Essential Oils of Umbelliferae (Apiaceae) Family Taxa as Emerging Potent Agents for Mosquito Control

Warm-humid areas around the globe constitute the cradle of humanity, providing their inhabitants the most favorable environments for living and agricultural production. In this “Garden of Eden”, which spreads within the globe’s temperate and tropical zones, is also thriving an annoying but dangerous daemon, the mosquito. This little devil constitutes the main vector of malaria and human encephalitis, both infectious diseases that account as major threats of public health (Becker et al., 2003). Recently, these threats have been spread to a broader geographical area, as a consequence of their vectors (Aedes sp., Anopheles sp. and Culex sp.) introduction into metropolitan areas of northern hemisphere, such as Chicago (Tedesco et al., 2010), New York (Peterson et al., 2006) and Paris (Delaunay et al., 2009). Since mosquito breeding habitats in both urban and rural areas are man-made (Imbahale et al., 2010), there are several restrictions limiting the efforts towards the development of an integrated vector management system. Todate, the history of evolutions of malaria vector interventions is directly connected with the mosquito control tools development, concerning either environmental modifications/manipulations or their chemical and/or biological control (Kilama, 2009).


Introduction
Warm-humid areas around the globe constitute the cradle of humanity, providing their inhabitants the most favorable environments for living and agricultural production.In this "Garden of Eden", which spreads within the globe's temperate and tropical zones, is also thriving an annoying but dangerous daemon, the mosquito.This little devil constitutes the main vector of malaria and human encephalitis, both infectious diseases that account as major threats of public health (Becker et al., 2003).Recently, these threats have been spread to a broader geographical area, as a consequence of their vectors (Aedes sp., Anopheles sp. and Culex sp.) introduction into metropolitan areas of northern hemisphere, such as Chicago (Tedesco et al., 2010), New York (Peterson et al., 2006) and Paris (Delaunay et al., 2009).Since mosquito breeding habitats in both urban and rural areas are man-made (Imbahale et al., 2010), there are several restrictions limiting the efforts towards the development of an integrated vector management system.Todate, the history of evolutions of malaria vector interventions is directly connected with the mosquito control tools development, concerning either environmental modifications/manipulations or their chemical and/or biological control (Kilama, 2009).
In respect the chemical control, a significant milestone was the DichloroDiphenyl-Trichloroethane (DDT) synthesis by Zeidler in 1874.The DDT success was followed by the fast introduction of numerous chlorinated hydrocarbons, which were used in massive amounts for the control of mosquito-borne diseases (Ray, 2010).Despite their efficiency, the use of organochlorines had severe environmental impacts which were publicly (and dramatically) addressed by Carson (1962) in Silent Spring, initiating the development of insecticide resistant mosquito populations.These undesirable characteristics, in combination with concerns on public health risks, derived from the organochlorine residues detected in humans and animals, led to their ban in early 70's.Thus, they were replaced by less persistent chemicals, such as organophosphates, pyrethroids and avermectin derivates, Essential Oils of Umbelliferae (Apiaceae) Family Taxa as Emerging Potent Agents for Mosquito Control 615 various commercially successful molecules like pyrethrins.Todate, the search for novel, potent and safer pesticides from this deposit has already provided several candidates, either as pure compounds and/or their extracts.Specifically, various organic acids such as lactic and orthophosporic acids (Chakraborty et al., 2010), alkaloids (Talontsi et al., 2011) and plant proteins (Chowdhury et al., 2008) have been identified as efficient mosquito control agents.Furthermore, several plants were used as the maternal material to produce bioproducts which were applied against mosquitoes with hopeful results (Shaalan et al., 2005;Sukumar et al., 1991).On the other hand, the plant derived Essential Oils (EOs) constitute a special category of natural products that exhibit the major advantage -for the mosquito control endeavor-of exhibiting an insect oriented mode of action with low penetrability to the ecosystems that does not affect larger animals.In addition, the natural diversity of their constituents addresses effectively the problem of resistance development (Isman, 2000).

Umbelliferae (Apiaceae) family: A source of potent natural agrochemicals
Many EOs originated from diverse plant families have been considered and studied as potential sources of natural agrochemicals.In this respect, previous research results on Umbeliferae (Apiaceae) family plant materials revealed the significant acaricidal activities of butylidenepthalides isolated from Angelica acutiloba Kitagawa var.sugiyame Hikino (Kwon & Ahn, 2002) and the similar activity of the EO of Foeniculum vulgare, attributed to the presence of p-anisaldehyde and (+,−)-fenchone in the EO (Lee, 2004).These EOs were practically inactive in fumigant toxicity tests against Lycoriella mali though they are known to contain the active monoterpenes -pinene and -pinene (Choi et al., 2006), which are common constituents of many Umbelliferae EOs.Methanolic extracts of Angelica dahurica, Cnidium officinale, and Foeniculum vulgare were also tested against the Coleoptera Lasioderma sericorne, Sitophilus oryzae and Callosobruchus chinensis exhibiting a moderate activity only the second extract (Kim et al., 2003a;Kim et al., 2003b).Other EOs of this family screened as inactive against coleoptera were originated from the species Anethum graveolens L., Apium graveolens Houtt., Coriandrum sativum L., Cuminum cyminum L. and Petroselinum sativum L. (Regnault-Roger & Hamraoui, 1994;Papachristos & Stamopoulos, 2002).On the contrary, the EOs of Pimpinella anisum L. and Cuminum cyminum L. displayed excellent ovicidal and insecticidal activities against the Tribolium confusum du Val and the Ephestia kuehniella Zeller (Tunc et al., 2000).In addition, the aqueous extract of Pimpinella anisum exhibited good repellent effect against the adults of sweet potato whitefly Bemisia tabaci (Ateyyat et al., 2007).
These rather controversial results are not connected with the impressive activities that Umbelliferae EOs were found to exhibit against the Diptera, with the EO of Ammi visnaga displaying -among 19 EOs-the most potent ovicidal activity against Mayetiola destructor (Lamiri et al., 2001).In addition, tests against Droshophila melanogaster of furanocoumarins and pthalides isolated from Angelica acutiloba Kitagawa var.sugiyame Hikino revealed the hypothesis that the insecticidal properties of the plant extracts are connected with the acetylcholinesterase inhibition (Miyazawa et al., 2004).Finally, alkylpthalides originated from Cnidium officinale Makino were tested as extremely effective against Droshophila melanogaster (Tsukamoto et al., 2005).

Umbelliferae (Apiaceae) family: A strong focal point for mosquito control
Table 1 summarizes the test results against various mosquito species reported for all extracts and EOs derived from plants belonging to the Umbeliferae family.Same table also contains the test results of fourteen EOs, which appear herein for the first time.Results indicate that the organic phase of the Cryoptaenia canadensis extract is the most active against fourth instars of Culex pipiens, leading to the isolation -from the extract-of the acetylated very toxic (LC 50 values lower than 10 mgl -1 ) molecules of falcarinol and falcarindiol (Eckenbach et al., 1999).The larvicidal properties of hexane soluble fraction of Apium graveolens seeds -a plant with pleasant aroma-and three isolated compounds (sedanolide, senkyunolide-N, senkyunolide-J) against Aedes aegypti mosquitoes highlighted sedanolide as very active (100% mortality at 50 mgl -1 , Momin & Nair, 2001).As a consequence, a gel containing 5% of the Apium graveolens hexane extract was developed, providing full protection to volunteers from mosquito bites for two hours (Tuetun et al., 2009), while the ethanolic formulations from the same plant also provided protection against Aedes aegypti.Another formulation containing the aforementioned hexane extract and 5% vanillin showed strong repellent activities against different mosquito species (Tuetun et al., 2005, see also Table 1 for details).The crude seed extract had no adverse effects on human volunteers skins when tested for several anti-mosquito properties (Choochote et al., 2004).This plant's EO exhibits potent larvicidal activity against two laboratory-reared mosquito species, the malaria vector Anopheles dirus and the vector of dengue Aedes aegypti (Pitasawat et al., 2007).Among all EOs tested for mosquito control, the most potent was derived from Foeniculum vulgare, which caused the highest mortality against Aedes albopictus (Conti et al., 2010) and moderate against Anopheles dirus and Aedes aegypti (Pitasawat et al., 2007).Main component of this EO is methyl chavicol (more than 43%), while its methanolic extract (trans-anethole chemotype) was moderately active against Aedes aegypti, the yellow fever mosquito (Orozco & Lentz, 2005).The hexane fraction from its fruit-derived parts showed 99% repellency against Aedes aegypti, while the other fractions (chloroform, ethyl acetate and water: 37, 37 and 17% respectively) were practically inactive (Kim et al., 2002).Repellency and toxicity were also studied against Culex pipiens (Trabousli et al., 2005), indicating that the EO of Foeniculum vulgare was the most effective, while the repellency assays revealed protection time for almost one hour when applied at concentration of 3%.
Among the methanolic extracts of 118 Euroasiatic plants, tested for their larvicidal effects against Culex quinquefasciatus, the species Ammi visnaga and Seseli pallasii were determined as two of the most toxic materials tested, with LC 50 values lower than 10 mgl -1 (Pavela, 2008(Pavela, , 2009)).On the other hand, the extracts of Angelica archangelica and Imperatoria ostruthium exhibited LC 50 values lower than 70 mgl -1 , while Seseli tortuosum and Ferula lancerottensis displayed moderate larvicidal activity (LC 50 values around 430 mgl -1 ).The only inactive Apiaceae plant tested was Ferula assa-foetida (LC 50 value higher of 1000 mgl - 1 ), with the EO of Ferula galbaniflua exhibiting the weakest activity against Culex quinquefasciatus and Anopheles stephensi (mortality level less than 14% of dead larvae after 48 hours, Amer & Mehlhorn, 2006a).The same authors also reported that Anopheles stephensi was the most resistant to dill (Anethum graveolens), while the Culex quinquefasciatus the more sensitive.Dill was also evaluated for persistency to larvicidal effects under different conditions for 1 month after the preparation of its solutions.In all cases (open, closed, in light or in dark) the EO was active only when was used immediately after preparation (Amer & Mehlhorn, 2006b).
Finally, an interesting result was obtained during the study of several EOs using coupled gas chromatography-electroantennographic detection (GC-EAD), on the hypothesis that compounds can be detected by the antennae of the yellow fever mosquito, Aedes aegypti.Thus, cumin aldehyde and cumin alcohol the Cuminum cynimum EO components were identified as such molecules.It must be noted that for both components, their EO (cumin oil) was also EAD-active (Campbell et al., 2011)

Greek Umbelliferae (Apiaceae) plants extract activities against Culex pipiens mosquitoes
The larvicidal activity of the EO obtained from the stem of Greek Foeniculum vulgare was determined against Culex pipiens larvae, while methyl chavicol was determined as its main component (more than 32%).Although the LC 50 value of methyl chavicol was more than 80 mgl -1 , the respective EO was determined as 2.1-fold more toxic (Manolakou et al., 2009).As a continuation of our ongoing efforts to exploit the use of natural products for the development of environmentally friendly means for the mosquito population control, our interest was stimulated on the investigation of Umbeliferae (Apiaceae) plants EOs.In this context, we report herein the chemical composition and larvicidal activity results for 14 EOs originated from different taxon obtained during Greek Umbelliferae biodiversity studies (Table 1).(Pimenov & Leonov, 1993;Tutin et al., 1968).

Essential oils isolation
The freshly collected plant materials (steams, leaves and flowers) were washed thoroughly, chopped off finely and subjected to steam distillation in a Clevenger-type apparatus, using the Microwave Accelerated Reaction System (MARS 5) at 1400 W for 40 min with 3 L of H 2 O in order to obtain their EOs.The resulting oils were dried over anhydrous sodium sulphate and stored at 4 o C. The EO yield of each plant is included in Table 3.  Table 3. Essential oils yields.

Gas Chromatography-Mass Spectrometry (GC-MS) analyses
Gas Chromatography (GC).All GC analyses were carried out on a Agilent Technologies 7890A gas chromatograph, fitted with a HP 5MS 30m x 0.25mm x 0.25μm film thickness capillary column and FID.The column temperature was programmed from 60 to 280 °C at a initial rate of 3 °C/min.The injector and detector temperatures were programmed at 230 and 300 °C, respectively.Helium was used as the carrier gas at a flow rate 1 ml/min.
Gas Chromatography-Mass Spectrometry (GC-MS).The GCMS analyses were performed on the same instrument using the Agilent 5957C, VL MS Detector with Triple-Axis Detector system operating in EI mode (equipped with a HP 5MS 30m x 0.25mm x 0.25μm film thickness capillary column), using He (1 ml/min) as the carrier gas.The initial temperature of the column was 60 °C.The column was heated gradually to 280 °C with a 3 °C/min rate.The identification of the compounds was based on comparison of their retention indices (RI) (Van den Dool & Kratz, 1963), obtained using various n-alkanes (C9-C24).Also, their EImass spectra were compared with the NIST/NBS and Wiley library spectra and the literature (Adams, 1995;Massada, 1976).Additionally, the identity of the indicated phytochemicals was confirmed by comparison with available authentic samples.

Mosquito rearing
A colony of the species Culex pipiens biotype molestus is maintained for more than 25 years in the laboratory of Entomology of the Benaki Phytopathological Institute, Kifissia, Greece.Adult mosquitoes are kept in wooden framed cages (33x33x33 cm) with a 32x32 mesh at 25±2 o C, 80±2% relative humidity and photoperiod of 14:10 (L:D) h.Cotton wicks saturated with 10% sucrose solution are used as food source.Females lay eggs in round, plastic containers (10 cm diameter x 5 cm depth) filled with 150 ml of tap water.Egg rafts are removed daily and placed in cylindrical enamel pans (with diameter of 35 cm and 10 cm deep), in order to hatch.Larvae are reared under the same conditions of temperature and light and are fed daily with baby fish food (TetraMin, Baby Fish Food) at a concentration of 0.25 gl -1 of water until pupation.Pupae are then collected and introduced into the adult rearing cages.

Larvicidal bioassays
Stock solutions of EOs tested were prepared in ethanol and maintained in a freezer as 1% mgl -1 solutions.They were dissolved in double distilled water to produce solutions of the tested materials in concentrations ranging from 5 to 150 mgl -1 .Prior to biological determinations the toxicity of each EO was evaluated (data not shown).
The larval mortality bioassays were carried out according to the test method for larval susceptibility, proposed by the World Health Organization (WHO, 1981).Twenty 3 rd to 4 th instar larvae of the species Culex pipiens biotype molestus were collected from the colony, placed in a glass beaker with 250 ml of aqueous suspension of the tested material at various concentrations and an emulsifier was added in the final test solution (less than 0.05%).Four replicates were made per each concentration and a control treatment with tap water and emulsifier was also included.Beakers with larvae were placed at 25±2 o C, 80±2% relative humidity and photoperiod of 14:10 h (L:D).

Data analysis
Larvicidal effect was recorded 48 h after treatment.Data obtained from each dose-larvicidal bioassay (total mortality, mgl -1 concentration in water) were subjected to probit analysis in which probit-transformed mortality was regressed against log 10 -transformed dose; LC 50 , LC 90 values, and slopes were calculated (SPSS 11.0).

Phytochemical analysis
Fourteen distinct Umbeliferae taxa (twelve genera) are studied herein, one of which is endemic to Greece (Thamnosciadium Hartvig).It must be noted that there are no literature reports and studies on the EOs and their chemical compostitions for the material obtained (SP) and Smyrnium rotundifolium Miller (SR).In addition, the discussion section on the related taxa EOs compositions includes ten (out of twelve) genera studied herein, since there are also no previous reports on the composition of EOs obtained from Conium L. and Thamnosciadium Hartvig genera.
In total seventy phytochemicals, representing 76.64 to 99.83 % of the respective EOs samples have been identified as their constituents using combined GC and GC/MS analyses and in certain occasions verified by NMR studies.The detailed qualitative and quantitative analytical data of the main constituents of steam volatiles (and their respective retention indices) are presented in Table 4.
The EOs of Pimpinella L. have been thoroughly studied, mainly because the application of their several taxa as culinary herbs and/or spices.Though the EOs of fourteen ( 14) taxa were studied, only one (Tabanca et al., 2005) refers to PP (Pimpinella peregrina L.) and none to PT (Pimpinella tragium ssp tragium VIII) and PR (Pimpinella rigidulla Boiss.& Orph.H. Wolf).
Major components of the EO of Scaligeia cretica (Miller) Boiss (SC) are -pinene, -farnesene and germacrene D, which have also been detected in previous studies on the EOs of Scaligeria DC.In this respect, the EO of S. lazica contains -farnesene as major and -pinene, germacrene D as minor components (Baser et al., 1993).On the contrary, the EO of S. tripartite contains -farnesene and germacrene D as minor compounds, while -pinene is absent (Tabanca et al., 2007).Compounds assayed herein and never reported before in the EOs of Scaligeria DC are -terpineol, -elemene and -humulene.

Larvicidal assays
The investigated EOs were evaluated ─for the first time─ in respect to their larvicidal activities against 3 rd -4 th instar larvae of Culex pipiens.The relative results expressed as the respective LC 50 and LC 90 values are included in Table 5.Among the EOs tested only two were rather inactive (AS and PP, displaying LC 50 values above 150 mgl -1 ), while the EOs of SC and SP were moderately active displaying LC 50 values above 100 mgl -1 (111.99 and 122.54 mgl -1 respectively).

Angelica sylvestris >150
Pimpinella peregrina >150 a LC values are expressed in mgl -1 and they are considered significantly different when 95% CL fail to overlap.

a
Comparison of mass spectra with MS libraries and retention times b Comparison of experimental RI with reported RI c Comparison with authentic compounds RI: Retention indices calculated against C8 to C24 n-alkanes on the HP 5MS column.

Table 2 .
A voucher specimen of each plant is deposited in the herbarium of the Agricultural University of Athens, Athens, Greece.

Table 4 .
Chemical constituents of the essential oils tested.

Table 5 .
LC 50 and LC 90 values for the tested essential oils against larvae of Culex pipiens biotype molestus.Integrated Pest Management and Pest Control -Current and Future Tactics 628The EO derived from the endemic in Greece plant Athamanta densa was determined as the most active since displayed the highest toxicity against mosquito larvae, with LC 50 value 10.15 mgl -1 .The EO tested contains a series of compounds which were not found in the other EOs tested, such as bisabolene and the unidentified compounds C 14 H 30 O, C 12 H 25 O 2 N and C 13 H 27 O 2 N, which have to study more thoroughly in order to determine their activities.The remaining EOs (PR, TJ, PT, PN, CH, LP, FN, SR and PO) displayed LC 50 values ranging from 40.31 to 86.46 mgl -1 .No significant relationship between toxicity and phytochemical content was detected. www.intechopen.com