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Repellent Potential of Tageteserecta L. and Callistemon brachyandrus Lindl. Against Mosquito Larvae for Formulation of Herbal Repellent Compounds

P.Pavitha1 and S.Poornima2
  1. UG Scholar, Department of Biotechnology, K.S.Rangasamy College of Technology, Tiruchengode, Namakkal District, Tamil Nadu, India
  2. Assistant Professor, Department of Biotechnology, K.S.Rangasamy College of Technology, Tiruchengode, Namakkal District, Tamil Nadu, India
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To avoid harmful effects of commercially available mosquito repellent creams in market, an attempt has been made to prepare herbal formulations from natural products such as flowers of Tagetes erecta L. and leaves of callistemon brachyandrus Lindl. These are ornamental plants, flourishing in India. All parts of the plant, from root to seed possess a multitude of phytochemical secondary metabolites and also responsible for repellent activity. To evaluate the repellent potential, phytochemical studies, larvicidal activity, smoke toxicity and repellent activity tests were performed against Anopheles stephensi, Culex infulus and Aedes agepyti. Water extract of Tagetes erecta L. showed of mortality rate of 40%, 40%and 50% and Callistemon brachyandrus Lindl. water extract showed 20%, 13.33% and 40% mortality rate against Anopheles stephensi, Culex infulus and Aedes agepyti respectively. These plant extracts were used to formulate herbal repellent compounds such as incense log and cream. Repellent activity test for cream formulation showed 89.87%, 87.5% and 90% protection and smoke toxicity test for incense log showed 66.25%, 70% and 67.5% protection against Anopheles stephensi, Culex infulus and Aedes agepyti respectively


Tagetes erecta L., Callistemon brachyandrus Lindl., larvicidal activity, formulation repellent compound, repellent activity, smoke toxicity.


Mosquito borne diseases are the major sources for death in developing countries. Anopheles stephensiliston is a major insect vector in India as well as some other countries in the world has been shown directly responsible for about 40-50% of the annual malarial incidence [1],[2].Control of mosquitoes is something important in the present day with increase in number of mosquito borne illnesses. Malaria kills 3 million people each year, including one child every 30 second [3]. Although insect borne diseases are responsible for major health problems in tropical and subtropical climate level. Several mosquito species belonging to genera Anopheles, Culex and Aedes are vectors responsible for the pathogens of various diseases like malaria, filariasis, Japanese Encephalitis, Dengue fever and yellow fever. Most parasitic disease is the tropical, indentifying globalization and climatic changes are increasing the risk of contracting arthropod-borne illnesses [4],[5].One example is West Nile virus; current federal statistics indicate that it will be responsible for thousands of cases of neuroinvasive disease in North America over the next several years [6]. DEET (N,N-diethyl-meta-toluamide) is the active ingredient in many insect repellent products. DEET has been found to inhibit the activity of a central nervous systemenzyme, acetylcholinesterase, in both insects and mammals. This enzyme is involved in the hydrolysis of the neurotransmitter acetylcholine, thus playing a role in the function of the neurons which control muscles[7]. Plants are considered as a rich source of bioactive chemicals and they may be an alternative source of mosquito control agents. There are a number of suitable alternatives to using topical pesticides on human skin [8]. These types of products are completely non-toxic, with no side effects, and are just as effective as dangerous chemical pesticides. The only difference is that they may need to be applied more often.Many mosquito repellents and insect repellents are used to control the mosquitoes. Unfortunately these are loaded with chemicals like propoxur, dichlorvos and transfluthrin which are harmful to human beings. This is true of any insect repellent, anywhere in the world. Because of this fact; people are seeking natural ways to repel mosquitoes[9]. One natural and effective way to repel mosquitoes is the use of mosquito repellent plants.


In this study of bioassay of herbal mosquito repellent formulated from the essential oil of plants., the repellency activities of four formulated herbal mosquito repellents of some selected MAPPs Nigeria were evaluated. The essential oils were extracted by hydrodistillation method at 500C using all glass clevenger apparatus. These were then stored at 40C in refrigeration and it graded concentration (6%, 8%, 10% and 12%) of the essential oils in a complex solution of polyethylene glycol, ethanol and water were prepared and their mosquito repellency activities was carried out in the laboratory against standard kisumu strain of Anopheles gambiae. This study showed that both 8% and 10% formulation have the most promising activity exhibiting 68-95% repellency activities lasting for 2 hours.The standard exhibited 75-100% repellency activity which also lasted for 2hours as compared to 8% and 10% formulations. This study demonstrated the potential use of essential oils from medicinal plants as mosquito repellent[10]. Repellent activity of Ageratum houstonianumMill. (Asteraceae) leaf extracts against Anopheles stephensi, AedesaegyptiandCulexquinquefasciatus(Diptera: Culicidae was determined for hexane, ethyl acetate and methanol crude extracts of Ageratum houstonianum(A. houstonianum) leaves against adult Anopheles stephensi,Aedes: aegypti(Ae. aegypti) andCulexquinquefasciatus(Cx. quinquefasciatus)offixed concentration of Bioassay on laboratory reared Swiss albino mice by topical application at a 0.01 per cent with coconut oil as a base. Crude leaf extracts of A.houstonianumin combination with coconut oil repelled vector mosquitoes. Amongst the three extracts, methanol extract gave the maximum protection of 95.0% against Cx. quinquefasciatus. Hexane and ethyl acetate extracts gave a maximum of 93.4% protection against An. stephensi.finally they conformed The crude extracts of A. houstonianumleaves in combination with coconut oil showed repellent activity with repellent quotient ranging from 0.6 to 0.9 [21]. Larvicidal and pupicidal potential of the methanolic extracts from Moringaoleifera (M. oleifera) plant seeds against malarial vector Anaphelesstephensi (A. stephensi) mosquitoes at different concentrations such as 20, 40, 60, 80 and 100ppm were studied. They found out larvicidal activity of M. oleifera exhibited in the first to fourth instar larvae of the A. stephensi, and the LC50 and LC90 values were 57.79ppm and 125.93ppm for the first instar, 63.90ppm and 133.07ppm for the second instar, 72.45ppm and 139.82ppm for the third instar, 78.93ppm and 143.20ppm for the fourth instar, respectively. They found out the phytochemicals derived from M. oleifera seeds extracts are effective mosquito vector control agents [11]. Essential oil of five plant species Centuellaasiatica, Ipomoea cairica L., Momordicacharantia L., Psidiumguajava L. and Tridaxprocumbens L. for their topical repellency effect of malarial vector Anopheles stephensi was studied. They have used different concentration for repellency test such as 2, 4 and 6%. In this study, clear doseresponse relationships were established in all essential oils, with higher concentration of 6% provided high repellency effect [20].


Sample Collection
The plant Tagetes erecta L. was collected from Sirumalai, Dindigul District, Tamil Nadu. The plantCallistemonbrachyandrus Lindl.was collected from the area around K.S.Rangasamy college of Technology, Tiruchengode, Namakkal District, Tamil Nadu. They were identified at Tamil Nadu Agriculture University, Coimbatore. The plants were allowed to dry under shadow condition. The dried plant materials were powdered by an electrical blender. The powder plant material was stored at 40C for further use [10]. Mosquito Larvae Collection The mosquito larvae of Anopheles Stephensi, Culex infulus and Aedes agepyti larvae of IV pupae were collected from Southern Indian Branch, National Center for Disease Control, Mettupalayam. The mosquito larvae were maintained at room temperature. The larvae were fed with dog biscuits and yeast at 3:1 ratio [11]. Phytochemical Screening Dried and powder form of leaf of Callistemon brachyandrus Lindl. and flower of Tagetes erecta L. were subjected to quantitative phytochemical screening for the identification of various classes of active chemical constituents such as tannins, alkaloids, cardiac glycosides, saponins, reducing compounds and anthraquinones by the methods used in [12]-[14]


Formulation of Repellent Compounds
Flower of Tagetes erecta L. and leaves of Callistemon brachyandrus Lindl. ethanol extracts were allowed to evaporation at room temperature and residue was collected that was used to formulate insense log. This product was used to smoke toxicity assay. The mosquito repellent insense log were prepared following the method, 10g of charcoal powder, 3g of white wood powder, 4g of joss powder. All the three were thoroughly mixed with distilled water to form a semisolid paste. Insense log (approximately 3.48±0.2 g weight) was prepared manually by using moulding techniques and shade dried. The control insense log was prepared without the plant ingredient. Pure petroleum jelly (22.37 g) was accurately weighed into a 250 ml wide –mouth glass beaker and heated in water bath at temperature of about 500C. At this stage, 1.73 g of Tagetes erecta L. and Callistemon brachyandrus Lindl.powder (residue) obtained in ethanol extraction was added and mixed well. The mixture, in a screwed, covered bottle, was stirred properly to ensure that the sample was uniformly mixed with the molten stage of cream. The product is then taken for repellency test [15] & [16], [23].


Test for Larvicidal Activity
Anopheles Stephensi, Culex infulus and Aedes agepyti larvae were used to test the larvicidal activity of Leaves of Callistemon brachyandrus Lindl. and flowers of Tagetes erecta L. 30ty IV instar larvae of all three mosquito family were kept in 50ml glass beaker containing 40 ml of dechlorinated water (control) and different concentration of both sample was prepared (20,40,60,80 and 100ppm) from stock solution. Three replicates for each concentration were set up. The control mortality rate was corrected by using Abbott's formula [17] – [19]. Abbott formula is,
Repellent Activity
Repellent activity of mosquito repellent cream was tested with human volunteers in South Indian Branch, National Centre for Disease Control. For the repellent activity of plant extracts percentage protection in relation to dose method was adopted. Three to four days old blood starved female adult mosquitoes (40) were kept in a net cage of 60cm×40cm×100cm and window 20cm×20cm. The arms of tested person were cleaned with ethanol. After air drying the arm only of the dorsal side of the skin on each arm was exposed and the alcohol served as control. The plant extract containing cream was applied on arms skin of volunteer. The control and treated arms were introduced simultaneously into the cage. The number of bites was counted over 5 min every 60 min, from12.50pm to 6:50pm. The experiment was conducted three times. The percentage protection was calculated by the following formula [20] & [21].
Smoke Toxicity Test
The natural repellent incense log (control I) was prepared by the above procedure and that was used for smoke toxicity test. The experiments were conducted in a glass chamber of 80cm X 40 cm X 60 cm. A window of 30 cm X 10 cm was situated at mid bottom of one side of the chamber. Hundred of three or four days' old blood starved adult female mosquitoes, fed with sucrose solution, were released into the chamber. The experimental chamber was tightly closed. The experiment was repeated three times on separate days, including control mosquitoes of the same age groups. The data were pooled and average values were subsequently used for calculations. Controls were maintained in two sets. One set was run with incense log lacking the active ingredient of plant powder (control II), the other was a commercial coil which was used for positive control to compare the effectiveness of plant coils. After the experiment got over, fed and unfed (active and dead) mosquitoes were counted. The protection given by the smoke from incense log against the biting of adult mosquito was calculated in terms of percentage of unfed mosquitoes due to treatment. The % of protection was calculated for unfed mosquitoes by using the following formula,[22] & [11].


Phytochemical Analysis
The phytochemical and elemental analysis of leaf of Callistemon brachyandrus Lindl.and flower of Tagates erecta L.showed the presence of useful classes of chemical compounds suchas saponins, tannins, alkaloids and reducing compounds. Table 1 shows the phytochemical results of Tagetes erecta L. and Callistemon brachyandrus Lindl.
Mortality rate
`The results of mortality rate of Anopheles stephensi, Culex infulus and Aedes agepyti at different concentration of water extract were presented in Table 2. The 100ppm concentration solution showed % mortality of 40%, 40% and 50% in Anopheles stephensi, Culex infulus and Aedes agepyti respectively. Table3.shows The 100ppm concentration of solution showed % mortality of 20%, 13.33% and 40% in Anopheles stephensi, Culex infulus and Aedes agepyti respectively.
Formulation of Repellent Compounds
Repellent compounds were prepared such as incense log and cream as said in the procedure. The compounds were tested to assess their efficacy against mosquito by repellent activity and smoke toxicity tests.
Repellent activity and smoke toxicity test
Table 4 shows the repellent activity test conducted with mosquito repellent with sample (Control I), cream without sample (Control II) and already available commercial cream against Anopheles stephensi, Culex infulus and Aedes agepyti. The control I showed effective result of % protection of 89.87%, 87.5% and 90% against Anopheles stephensi, Culex infulus and Aedes agepyti. and it is comparable with available cream in market. Table 5 showed smoke toxicity of three types of repellent products used in smoke toxicity test such as commercial coil, insense log with sample (Control I) and incense log without sample (Control II). Control I showed 66.25%, 70% and 67.5% protection against Anopheles stephensi, Culex infulus and Aedes agepyti respectively. \


To overcome the harmful effects of commercially available mosquito repellent formulations, herbal formulations are prepared using the flowers of Tagetes erecta L. and leaves of Callistemon brachyandrus Lindl. Phytochemical such as alkaloids, flavonoids and monoterpenes are known their mosquito repellent activity. The phytochemical study for Tagetes erecta L. showed positive results for tannins, saponins test, alkaloids test, reducing compound test and cardioglycosides test and showed negative result on anthraquinones. Callistemon brachyandrus Lindl. Showed positive result on tannins test, saponins test, alkaloids test, reducing compound test and cardioglycosides test and showed negative result on anthraquinones. Tagetes erecta L. showed 40% of mortality rate against Anopheles stephensi and Culex infulus. So, this plant source has maximum larvicidal activity against Anopheles stephensi and Culex infulus. Callistemon brachyandrus Lindl.showed maximum mortality rate 13.33% against Culex infulus. So, this plant source has maximum larvicidal activity against Culex infulus. The formulations such as incense log and cream showed better repellent activity and the smoke toxicity test also supports the efficacy of the products in terms of percentage protection. Incense log (Sambrani) showed maximum protection of 90% against Aedes agepyti and 89.87% against Anopheles stephensi and it is comparable with the commercial cream. Smoke toxicity test of incense log showed maximum percentage protection of 67.5 against Aedes agepyti.
The results of present investigation revealed that flowers of Tagetes erecta L. and leaves of Callistemon brachyandrus Lindl. showed potential larvicidal activity against Anopheles stephensi, Culex infulus and Aedes agepyti. These herbal products are eco-friendly, non-toxic and possess no side effects; the formulation process is also quite simple.


We thank Department of Biotechnology, K.S.Rangasamy College of Technology for supporting this study.


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