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Adulticidal activity of essential oil of Lantana camara leaves against mosquitoes.

Mosquitoes constitute a major public health problem as vectors of serious human diseases like malaria, filariasis, Japanese encephalitis, dengue fever, chikungunya and yellow fever (1) cause substantial 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 vectors (2). Development of insect resistance to synthetic pesticides such as malathion, DDT, deltamethrin and even bio-pesticides such as Bacillus thuringiensis (3,4), high operational cost and environmental pollution have created the need for developing alternative approaches to control vector-borne disease (5). Plants products are emerging as a potential source of mosquito control and among them essential oils have special interest due to their insecticidal properties (6,7).

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 India (8,9). 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 agent (10), roots for the treatment of malaria, rheumatism, and skin rashes (11). Several tri- terpenoids, flavonoids, alkaloids, and glycosides isolated from this plant are known to exert diverse biological activities (9). Extract from the leaves of L. camara possessed larvicidal activity (12) while extract from flowers of the plant showed repellent activity against mosquitoes (13,14).

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 hours (14). 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 [+ or -] 2[degrees]C 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.

Adulticidalbioassay: 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 method (15). 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 [cm.sup.2]) as described earlier (16). 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/[cm.sup.2]) 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 formula (17).

% mortality = % test mortality - % control mortality / 100 - % control mortality X100

[LD.sub.50], [LD.sub.90] with their 95 per cent confidence limits of the oil were determined using Log probit analysis test (18).

Further studies on adulticidal activity of the essential oil was carried out on 0.208 mg/[cm.sup.2] 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/[cm.sup.2] was determined by multiplying the [LD.sub.50] value with a factor of four for testing of adulticidal activity against mosquitoes (18). 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 [cm.sup.2]) and tested for adulticidal activity as described above. Number of mosquitoes knocked down 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 ([KDT.sub.50] and [KDT.sub.90]) values were determined using log probit analysis (19).

Persistence: Persistence of essential oil on 0.208mg/ [cm.sup.2] impregnated test paper stored at 4 and 26 [+ or -] 2[degrees]C was studied at weekly interval for 49 days. Twenty Ae. aegypti female were exposed to the impregnated paper (dose 0.208 mg/[cm.sup.2]) and adulticidal activity was evaluated. Per cent mortality was determined at weekly intervals. After evaluation, the impregnated papers were stored at 4 and 26 [+ or -] 2[degrees]C till further evaluation of adulticidal activity.

Stability: The essential oil was stored at 26 [+ or -] 2[degrees]C 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 [cm.sup.2]) was impregnated with the test fraction at the concentration of 0.208 mg/[cm.sup.2] during the study. Adulticidal activity was evaluated at 26 [+ or -] 2[degrees]C 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 (im in thickness). Helium was used as a carrier gas (1.1 ml/min). GC oven programme comprised of an initial temperature 70[degrees]C (4 min) to 220[degrees]C at 4[degrees]C /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 [micro]l 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 literature (20).

Results

Steam distillation of L. camara leaves yielded 0.4 per cent (w/w) yellow coloured oil with aromatic smell. [LD.sub.50] values of the oil were 0.06, 0.05, 0.05, 0.05 and 0.06 mg/[cm.sup.2] while [LD.sub.90] values were 0.10, 0.10, 0.09, 0.09 and 0.10 mg/[cm.sup.2] 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/[cm.sup.2] impregnated papers and results were compared with 0.05 per cent deltamethrin paper. [KDT.sub.50] and [KDT.sub.90] 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. [KDT.sub.50] and [KDT.sub.90] 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 4[degrees]C showed more adulticidal activity for longer period of time than paper stored at 26 [+ or -] 2[degrees]C (Table III).

Hundred per cent mortality of An. stephensi was observed during the storage period at 26 [+ or -] 2[degrees]C. [KDT.sub.50] value was 11 and 13 min at 0 day and 6 months storage period respectively against An. stephensi (Table IY).

The essential oil stored in closed vial for six months at room temperature also showed 100 per cent mortality Number of each replicate 3; Storage temperature 26 [+ or -] 2[degrees]C 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%), [alpha]-humelene (8.22%), and germacrene-D (7.41%) were 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 workers (3,21,22). L. camara is reported to possess insecticidal activity against stored grain pest, vegetable crops pest, mosquito larvae and antifungal, repellent, and other biological activities (9-14). 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 [KDT.sub.50] value of 0.44 per cent (23).

Yang and co-workers (3) have evaluated adulticidal activity of five essential oils against Cx. quinquefasciatus. Ethanol extract of Apium graveolence exhibited adulticidal activity against Ae. aegypti with [LD.sub.50] and [LD.sub.90] values of 6.6 mg/[cm.sup.2] and 66.4 mg/[cm.sup.2], (24). Essential oil of L. camara leaves showed more adulticidal activity against mosquitoes compared to earlier reports (3,23,24) and almost all mosquitoes showed signs of paralysis at exposure to 0.208 mg/[cm.sup.2] 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 death (24).

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-workers (22) 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-workers (25) have identified germacrene-D (15.85%), [beta]-caryophyllene (12.35%), [alpha]-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%), [alpha]-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 earlier (25-28).

Some of the major components of essential oil of L. camara identified in the present study have been reported to exhibit insecticidal properties. [beta]-caryophyllene isolated from the plant Thymus capitatus showed good adulticidal activity against Cx. pipiens (21). Essential oil of Ocimum americanus and 0. ratissium contains caryophyllene as main constituent possessed larvicidal activity against Ae. aegypti (29). Farnesol and germacrene-D have been reported to have pesticidal activity (30). Eucalyptol is the main constituent (15-57%) of essential oil of eucalyptus, which exhibited pesticidal activity (31).

Essential oils are usually safe to humans and the environment (3). 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 compounds (32,33).

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.

Received August 1, 2008

References

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

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

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
Table I. Adulticidal activity of essential oil of Lantana
camara against mosquitoes

Mosquito species Adulticidal activity (mg/[cm.sup.2])

 [LD.sub.50] 95% CL

Ae. aegypti 0.06 [+ or -] 0.01 0.05-0.07
Cx. quinquefasciatus 0.05 [+ or -] 0.01 0.04-0.06
An. culicifacies 0.05 [+ or -] 0.01 0.04-0.06
An. fluviatilis 0.05 [+ or -] 0.01 0.04-0.06
An. stephensi 0.06 [+ or -] 0.01 0.05-0.07

Mosquito species Adulticidal activity (mg/[cm.sup.2])

 [LD.sub.90] 95% CL

Ae. aegypti 0.10 [+ or -] 0.03 0.06-0.14
Cx. quinquefasciatus 0.10 [+ or -] 0.02 0.07-0.12
An. culicifacies 0.09 [+ or -] 0.02 0.07-0.11
An. fluviatilis 0.09 [+ or -] 0.01 0.08-0.10
An. stephensi 0.10 [+ or -] 0.02 0.08-0.12

CL, Confidence limit; Mosquitoes was exposed for 1 h and mortality
was recorded at 24 h recovery period; Values are mean [+ or -] SD
(No. of replicates = 5)

Table II. Adulticidal activity of essential oil of Lantana camara :
against mosquitoes on 0.208 mg/[cm.sup.2] impregnated paper

Test materials Mosquito species Knockdown time *
 (Mean [+ or -] SD) min

 KDT50 KDT90

Essential oil Ae. aegypti 20 [+ or -] 2 35 [+ or -] 3
 Cx. quinquefasciatus 18 [+ or -] 1 28 [+ or -] 2
 An. culicifacies 15 [+ or -] 2 25 [+ or -] 2
 An. fluviatilis 12 [+ or -] 2 18 [+ or -] 2
 An. stephensi 14 [+ or -] 2 23 [+ or -] 3

 Ae. aegypti 11 [+ or -] 1 16 [+ or -] 2
Deltamethrin Cx. quinquefasciatus 10 [+ or -] 2 25 [+ or -] 3
 An. culicifacies 10 [+ or -] 2 18 [+ or -] 2
 An. fluviatilis 9 [+ or -] 2 15 [+ or -] 3
 An. stephensi 10 [+ or -] 2 17 [+ or -] 2

Test materials Mosquito species %
 % knock- mortality at 24 h
 down
 in 1 h

Essential oil Ae. aegypti 100 93.3 [+ or -] 2.5
 Cx. quinquefasciatus 100 95.2 [+ or -] 3.0
 An. culicifacies 100 100
 An. fluviatilis 100 100
 An. stephensi 100 100

 Ae. aegypti 100 100
Deltamethrin Cx. quinquefasciatus 100 100
 An. culicifacies 100 100
 An. fluviatilis 100 100
 An. stephensi 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/[cm.sup.2] impregnated
paper

 Per cent mortality of Ae .aegypti *
Days of
observation Paper stored at 40C Paper stored at 26 [+ or -] 20C
 (Mean [+ or -] SD) (Mean [+ or -] SD)

0 100 100
7 93.5 [+ or -] 4.0 86.0 [+ or -] 5.0
14 90.0. [+ or -] 6.0 63.5 [+ or -] 4.0
21 86.4 [+ or -] 4.5 46.4 [+ or -] 3.0
28 70.0 [+ or -] 3.2 40.0 [+ or -] 3.5
35 65.6 [+ or -] 4.8 38.2 [+ or -] 5.0
42 56.0 [+ or -] 5.8 23.0 [+ or -] 4.0
49 44.8 [+ or -] 5.8 13.0 [+ or -] 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.
stephensi

Months of Knockdown Knockdown
extraction time (Mean [+ or -] SD) in
 min hour 1

 [KDT.sub.50] [KDT.sub.90] exposure (%)

0 11 [+ or -] 1 16 [+ or -] 2 100
1 11 [+ or -] 1 17 [+ or -] 1 100
3 12 [+ or -] 2 17 [+ or -] 3 100
6 13 [+ or -] 1 18 [+ or -] 3 100

Months of Mortality at
extraction 24 h
 recovery
 period (%)

0 100
1 100
3 100
6 100
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Author:Dua, V.K.; Pandey, A.C.; Dash, A.P.
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Mar 1, 2010
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