopheles culicifacies Giles, An. fluviatilis James and An. stephensi Liston mosquitoes.

Material & Methods

Sample preparation: Leaves of L. camara were collected from the field in district Hardwar, India. The leaves were washed with distilled water to remove dust particles and essential oil from the leaves was obtained by simultaneous hydro-distillation followed by the extraction of the distillate using n-hexane as organic phase for five hours14. Solvent was removed by vortex evaporator.

Test mosquitoes: Laboratory colonies of different species of mosquitoes were reared continuously for several generations in a laboratory free of exposure to pathogens and insecticides. They were maintained at 26 ± 20C and 60-80 per cent relative humidity in the insectory of the National Institute of Malaria Research, Field Unit, Haridwar. Larvae were fed on a mixture of

commercial dog biscuit and yeast powder (3:2 ratio) as nutrient. Adult mosquitoes were reared in humidified cages and fed with 10 per cent glucose. Female mosquitoes were periodically blood-fed on rabbits for egg production.

Adulticidal bioassay: Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluviatilis and An. stephensi mosquitoes were selected for the testing of adulticidal activities. Adulticidal bioassay was performed by WHO method15. Appropriate concentrations of the essential oil of L. camara were dissolved in 2.5 ml of acetone and applied on Whatman no. 1 filter papers (size 12 x15 cm2) as described earlier16. Control papers were treated with acetone under similar conditions. Adulticidal activity of the oil was evaluated at seven concentrations (0.01, 0.03, 0.07, 0.13, 0.20, 0.27 and 0.37 mg/cm2) to produce a range of mortality from 10 to 100 per cent along with control.

Twenty female mosquitoes (2-5 days old glucose fed, blood starved) were collected and gently transferred into a plastic holding tube. The mosquitoes were allowed to acclimatize in the holding tube for 1 h and then exposed to test paper for 1 h. At the end of exposure period, the mosquitoes were transferred back to the holding tube and kept 24 h for recovery period. A pad of cotton soaked with 10 per cent glucose solution was placed on the mesh screen. Mortality of mosquitoes was determined at the end of 24 h recovery period. Per cent mortality was corrected by using of Abbott’s formula17.

% test mortality - % control mortality% mortality = X100

100 - % control mortality

LD50, LD90 with their 95 per cent confidence limits of the oil were determined using Log probit analysis test18.

Further studies on adulticidal activity of the essential oil was carried out on 0.208 mg/cm2

impregnated paper along with 0.05 per cent deltamethrin impregnated paper used as positive control against female Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluviatilis and An. stephensi. The application dose of 0.208 mg/cm2 was determined by multiplying the LD50 value with a factor of four for testing of adulticidal activity against mosquitoes18. Essential oil (37.5 mg) was dissolved in 2.5 ml acetone (1.5% w/v) and applied on Whatman no. 1 filter paper of size (12x15 cm2) and tested for adulticidal activity as described above. Number of mosquitoes knocked down

DUA et al : MOSqUITOCIDAL ACTIvITy Of L. CAMARA ESSENTIAL oIL 435

in the exposure tube was recorded at 3 min interval period till the last mosquito was knocked down. At the end of exposure period, mosquitoes were transferred to holding tube and kept for 24 h. Knock down time (KDT50 and KDT90 ) values were determined using log probit analysis19.

Persistence: Persistence of essential oil on 0.208mg/cm2 impregnated test paper stored at 4 and 26 ± 20C was studied at weekly interval for 49 days. Twenty Ae. aegypti female were exposed to the impregnated paper (dose 0.208 mg/cm2) and adulticidal activity was evaluated. Per cent mortality was determined at weekly intervals. After evaluation, the impregnated papers were stored at 4 and 26 ± 20C till further evaluation of adulticidal activity.

Stability: The essential oil was stored at 26 ± 20C in closed vial up to six months and stability of the fraction was determined at 0, and 1, 3 and 6 months time intervals. Whatman no. 1 filter paper (size 12 x 15 cm2) was impregnated with the test fraction at the concentration of 0.208 mg/cm2 during the study. Adulticidal activity was evaluated at 26 ± 20C and 60 to 80 per cent relative humidity.

Gas chromatographic-mass spectrometric (GC-MS) analysis: The GC-MS analysis for the separation and identification of the essential oil was carried using a Shimadzu GC-2010 gas chromatograph coupled to a qP 2010 mass selective detector (Simadzu Corporation, Kyoto, Japan) with capillary column BP-20 (30 m in length, 0.25 mm internal diam. and 0.25 µm in thickness). Helium was used as a carrier gas (1.1 ml/min). GC oven programme comprised of an initial temperature 700C (4 min) to 2200C at 40C /min and held at the final temperatures for 5 min. The essential oil of L. camara was diluted in 1.0 ml dichloromethane and 0.25 µl of the resulting solution was injected for analysis. The identification of the compounds was

performed using a mass spectral data base search (NIST, WELY and SZTERP software library of mass spectra) and spectra reported in literature20.

Results

Steam distillation of L. camara leaves yielded 0.4 per cent (w/w) yellow coloured oil with aromatic smell. LD50 values of the oil were 0.06, 0.05, 0.05, 0.05 and 0.06 mg/cm2 while LD90 values were 0.10, 0.10, 0.09, 0.09 and 0.10 mg/cm2 against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluvialitis and An. stephensi respectively (Table I).

The essential oil was further investigated against different mosquitoes species on 0.208 mg/cm2

impregnated papers and results were compared with 0.05 per cent deltamethrin paper. KDT50 and KDT90 values of the essential oil were 20, 18, 15, 12 and 14 min and 35, 28, 25, 18 and 23 min against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluvialitis and An. stephensi with their per cent mortality of 93.3, 95.2,100, 100 and 100 per cent respectively. KDT50 and KDT90 values of 0.05 per cent deltamethrin impregnated papers were 11, 10, 10, 9 and 10 and 16, 25, 18, 15 and 17 against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluviatilis and An. stephensi respectively with 100 per cent mortality (Table II).

Mortality was 86.4 and 46.4 per cent respectively at week three, while mortality at week seven was 44.8 and 13.0 per cent respectively. Papers stored at 40C showed more adulticidal activity for longer period of time than paper stored at 26 ± 20C (Table III).

Hundred per cent mortality of An. stephensi was observed during the storage period at 26 ± 20C. KDT50 value was 11 and 13 min at 0 day and 6 months storage period respectively against An. stephensi (Table Iv).

The essential oil stored in closed vial for six months at room temperature also showed 100 per cent mortality

Table I. Adulticidal activity of essential oil of Lantana camara against mosquitoesMosquito species Adulticidal activity

(mg/cm2)LD50 95% CL LD90 95% CL

Ae. aegypti 0.06 ± 0.01 0.05 - 0.07 0.10 ± 0.03 0.06 - 0.14Cx. quinquefasciatus 0.05 ± 0.01 0.04 - 0.06 0.10 ± 0.02 0.07 - 0.12An. culicifacies 0.05 ± 0.01 0.04 - 0.06 0.09 ± 0.02 0.07 - 0.11An. fluviatilis 0.05 ± 0.01 0.04 - 0.06 0.09 ± 0.01 0.08 - 0.10An. stephensi 0.06 ± 0.01 0.05 - 0.07 0.10 ± 0.02 0.08 - 0.12CL, Confidence limit; Mosquitoes was exposed for 1 h and mortality was recorded at 24 h recovery period; Values are mean ± SD (No. of replicates = 5)

436 INDIAN J MED RES, MARCH 2010

present in major amounts and contributed 42.75 per cent of the total. Farnesol (5.32%), bicyclogermacrene (3.65%), sesquilavandulol (3.48%), caryophyllene oxide (2.98%), 1-H-cycloprop azulen-7-ol, decahydro-1, 1, 7-trimethyl-4-methylene (2.95%), 3-cyclohexene-1ol, 4-mehtyl-1-(1-mehtylehtyl) (2.90%), davanone (2.88%), contributed in the range of 2.88-5.32 per cent amounting to 24.16 per cent. Twelve compounds identified in minor amounts with the range of 1.0-2.45 per cent and contributed 15.78 per cent while another 22 compounds were present in traces (<1.0) and their total contribution was 17.31 per cent.

Discussion

Insecticidal properties of essential oils against adult mosquitoes have been reported by many workers3,21,22. L. camara is reported to possess insecticidal activity against stored grain pest, vegetable crops pest, mosquito larvae and antifungal, repellent, and other biological activities9-14. In the present study the oil extracted from L. camara leaves showed adulticidal activity against different mosquitoes. Adulticidal activity of the oil was highest against An. fluviatilis followed by An. culicifacies, Cx. quinquefasciatus, An. stephensi and Ae. aegypti. Cedar wood oils possessed potential insecticidal activity against An. stephensi with KDT50 value of 0.44 per cent23.

Yang and co-workers3 have evaluated adulticidal activity of five essential oils against Cx. quinquefasciatus. Ethanol extract of Apium graveolence exhibited adulticidal activity against Ae. aegypti with LD50 and LD90 values of 6.6 mg/cm2 and 66.4 mg/cm2,24. Essential oil of L. camara leaves showed more adulticidal activity

Table II. Adulticidal activity of essential oil of Lantana camara against mosquitoes on 0.208 mg/cm2 impregnated paperTest materials Mosquito species Knockdown time*

(Mean ± SD) min% knock-

downin 1 h

%mortality at 24 h

KDT50 KDT90

Essential oil Ae. aegypti 20 ± 2 35 ± 3 100 93.3 ± 2.5Cx. quinquefasciatus 18 ± 1 28 ± 2 100 95.2 ± 3.0An. culicifacies 15 ± 2 25 ± 2 100 100An. fluviatilis 12 ± 2 18 ± 2 100 100An. stephensi 14 ± 2 23 ± 3 100 100

DeltamethrinAe. aegypti 11 ± 1 16 ± 2 100 100Cx. quinquefasciatus 10 ± 2 25 ± 3 100 100An. culicifacies 10 ± 2 18 ± 2 100 100An. fluviatilis 9 ± 2 15 ± 3 100 100An. stephensi 10 ± 2 17 ± 2 100 100

*Number of each replicates: 5; Mosquitoes were exposed for 1 h and mortality was recorded at 24 h recovery period

Table III. Persistence of adulticidal activity of essential oil of L. camara against Aedes aegypti on 0.208 mg/cm2 impregnated paperDays of observation

Per cent mortality of Ae .aegypti*

Paper stored at 40C(Mean ± SD)

Paper stored at 26 ± 20C(Mean ± SD)

0 100 1007 93.5 ± 4.0 86.0 ± 5.014 90.0. ± 6.0 63.5 ± 4.021 86.4 ± 4.5 46.4 ± 3.028 70.0 ± 3.2 40.0 ± 3.535 65.6 ± 4.8 38.2 ± 5.042 56.0 ± 5.8 23.0 ± 4.049 44.8 ± 5.8 13.0 ± 4.5*Number of each replicate: 5; Mosquitoes were exposed for 1 h and mortality was recorded at 24 h recovery period

Table IV. Stability test of essential oil of L. camara against An. stephensiMonths ofextraction

Knockdowntime (Mean ± SD)

min

Knockdown in

hour 1 exposure

(%)

Mortality at 24 h

recovery period

(%)KDT50 KDT90

0136

11 ± 111 ± 112 ± 213 ± 1

16 ± 217 ± 117 ± 318 ± 3

100100100100

100100100100

Number of each replicate 3; Storage temperature 26 ± 20C

of An. stephensi which implies that the essential oil was stable at room temperature.

GC-MS analysis of the essential oil showed 45 peaks. Caryophyllene (16.37%), eucalyptol (10.75%), α-humelene (8.22%), and germacrene-D (7.41%) were

DUA et al : MOSqUITOCIDAL ACTIvITy Of L. CAMARA ESSENTIAL oIL 437

against mosquitoes compared to earlier reports3,23,24 and almost all mosquitoes showed signs of paralysis at exposure to 0.208 mg/cm2 impregnated paper within 10 to 15 min, and at the end of 1 h exposure all mosquitoes become inactive. At 24 h holding period per cent mortality ranged from 93 to 100 per cent against all test mosquitoes. The symptoms observed in adult mosquitoes were similar to those caused by nerve poisons i.e., excitation, convulsion, paralysis and death24.

Studies on persistence of essential oil of L. camara on impregnated paper revealed that it possessed more adulticidal activity for longer period stored at low temperature. This may be due to low volatile nature of oil. Panella and co-workers22 evaluated adulticidal activity of 15 natural products isolated from essential oils of yellow cedar against Ae. aegypti, five of which demonstrated residual activity for up to six weeks. Stability test of essential oil against An. stephensi during six months observation revealed that the oil was stable at room temperature.

Sundufu and co-workers25 have identified germacrene-D (15.85%), β-caryophyllene (12.35%), α-humelene (9.31%) and germecrene (6.19%) as major constituents in the essential oils of leaves of Lantana camara in South China. In the present study we have identified caryophyllene (16.37), eucalyptol (10.75%), α-humelene (8.22%) and germecrene (7.11%) as major constituents. Chemical variations in the composition of the essential oil of L. camara has been also reported earlier25-28.

Some of the major components of essential oil of L. camara identified in the present study have been reported to exhibit insecticidal properties. β-caryophyllene isolated from the plant Thymus capitatus showed good adulticidal activity against Cx. pipiens21. Essential oil of Ocimum americanus and 0. ratissium contains caryophyllene as main constituent possessed larvicidal activity against Ae. aegypti29. Farnesol and germacrene-D have been reported to have pesticidal activity30. Eucalyptol is the main constituent (15-57%) of essential oil of eucalyptus, which exhibited pesticidal activity31.

Essential oils are usually safe to humans and the environment3. Insecticides of plant origin are expected to be target selective and biodegradable leading to fewer harmful effects on human and other animals and are environmentally safe as compared to synthetic compounds32,33.

In the present investigation, essential oil obtained from the leaves of L. camara showed adulticidal activity

against important vectors of malaria (An. culicifacies, An. stephensi), filariasis (Cx. quinquefasciatus) dengue, dengue haemorrhagic fever, yellow fever and chikungunya (Ae. aegypti). The present finding may be utilized for the development of plant-based pesticides as supplementary to synthetic insecticides.

Acknowledgment The authors acknowledge the financial support from the Integrated Disease Vector Control of Malaria Project to perform this study.

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Reprint requests: Dr V.K. Dua, Scientist F & officer-in-Charge, National Institute of Malaria Research, Industrial Malaria Unit Health Centre, Sector III, BHEL, Ranipur, Haridwar 249 403, Uttaranchal, India e-mail: vkdua51@gmail.com

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