World Health Organization revealed that 3.9 billion people inhabiting 128 countries at risk of dengue infection in the World [1]. At present, WHO reports have also indicated, 47 countries are at the risk of yellow fever while 60 countries 60 countries are effected by Chikungunya. A modelling study by them estimated the occurrence of 84000-170000 severe cases of yellow fever in Africa and America leading to 29000-60000 deaths in 2013. Outbreaks of Zika virus disease have been recorded in Africa, the America, Asia and Pacific which is no increase every year [1]. In India, dengue fever and Chikungunya are progressively becoming the imperative Public Health Problem[2].Mosquito borne diseases such as dengue haemorrhagic fever, malaria, filariasis are harmful to human and they are a public health problem in Myanmar [3].The official records of the Union Health Ministry  reported considerable increase in the number of cases in last few years in the country especially in Yangon, Mandalay, Mawlamyaing and Hpa-an [4](MOHS 2020)Billions of people, primarily in tropical countries, are at risk from such diseases which are transmitted by three different genera of mosquitoes as Aedes, Culix and Anopheles species [5]. Aedes aegypti is one of the major vectors for Dengue Fever (DF), Dengue Haemorrhagic Fever (DHF), Chikungunya and Yellow fever. Culex tritaeniorhynchus transmit Japanese encephalitic and Culex quinquefasciatus is a major vector of filariasis. Anopheles dirus and An. minimus are major vector of malaria in Myanmar [6]. Mosquito vectors are generally collected by insecticides, bio-control agent and insect growth regulators. To control mosquitoes and mosquito-borne diseases, which have a worldwide health and economic impacts, synthetic insecticide-based interventions are still necessary, particularly in situations of epidemic outbreak and sudden increases of adult mosquitoes [7]. However, indiscriminate use of conventional insecticides is fostering multifarious problems, 85 to 90% of applied pesticides never reach target organisms. Mosquito control approach has been almost completely based on synthetic organic insecticides. It can cause chemical haxard. Synthetic insecticides are toxic and adversely affect the environment by contaminating soil, water and air. Therefore, there is a need to alternative ways for environmental safety, biodegradable, low cast and indigenous methods for vector control. In attempt to overcome these problems, great emphasis has been recently placed on the research and development of mosquito control using natural plant products. Many active insecticidal materials have been derivied from plant sources, i.e. nicotine, pyrethrins and rotenones. Orange, Lemon and Kaffir lime leaves and fruit extracts have been used as pest control against rice weevil Sitophilus oryzae infestation in stored rice and mosquito repellent as well as larvicide [8-10].

Among the plants investigated to date, one showing enormous potential is the Ocimum family [11]. Ocimum plants such as Ocimum basilicum L is a familiar through use of their leaves for culinary purposes and traditional medicine [8,12]. In Asian countries as China, India, Myanmar, Thailand, Malaysia and Indonesia, have been used its leaves, fruits barks and roofs as traditional medicine and apart from use as food flavoring and appetizer. In Myanmar, essential oil extract of Ocimum basilicum L leaves and also powder of leaves, fruits and seed, barks and roots are very useful for making pain release traditional medicines. It chemically contains a low percentage of volatile and essential oils and is used in the conventional medicine to alleviate pain, treat stress, mitigate vomiting and even as an insect repellent.  Ocimum basilicum L plants are available in many parts of the country although the plants are abundantly present in hilly areas of Myanmar, India, Cylone, several Mediterranean countries, many parts of Asia, Africa and the Pacific Islands [9,13]. It chemically contains a low percentage of volatile and essential oils and is used in the conventional medicine to alleviate pain, treat stress, mitigate vomiting and even as an insect repellent [9,14]. Ocimum basilicum L Leaves can began to be harvested any time after the plants have reached a height of 15-20 cm. Chemical constituents of Ocimum basilicum Lleaves fresh is yield essential oil containing small amount of estragol, eucalyptol, ocimene, linallol acetate, eugenol, 1-epibucyclosesquiphellandrene, menthol, menyhone, cyclohexanol, Cyclohexanone, myrcenol and nerol [15]. Essential oils have also been reported to possess antibacterial, antifungal, mosquito larvicidal and repellent activities [2,16,17].

However, there is no derivative of Ocimum basilicum L leaves essential oil on Aedes mosquitoes has been mentioned as an insecticidal agent in Myanmar. Therefore, investigation on larvicidal, ovicidal and repellent action of Ocimum basilicum L leaves essential oil extract to support the future vectors control activity in environmental sound manner.

Mosquito Larvae Collection

Dagon Myothit North Township strain of Aedes aegypti mosquito larvae and adult Aedes mosquitoes emerged from pupae were reared in laboratory of Medical Entomology Research Division, Department of Medical Research. Larvae were feed on DMR larva food. Adult were provided with 10% sucrose solution and 8weeks old mouse for blood meal. Mosquitoes were held at 26±2°C,65-75% relative humidity with a photo period of 12 –hours light and 12-hours dark. Laboratory reared larvae and mosquitoes were used for testing insecticidal properties of stream distillated essential oil of fresh Ocimum basilicum L leaves which were collected from Mingaladon Township Yangon Region.

Species Identification of Mosquitoes

Larvae and adult mosquitoes emerged from larval urvey were identified by morphological methods according to the key of Rampa and Prachong [18].

Collection and Preparation of Essential Oil Extraction from Ocimum Basilicum L Leaves

Ocimum basilicum L leave Myanmar name Pinsein was collected from Mingaladon Township, Yangon Region. A total of 10 Kelo grams of Ocimum basilicum L leave was cleand and 300 grams of Ocimum basilicum L leave were extracted with 1000nl distilled water by stream distillation method at 100°C for 3 hours. Complete removal of the solvent from the exteact was accomplished in glass rotary evaporator. The resulting 3.39gm of essential oil was obtained from 300gm of fresh Ocimum basilicum L leave. The essential was stored at 4°C in refrigerator until use. The extraction was done in Department of Pharmacology, Institute of Medical Technology, Yangon.

Larvicidal Testing Procedure

Based on preliminary tests, further dilutions were prepared with distilled water. Different concentration of Ocimum basilicum L leave essential oil as 0.1g,1.05g.1.025g,0.0125g and 0.00625 g were prepared freshly in 100ml each of distilled water in 150ml plastic cups. Fifty (50) each 3^rd and 4^th instars Aedes aegypti larvae were put into different concentrations and negative control test was done simultaneously. Detail testing was done according to standard method [19]. Larvae were exposed 24 hours for each replication in different concentrations in laboratoty at 27-29°C and 70% -80% relative humidity. Five replicates were carried out and knockdown was checked and recorded after 60 minutes’ exposure period and mortality was checked and recorded after 24 hours of exposure periods. Knockdown and dead larvae were identified when the larvae failed to move after probing with a needle in the thorax region of the body. Lethal concentration LC50 and LC90 values for 95% condidential limits were calculated by following formula [20].

X2=(O-E)2/Ex(100-E) (X2= Chi squire, O=Observed value, E=expected value, O-E=Observed minus expected)

Repellent Activity Testing

The repellent study was followed the method of WHO (19). Five days old blood staved female Aedes aegypti mosquitoes 60 each were kept in two steel net cages (59x59x59cm) one for male and one for female volunteers. Aedes aegypti is a day tim time biter; therefore, the tests were done between 08:00 hour and 16:00 hour. Evaluation tests were carried out in a (12x15x15ft) room at 24-28°C and relative humidity of 70-85%. The volunteers had no contact with lotions, perfumes, or perfumed soaps on the day of assay. The arms of volunteers, one ml of ethyl alcohol 95% diluent used in the preparation of the test repellent in applied evenly using a pipette to average 372.375cm2 (Four volunteers-two males +two female) of forearm skin between the wrist and elbow and allowed to dry for 1 minute. Before insertion of arm into the cage containing 60 Aedes female mosquitoes, the hands are protected by plastic gloves to protect mosquito bite. The first step, ethyl alcohol applied forearm was inserted into the cage and counted the number of mosquitoes that land on skin during 30-secind period. The control forearm was carefully withdrawn and this arm was then treated with one ml of 0.0125g/ml of Ocimum basilicum L leaves essential oil solution and allowed to dry. The treated arm was placed in the cage for another 30 second period and onserved for mosquito landing. This procedure was repeated for each additional incremental of Ocimum basilicum L leaves essential oil dose. The tests were carried out one after the other without delay and Ocimum basilicum L leaves essential oil dose at each test was calculated as the sum of the doses applied to arrive at the cumulative dose for each test. Test was proceeding when the mosquito landing rate on the exposed forearm was less than 10 females in 30 second. Two trained technicians were recorded the number of landings. At the conclusion of the dose response experiment, 1ml of ethyl alcohol was applied on the other forearm and allowed to dry. This forearm was inserted in the cage for 30 second to verify that the number of landings was more than 10 per 30 second as was observed at the beginning of the experiment. Protection (P) was expressed as a proportion of the number of mosquito landing on treated arm (T) in relation to the number of landings on the control arm © of the same individual. Where C is the average of landing mosquitoes on two un treated arms.

P+1-(T/C)=(C-T)/C 

Estimation of Complete Protection Time

The complete protection time of Ocimum basilicum leaves essential oil was determined 99 to 100% protection dose 0.0125g/ml, 0.025g/ml, 0.05g/ml and 0.1g/ml were using on average 372.375cm² areas (another 2 male and 2 female volunteers) of forearm skin between the wrist and elbow individually. The protection test was followed by the procedure described as above. Before testing two mosquito cages (size 59x59x59cm) each containing 60 non blood fed 5 days old Aedes aegypti female mosquitoes were normally used. One cage was used for testing female volunteers and another cage was used for testing male volunteers. Before testing the arms of volunteers, one ml of ethyl alcohol 95% diluent used in the preparation of test of Ocimum basilicum leaves essential oil repellent in applied evenly using pipette to average 372.375cm² of forearm skin between the wrist and elbow and allowed to dry 1 minute. Before insertion of arm into the cage containing 60 Aedes female mosquitoes. The hands are protected by plastic gloves to protect mosquitoes bite.  The first step, ethyl alcohol applied forearm was inserted in to the cage and counted the number of mosquitoes that land on the skin during 3 minute period. The control forearm was carefully withdrawn from the cage. Then 0.1g of Ocimum basilicum leaves essential oil was prepared in one ml of ethyl alcohol solution was applied evenly on average 372.375cm² of another forearm skin between the wrist and elbow. The treated arm was placed in the cage for 3minute period and observed for mosquito landing.

After 30minutes, the Ocimum basilicum leaves essential oilrepellent treated arm was inserted again into the cage and exposed for 3 minutes to determine landing activity. This procedure was repeated at 30 minute intervals for240minutes and the procedure was used consistently throughout the experiment. Test was repeated 4 times for 2 males and 2 females’ volunteers. The mosquitoes that landed on the hand were recorded and then shaken off before imbibing any blood. The mosquitoes that landed on the hand were recorded and then shaken off before imbibing and blood. Test was proceeding when the mosquito landing rate on the exposed forearm was less than 80% within 3 minutes. The end of the experiment, ethyl alcohol applied forearm was inserted into the cage and counted the number of mosquitoes that land on the skin during 3-minute period to verify that the number of landings was same or not observed at the beginning of the experiment. The control forearm was carefully withdrawn from the cage. The average of landing mosquitoes on two untreated arms was used to calculate the percentage protection of mosquito bite. Complete protection time was estimated after experiment. Same procedure was done for BPI product Rapito oil (mosquito repellent) dose 0.1g/ml and 1ml of pure 50% oil dose and one ml of 50% Ocimum basilicum leaves essential oil in ethyl alcohol.                            

Data Analysis Plan

Data entry and proceeding was made using Microsoft Excel software. The average larval mortality data were subjected to probit analysis for calculating LC50 and LC90 values and other statistics at 95% confidence limits of upper confidence limit, and Chi-squire values were calculated using the dose –effect probit analysis (20)¹⁶. Results with p<0.05 were considered to be statistically significant.

Table 1: Knockdown and mortality effect of different dilution of essential oil of Ocimum basilicum leaves essential oil against 3^rd and 4^th in star Aedes aegypti larvae.

Table 1.shows that effective knockdown effect of Aedes aegypti larvae were found 96.80% knockdown at 0.1g in 100ml dilution of essential oil. Lowest knockdown effect 10% was found at 0.00625g dilution of essential oil. Highest mortality of Aedes aegypti larvae was found 98.40% at 0.1g essential oil dilution and followed by 81.20% at 0.05g dilution of Ocimum basilicum leaves essential oil. Lowest mortality effect was found 16.40% of larvae at 0.00625g dilution of essential oil.

Table 2: LC₅₀ and LC₉₀ values of Ocimum basilicum leaves essential oil on 3^rd and 4^thinstar Aedes eagypti larvae.

X2=Chi squire, P=propoablety, df= degree of freedom.

Table 2. showed that dose effect analysis of LC50 and LC90 values of Ocium basillicum leaves essential oil against 3^rd and 4^th instars

 Aedes aegypti larvae were found 0.0178g for 50% mortality and 0.0576g for 90% mortality respectively P<0.05, X²=0.0620.

Table 3: Experiment of successive Ocimum basilicum leaves essential oil doses applied to arrive at a cumulative dose for Aedes aegypti.

Successive cumulative dose of Ocimum basilicum leaves essential oil on average 372.375±13.4374cm² area of arm for 100% protection of Aedes aegypti mosquito landing to probe the skin was 0.1g/ml or 0.000267g/cm².

Table 4: Repellency test of Ocimum basilicum leaves essential oil doeses on 4 volunteers Arms against Aedes mosquitoes in Laboratory.

Table 4 shows that repellency activity of complete protection time of (Ocimum basilicum leaves essential oil dose 0.1g/ml or 0.0003g/cm²   provided 83.70% protection for 240 minutes against Aedes aegypti mosquitoes and over 90% protection was found for 180 minutes, over 98% protection was found 90 minutes and 100% protection was observed 60 minutes. Ocimum bosilicum leaves essential oil was found to be very effective repellency on Aedes mosquitoes.

Table 5: Repellency test of 0.1g/ml dose of Rapito (BPI product)/EtOH on 4 volunteers Arms in laboratory.

Table 5 Shows that repellency activity of complete protection time of Rapito oil 0.1g/ml or 0.000267g/ml provided over 80% protection for 240minutes against Aedes aegypti mosquitoes, over 90% protection was found 210 minutes, over 95%protection was found to be 120minutes and 100% protection was found 60 minutes BPI product Rapito oil was found to be very effective repellency on Aedes mosquitoes.

Table 6: Repellency test of 50% Ocimum basilicum leaves essential oil (50ml oil + 50ml EtOH (v/v) 0.5g/ml each on 4 volunteers Arms in laboratory.

Table 6 shows that repellency activity of complete protection time of 50% Ocimum bosilicum leaves essential oil 0.5g/ml or 0.000267g/ml provided 83.06% protection for 240minutes against Aedes aegypti mosquitoes, over 90% protection was found 210 minutes, over 95%protection was found to be 150minutes and 100% protection was found 60 minutes.  50% Ocimum bosilicum leaves essential oil was found to be very effective repellency on Aedes mosquitoes.

Table 7: Repellency test of 50% Rapito oil (50ml + 50ml EtOH V/V) 0.5g/ml each on 4 volunteers Arms in laboratoey.

Table 7 shows that repellency activity of complete protection time of 50% Rapito oil dose 0.5g/ml or 0.0003g/cm² provided 87.82% protection for 240 minute against Aedes aegypti mosquitoes. Over 80% protection was found for 240 minutes, over 90% protection was found for 210 minutes, over 95% protection was found for 180 minutes and 100% protection was observed 90 minutes. 50% Rapito was found to be very effective repellency on Aedes mosquitoes.

The main vector of dengue virus in Myanmar is Aedes aegypti. Rapid, poorly planned urbanization in association with weak regulatory policies for discharge of solid waste has resulted in the accumulation of solid waste which in turn results in the accumulation of discarded containers in most developing countries. Poor water supply created a lot of water storage containers for storing water for multiple used. These accumulations have favored the establishment and geographic spread of Aedes aegypti mosquitoes. It discarded largely because of it antropophilic feeding behavior, resting behavior inside houses and its capability to exploit most water holding containers for breeding [21]. Dengue Hemorrhagic Fever (DHF) is one of the global health concerns in tropical area and an endemic disease in more than 110 countries in the worlds [22]. Approximately 2.5 billion people live in endemic countries of which about 1.8 billion (more than 70%) in Southeast Asia and the Western Pacific Region [23,24,25]. Aedes aegypti is a main vector of dengue virus in urban areas and Aedes albopictus is rural area vector. 

Mosquitoes are a serious threat to public health transmitting several dangerous diseases for over two billion people in the tropic. Insecticide residues in the environments as a result of chemical insecticide usage have turned the researcher’s attention towards natural products. In the past year, the plant kingdoms synthesize a variety of secondary metabolite which plays a vital role in defense of plants against insect/mosquitoes. Mosquito repellents may be one of the most effective tools for protecting human from vector borne diseases and nuisance caused by mosquitoes. Natural products are safe for humans when compared to that of synthetic compounds. According to Bowers et al., [26] the screening of locally available medicinal plants for mosquitoes control would generate local employment, reduce dependence on expensive imported products and stimulate local efforts to enhance public health. Different parts of the plants contain a complex of chemicals with unique biological activity [27,28] which is thought to be due to toxins and secondary metabolites which act as mosquitocidal agent [29]. Smoke is still, the most widely used common methods of repelling biting insects that is used throughout the World. Most households in the developing world rely on personal protection measures of limited effectiveness, such as burning mosquito coils or leaves and peels. The bundles of dried Artemisia vulgris L., dry organic and lemon peels were burned to repel biting insect since it contains insect repellents that can be released from the plant by combustion [30].

Bio-larvicidal compounds are presented in the extract of Ocimum basilicum leaves essential oil. Ocimum basilicum leaves essential oil concentration 0.1g/100ml water found 96.80% knockdown and 98.40% mortality and LC₅₀ and LC₉₀ values were found 0.0178g and 0.0576g. Same larvicidal effect has been found Orange, Lemon and Orange and lemon mixture of essential oil and laboratory larvicidal experiment indicate that the higher essential oil concentration found to be the higher the larval mortality, the essential oil of Orange at concentration of 0.01g/100ml water caused 98.33% mortality rate of Ae. aegypti larvae and Lemon essential oil at concentration 0.01g/100ml water caused 100% mortality rate of Ae. aegypti larvae as well as Orange and Lemon essential oil mixture at the concentration 0.01g/100ml water caused 96.67% mortality of 3^rd and 4^th instar Ae. aegypti laevae [31]. This finding revealed that Lemon essential oil (0.01g/100ml) was found 100% mortality had higher potency than the present Ocimum basilicum leaves essential oil 0.1g/100ml). Maung Maung Mya and his associates revealed that Citrus hystrix DC (Kaffir lime) leaves, fruit and fruit peel extracts were found to e very effective and high mortality against 3^rd and 4^th instar larvae 2.4% and 2.1% concentrated Citrus hystrix DC leaves extract in distilled water caused 99.5% and 85.5% mortality and LC₅₀ and LC₉₀ values were 1.73% and 2.08% concentrations for leaves extract.0.0138g and 0.0515g for fruit extract, 0.0142g and 0.0522g for peel extract [9,32]. Third and fourth instars Ae. aegypti larvae from Pindaya Township were more susceptible, 100% mortality at 0.003125g and 0.00625g in Citrus curantifolia and Citrus maxima than North Dagon strain (97-100% mortality at 0.00625g) to Citrus maxima and Citrus curantifolia peel essential oil extracts [10]. 

Singhi et al., [33] have reported that the latex of Citrus procera has shown larvicidal efficacy against all three important vector species as Ae. aegypti, An. stephensi, and Cx. quinquefqsciatus in India. Other researcher evaluated that larvicidal activity of Plumbago zeylanica and Cestrum nocturnum extracts on Ae. aegypti; the LC₅₀ values of both medicinal plants were less than 50ppm. The larvicidal stability of the extracts at five constant temperatures (19°C, 22°C,25°C,28°C, and 31°C) evaluated against 4^th instars larvae revealed that toxicity of both plant extracts increases in temperature [34]. Halim reported that the insecticidal activity of Zingiber officinale against the larval maturation, and adult emergency of Anopheles pharoensis 3^rd stage showed 100% larval mortality rate and at 0.2% and 0.1% caused mortality of 66.7% respectively [35]. The ethanolic extract of whole plant Leucas aspera on the 1^st to 4^th instar larvae and pupae values of LC₅₀ 1^st instar was 9.695%, 2^nd instar was 10.272%, 3^rd instar was 10.823% and 4^thinstar was 11.303% and pupae was 12.732% respectively against An. stephensi [36]. in the present study, Ocimum basilicum leaves essential oil 0.1g/100ml caused 98.40% mortality against 3^rd and 4^th instar Ae. aegypti larvae. Although other researcher revealed that among the two Citrus cultivars tested as larvicide against Ae.albopictus , Valencia late (Citurs sinensis) was the best in terms of LC₅₀ (297ppm ) percent mortality (97%) and LT₅₀ (18.49hours) than  freutrall early (Citrus reticulate) with LC₅₀ (377.4ppm), percent mortality (88%) and LT50 (31hours), While nomilin gave lowest LC₅₀ (121.04ppm) than limonin (382.22ppm) after 72hours of exposure. Valencia late also had more limonin and nomilin (377µg/ml and 21.19µg/ml) than freutrall early 5.29µg/ml and 3.89µg/ml) respectively [37]. Other study of bio-larvicide activity testing of Orange peel, Lemon peel and Orange and Lemon peel essential oil mixture were analyzed and found that LC₅₀ and LC₉₀ values of lemon essential oil 0.001g and 0.004g found highest bio-larvicide property against 3^rd and 4^th instar Aedes larvae was found to be more toxic and effective as bio-larvicide [31,38]. The essential solution made between those concentrations interval is effective and would be able to kill 90% 3^rd instar Ae. aegypti larvae. Yadav and associates have reported the methanol, Chloroform and ether extracts of Euphorbia tirucalli latex and stem bark were evaluated for larvicidal activity against laboratory reared larvae of Cx.quinquefasciatus. Sharma et al., 2005(39) reported that the acetone extract of Nerium indicum and Thuja oriertelis has been studied with LC₅₀ values of 200.87, 127.53, 209.00 and 155.97ppm against 3^rd instar larvae of An. stephensi and Cx. quinquefasciatus, respectively. Lyophilized powders of purified CytlA crystal of B. thuringiensis were much more toxic yielding a 50% LC50 of 11.332g/L, respectively [40] . Non polar extract fraction from Citrus hystrix is more toxic and is an Effective bio-larvicide with LC₉₀ =2885ppm compared with polar extract fraction from Citrus hystrix which has an LC₉₀ = 3180ppm [41].

The mosquito sensitivity to repellents varies among Aedes, Anopheles and Culex mosquitoes [42,43]. A large number of plant extracts have been reported to have mosquitocidal or repellent against mosquito vectors, but very few plant products have shown practical utility for mosquito control. Some of the plants that have been tested against mosquito larvae in India are Cleome viscosa, Ocimum basilicum, Vitex negundo,Delonixregia, Oligo chaetaramosa, Azadirachta indica,Ouassia amara, Anacardium occidentale,Thevetianerii folia etc. Natural products are preferred because of their bio-degradability and less toxic compared to the synthetic ones [44].

The genus Citrus of the family Rutaceae includes about 17 species distributed throughout the tropical and temperate region [45,46]. Citrus oils are mixtures of vary volatile components as terpenes and oxygenated compounds [47]. Limonene, a monoterpene, is the major component of lime and other related Citrus essential oils [48]. Bourgou and his party observed that Citrus aurantifolia has the highest limonene composition which could be a reason for the Citrus aurantifolia’s high lethal activity for repellent of mosquitoes [49].

Acute Toxicity and Allergenicity (Irritation)

Acute Toxicity screening Ocimum bosilicum leaves essential oil extract was done with the dosage of 2000mg/kg and 5000mg/kg body weight in albino mice. The results shown no lethality of the mice was observed up to fourteen days’ administration. Each group of animals were also observed still alive and did not sho any visible symptoms of toxicity like restlessness, respiratory disorders, convulsion, aggressive activities, coma and death. The sample has been tested as per “DBT”, Guideline for toxicity and allergenicity of Ocimum bosilicum leaves essential oil was evaluated on the body of Rabbits in laboratory and result of the sample of Ocimum bosilicum leaves essential oil 0.1g/ml dose or 0.0003g/cm² was found to be ‘nonirritant’ to the skin of rabbits for 14 days’ trial when compared with the sample of control rabbits. Same result has been found the researchers from Sri Lanka and Myanmar they revealed that the Artemisia vulgaris extract, Orange, Lemon and Citrus hystrix DC, Citrus maxima and Citrus curantifolia peel essential oil extracts were tolerated well by mice over a period of 14 days (Assay of sub-chronic toxicity), showing no overt signs of toxicity or stress [10, 31,32, 50]  ) and results showed oral activity, non –toxicity, and a weed with a potential for a cheap source of plant-based antimalaral [50].

Present repellent study of Ocimum bosilicum leaves essential oil applied to arrive at a cumulative dose 100% protection dose was 0.1g/ml or 0.000267g/cm² better than Orange essential oil 0.16g/ml or 0.0005g/cm² and protection dose and protection time are same with the Citrus hystrix DC extract, although Orange 0.05g and Lemon 0.05g mixture of essential oil dose 0.08g/ml or 0.0003g/cm² gave over 80% protection of bite for 150minutes was lower than the present study [31,32]. The ethanolic extracts of the Orange peel (Citrus sinensis) was tested for the toxicity effect on the larvae of the yellow fever mosquito Ae. aegypti [51] susceptibility tests were carried out in Cx. quinquefasciatus larvae using peel oil extracts of Citrus aurantium, Citrus sinensis and Citrus limon [52]; volatile extracts of Citrus sinensis had insecticidal activity against mosquito, cockroach and housefly [53] Garcia and Desrochers observed that appreciable mortality only with high concentrations (1x107cells/ml) of B.thurin giensis var. israelensis [54].

Ansari et al., [55] reported that the peppermint oil gave 94.1% protection for 6 hours, while mylol oil give 95% protection for 7.2 hours. They also reported that mylol iol and peppermint oil gave 100% protection for 11hours against An. annularis. Mylol oil gave 95.4% protection for 8.7 hours for the Anopheles species, whereas the peppermint oil gave 86.3% protection for 8 hours. Phukan and Kalita [56] showed that Litsea salicifilia recorded 70% and 50% repellency for 3 and 4 hours, respectively, against Aedes aegypti, but they failed to show much activity against Cx. quinquefasciatus. Other researcher reported that the akin repellent test at 1.0,2.5 and 5.0 mg/cm² concentration gave the mean complete protection time ranged from 119.17 to 387.83 minutes against An. stephensi with the benzene, petroleum ether, ethyl acetate and methanol extracts of Citrullus vulgaris tasted [57]. Another study of Citrus reticulate (Orange peels) and Citrus limonum (Lemon) peels extracts have significant repellent activity against Ae. aegypti mosquitoes. The highest concentrations of 0.016g/ml and 0.04g/ml provided over 210 minutes protection in ethanol mixture [31]. Mensah and his party revealed that Citrus limom showed that the highest repellent activity of 95%, 92.5% Citrus aurantifolia and 82.5% for Citrus sinensis, the oils had very strong repellent activities against carpenter ants [58].

Although present study results showed that 0.1g/ml or 0.000267g/cm² of Ocimum basilicum leaves essential oil gave over 80% protection for biting of Aedes mosquitoes for 240 minutes over 90% protection for 180minutes and 100% protection for 60 minutes.

When compared with 1ml of came concentration of Ocimum basilicum leaves essential oil 0.1g/ml and 50%(V/V) and BPI product Rapito oil applied on volunteer’s hand and test for repellency found to be 100% protection for 60 minutes, over 90% protection for 180 minutes and over 80% protection for 240 minutes were the same for the duration of complete protection times at the dose of 0.1g/ml both Ocimum basilicum L and Rapito oil. Although 50%  Ocimum basilicum leaves essential oil gave 100% protection for 60 minutes while 50% Rapito found 100% protection for 90 minutes, Ocimum basilicum  oil found 98.70% protection of mosquitoes bite for 90 minutes it was slightly lower than Rapito repellent, over 90% protection and over 80% protection time  were same i.e. 91.53% and 92.31% protection for 210 minutes for over 90%protection and 85.59% and 87.82% protection for 240 minutes for over 80% protection for Ocimum basilicum oil and Rapito. Saw Htat Thuya Lin [31] revealed that Orange, Lemon and mixture of Orange and Lemon essential oil creams gave 100% protection of mosquito bite for 30 minutes over 90% protection of mosquito bite were observed 180 minutes for Orange, 210 minute for Lemon, 150 minutes for Orange and Lemon mixture and over 80% protection for 270 minutes was same with the present study. Both Citrus maxima and Citrus curantifolia fruits peel essential oil was found very effective, 80-100% protection of Aedes mosquito bite for 210 minutes [59].

One controlled study evaluated in India, the efficacy of a cream formulation containing 5% neem oil against Cx. quinquefasciatus and An.culicifacies.  the cream 4-5 g was applied to the exposed skin areas of human volunteers in Ghaziabad, India in the summer months of May/June and the monsoon months of August/September. Neem cream was found to offer 82% protection against Culex bites and 100% protection against Anopheles bites, as compared to untreated controls [60]. Mechanism of action of the essential oil extracts are not quite clear, but according to Koul et al., [61], some constituents of the essential oil interfere with octopaminergic nervous system are absent in human and fishes, thereby, giving, it its safety and selectivity.

The report of the Poonkodi [62], showed that the predominant compounds were linalolol 1.8-cineol geranyl, D germacrene-cadinene, Epi-cadinolein the essential oil of Ocimum basilicum L. leaves in the study of Bilal et al., [63] report that linalolol 18.9%, limolele 30.9% and beta- phellandrene 15.3% and other amount of terpenoids were present in the essential oil of Ocimum basilicum leaves. Therefore, most study revealed that linalolol as major constituent of essential oil of Ocimum basilicum L. Different in chemical composition in essential oil were depending on the geographical sources, extraction methods and different used of solvent, climate factors and harvesting time.

In conclusion the essential oil of Ocimum basilicum L. leaves essential oil was effective to control Ae.aegypti larvae and protect over 80% of mosquito bite for 240 minutes at 0.1g/ml and 1ml of 50% Ocimum basilicum L. leaves essential oil from Aedes mosquito bite in day time in laboratory. Although semi field trial in Dagon Myothit North observed that 100% protection from mosquito bite in day time by 4 hourly application of leaves essential oil. From these results study indicated that Ocimum basilicum L. leaves essential oil exhibit larvicidal, ovicidal and repellent activities against dengue vector Aedes aegypti mosquitoes. Further analysis to isolate the active compound for larval control is under way in the pharmacology research laboratory. More study is needed to elucidate the essential oil activities against a wide range of mosquito species. The active compound responsible for repellent activity should be identified, which could be used to control different mosquito species in the future. These results could encourage the search for new active natural compounds offering an alternative to synthetic repellents and insecticides from other medicinal plants.

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