Lavandula angustifolia Essential Oil Phyto-Compounds as Leads to Potential Mosquitocides

: Mosquitocidal activities of Lavandula angustifolia (L. angustifolia) different concentration of essential oil (EO) and its major phytocompounds (MPCs) tested against three important human vector mosquitoes (HVMs). The quantitative analyses of EO phyto-chemical compositions (PCCs) were analyzed by using Gas Chromatography-Mass Spectrometry (GC-MS). The HVMswere exposed to various concentrations of EO and its MPCs tested under laboratory condition by using standard protocols. Vector mortalities were subjected to log-probit analysis. Chemical compositions (CCs) of 47 compounds were identified and the MPCs of EO were Terpinen (38.0339%) followed by Linalool (34.4992%), Caryophyllene (6.1480%), Octanone (2.3906%) and Camphene (2.0989%). The maximum larval mortality was found in Linalool against the larvae of Aedes aegypti (Ae. aegypti) followed by Anopheles stephensi (An. stephensi) and Culexquinquefasciatus (Cx. quinquefasciatus) with the LC 50 values were 36.26, 36.81 and 37.49 ppm respectively. Ovicidal activity of the Linalool found most effective than other compounds. These results are clearly suggests that EO and its MPCs have the potential to be used as an ideal ecofriendly approach, traditional method for control of HVMs and this is the first report on the mosquitocidal activities of L. angustifolia EO of MPCs.


Introduction
Mosquito and mosquito-borne diseases (MBDs) are successfully spreading entire world with a disproportionate effect on children and adolescents, which are more responsible for significant global morbidity and mortality.Mosquitoes have been regarded as significant vectors for transmission of several diseases which bites cause skin irritation and allergic responses to humans and other blood yielding vertebrates.WHO has declared the mosquitoes as "number 1 enemy to public".MBDs are predominant in more than 100 countries which are worldwide infecting 70 million people annually and 40 million people suffered only in India [1][2][3][4][5].Aedes aegypti (Ae.aegypti) is a very serious day biting vector which are proliferating fresh water bodies in and around home land.Recently estimated more than 50 million people are threatening worldwide by dengue virus.These mosquitoes are also known to spread chikunguniya, dengue and yellow fever in Central and South America, West Africa and many states in India [6][7][8].Anopheles stephensi (An.stephensi) is the principal vector of malaria and one of the most predominant diseases in the tropical world which disturbs 350 to 500 million peoples and kills more than 1 million infants and young children annually [9][10].Culex quinquefasciatus (Cx.quinquefasciatus) vector of filariasis which created major public health hazard and challenging socioeconomic problem in many parts of tropical countries [11][12][13].HVMs control has been pushed to enter a new dispute to the public health practitioners as a result of amplified threat of synthetic chemicals insecticides grows rapidly resistance among the vector mosquitoes.Chemicals insecticides are non-biodegradable and causing many side effects to all living organisms [14,15].
Moreover, mosquito coils, mat, liquid vaporizer and other repellent agents containing synthetic pyrethroids and other organophosphorus compounds cause so many side effects, such as breathing problem, asthma, itching, headache, eye irritation and sneezing to the users [16].The recent negative impacts of chemical insecticides should be removed urgently and search efforts towards the development of new environment friendly, naturally available vector control methods by using realistic mediators.In search of new vector control approach, science has increased the investigations to plants in recent decades, so the plant kingdom is receiving renewed consideration as mosquitocides [17].Recently, most of the researcher would like to search the new environmental friendly pesticides practices therefore working on phytochemical derivative pesticides.India is one of the vast plant diversity country as well as medicinal plants that have been used in traditional medicine for several thousand years and nearly 8000 plants species are documented for medicinal and insecticidal properties around 50% of all the medicinal plants are present in India [18,19].Phytochemicals are environmental and former friendly, target-specificity, nondevelopment of resistance to pest, reduced number of applications, higher acceptability, suitability for rural areas, low cost, biodegradable, easy preparation and universally accepted.Botanicals pesticides are used for alternative to synthetic insecticides and phyto-compounds have been projected as a tool in future for pest control agents which are shown to function as general toxicant [20,16].Phytocompounds are extracted from different parts of the variety of plants.Lamiaceae family plants are good odorous, medicinal properties flora and it consists of more than 252 genus and 7000 species.Lamiaceae are most abundant in Asian continents.It has been extremely cultivated in India, China, Pakistan, Malaysia and Indonesia for using of essential oil production [21].In view of the recently increased interest in developing plant origin insecticides as an alternative to synthetic chemical insecticide for controlling HVMs, this present investigation was undertaken to assess the larvicidal and ovicidal potential of L. angustifolia EO MPCs against the medically important HVMs.

Test Organisms
The mosquitoes were separately reared in laboratory, Department of Zoology, Govt.Arts College, Nandanam, Chennai -35, Tamilnadu, India.The larvae were fed 3:1 ratio of dog biscuits and yeast powder.Adults were provided with 10% sucrose solution with honey and one week old chick for blood meal.Mosquitoes were held at (28±2)°C, 70-85% relative humidity (RH), with a photo period of 14 h light, 10 h dark.

Plant Material and Essential Oil Extraction
The fresh aerial parts (the collected plant parts were keenly watched and washed with dechlorinated water for 15min) of L. angustifolia were collected from a thick forest region of Malapuram District, Kerala, India.The plant materials were collected in the month of February 2018, which were allowed to more than 15 days for air and shade dried in room temperature and ground to a fine powder with the help of electric blender and sieved through kitchen strainer.Plant powder 500g was sequentially extracted through hydrodistilled in a Clevenger apparatus for 6 h.The distilled oil was dried, stored at refrigerator under 4°C and packed in air lock amphour bottle until further bioassay test.At the time of plants collection, two pressed voucher herbarium (NGAC: 3684) specimens were prepared and identified with the help of Plant Taxonomist, Department of Botany, Govt.Arts College, Nandanam, Chennai.

Gas Chromatography -Mass Spectrometry Analysis
GC-MS analysis of the L. angustifolia EOwas performed using a Perkin Elmer GC Claurus 500 system and Gas Chromatograph interfaced to a Mass Spectrometer (GCMS) equipped with an Elite-5MS (5% Diphenyl/95% Dimethyl poly siloxane), 30 x 0.25mm x 0.25m df.For GC-MS detection, an electron ionization system with ionization energy of 70 eV was used.Helium gas (99.9%) was used as a carrier gas at a constant flow rate of 1 ml/min.and an injection volume of 2 micro liter was employed (Split ratio of 1:10).Injector temperature of 250°C is maintained and Ionsource temperature was 280°C.The oven temperature was programmed from 110°C (isothermal for 2 min), with an increase of 10°C /min to 200°C, then 5°C /min to 280°C, ending with isothermal for 9 min.at 280°C.Mass spectra were taken at 70 eV; a scan interval of 0.5 seconds and fragments from 45 to 450 Da. were measured.Total GC running time was 36 min and the relative percentage amount of each component was calculated by comparing its average peak area to the total areas with Software adopted to handle mass spectra and Chromatogram was Turbo mass ver.5.2.0.

Larvicidal Activity
The larvicidal activity of EO MPCs was evaluated as per the globally acceptable protocol and previously described [22].The larvicidal activity was determined, based on the wide and narrow range tests.The selected EO MPCs tested ranging from 10 to 70ppm which prepared and they were tested against the freshly moulted (0-6 hrs) third instar larvae of selected mosquitoes.The EO MPCs were dissolved in 1 ml DMSO and then diluted in 249 ml of dechlorinated tap water to obtain each of the desired concentrations.The control was prepared using 1ml of DMSO in 249 ml of dechlorinated water.The twenty five third instar larvae were Leads to Potential Mosquitocides (previously monitored: disease free, hale and healthy and uniform sized) introduced carefully in 500ml of transparent, heat resistant and unbreakable plastic cups containing 250 ml of aqueous medium (249 ml of dechlorinated water +1ml of DMSO) and the required amount of EO MPC was added.The larval mortality was keenly observed every 2 h interval and finally recorded after 24 h of post treatment.The entire bioassay experiment five replicates were maintained and the study of LC 50 as well as LC 90 value was calculated by using probit analysis [23].

Ovicidal Activity
The ovicidal activity was slightly modified and followed the method of Su and Mulla [24].The eggs/egg rafts of selected mosquitoes were collected from vector control laboratory, Department of Zoology, Govt.Arts College, Nandanam, Chennai.The various concentrations EO MPCs ranging from 10 to 70 ppm and before treatment, the eggs/egg rafts of selected mosquitoes were counted individually with the help of hand lens.Freshly hatched eggs (100nos) were exposed to each concentration of chemical compositions until they hatched or died.Eggs exposed to DMSO in water served as control.After treatment, the eggs from each concentration were individually transferred into distilled water cups for hatching assessment after counting the eggs under a microscope.Each test was replicated five times.The hatchability was assessed 48 h post treatment.

Statistical Analysis
The average larval mortality data were subjected to probit analysis for calculating LC 50 , LC 90 , and other statistics at 95% confidence limits of upper confidence limit (UCL) and lower confidence limit (LCL), and chi-square values were calculated using the SPSS 12.0 version (Statistical Package of Social Sciences) software.Results with p<0.05 were considered to be statistically significant.

Chemical Analysis of L. angustifolia
The CCs of L. angustifolia aerial parts the retention indices and the percentage of the individual components are summarized in table 1

Larvicidal Activity of L. angustifolia
The results of the larvicidal activity of L. angustifolia EO MPCs against the third instar larvae of three important HVMsAe.aegypti,An.stephensi and Cx.quinquefasciatus are presented in table 2. Among five compounds tested, the maximum larvicidal potential was observed in Linalool against Ae.aegyptithan An.stephensi and Cx.quinquefasciatus with the LC 50 values were 36.26,36.81 and 37.49 ppm respectively.The chi-square values are statically significant at p≤0.05 level.The chi-square values in the bioassays indicated probably the heterogeneity of the test population.

Ovicidal activity of L. angustifolia
The % of egg hatchability of Ae.aegypti, An. stephensi and Cx.quinquefasciatus were tested with five different EO MPCs concentrations and the results are listed in table 3. The percent hatchability was inversely proportional to the concentrations of EO MPCs and directly proportional to the eggs.Among the five different EO MPCstested against Ae.aegypti, An. stephensi and Cx.quinquefasciatus, the Linalool compound of EO exerted zero hatchability (100% mortality) at 40, 50, 60 and 70 ppm, respectively.The MPC Linalool compound found most effective than any other compounds against eggs of three HVMs and control eggs showed the 100% hatchability.

Discussion
Plant products have been used as traditionally by human communities in many parts of the world against the vectors and the different species of insects.Phytochemicals explored from plant sources which has good mosquitocidal agents and transmission of MBDs at the individual as well as at the community level.Our results showed that the different compounds of L. angustifolia EO have significant larvicidal and ovicidal activities against Ae.aegypti, An. stephensi and Cx.Quinquefasciatus mosquitoes.The finding of the present investigation is comparable with earlier reports the larvicidal activities of the four MPCs D-terpinene, linalool, borneol and germacrene D tested against larvae of HVMs [25 -28].Similarly, Isoflavonoids from tubers of N. mitis had a larvicidal effect on malaria and filariasis vectors [29].Saponin isolated from A. aspera against Ae.Aegypti and Cx.quinquefasciatus [30].The presence of lantadene triterpenoids and furano naphtha quinones phytocompounds in Lantana species has the mosquito larvicidal properties [31].A study on larvicidal effects of B. mollis EO against Cx.quinquefasciatus [32].The EOs of L. turbinataand L. polystachyaagainst the larvae of HVM against Cx.quinquefasciatus [33].The oleic acid isolated from T. javanicumshowed potential mosquitocidal agent tested against Ae.aegypti and Cx.Quinquefasciatus [34] reported that which compares well with present findings.The larvicidal activities of A. hypogaea MPC 9, 12, octadecadienoic acid ethyl ester showed strong mosquitocidal activity [35].The sesquiterpene compound, Pytol isolated from L. camera and tested against the mosquitocidal activities of important HVMs [36].The larvicidal activity of cinnamon and other EOs were recorded maximum activities [37] and the similar trend was also recorded in the study.
Plant-borne compounds and the fractions were tested as larvicides, ovicides, and repellency against An.stephensi.The larvicides activity was tested by 11-octadecenoic acid, methyl ester compound against An.Stephensi with LC 50 values of 22.32ppm [16].The phytochemicals like tannins, triterpenes, phenolics, and alkaloids of different medicinal plants showed different biological activities [38][39][40][41][42][43].Several researchers reported phytochemical based experiments for exploring the insecticidal activity on mosquito vectors [44,45].Different parts of the Citrus plant (fruits, seeds, roots and leaves) have been tested for their use asmosquitocidal components [46,47] which are similar to the present investigation.Since there is no previous record of literature available about the mosquitocidal activity of L. angustifolia EO MPCs of these present investigations serve as firsthand information.The finding of the present investigation revealed that the L. angustifolia EO MPCs possessed remarkable larvicidal and ovicidal activities against selected vector mosquitoes.

Conclusion
The finding of the present study reveal that L. angustifolia EO MPCs offers potential bio control agent for larvicidal and ovicidal against medically important vector mosquitoes Ae. aegypti, An. stephensi and Cx.quinquefasciatus.

Table 2 .
Larvicidal activity of L. angustifolia EO MPCs against freshly molted third instar larvae of selected mosquitoes.
Mortality of the larvae observed after 24h of exposure period.* Significant at P <0.05 level.LC50=Lethal Concentration brings out 50% mortality and LC90=Lethal Concentration brings out 90% mortality.LCL=Lower Confidence Limit; UCL=Upper Confidence Limit.Mathalaimuthu Baranitharan et al.: Lavandula angustifolia Essential Oil Phyto-Compounds as Leads to Potential Mosquitocides

Table 3 .
Ovicidal activity of L. angustifolia EO MPCs against the eggs of selected mosquitoes.
Values represent mean ± S. D. of five replications.Eggs in control groups were sprayed with no phytochemicals.NH -No hatchability (100% mortality)