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LarvicidalactivityagainstAedesaegyptiofFoeniculumvulgareessentialoilsfromPortugalandCapeVerde

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Larvicidal Activity Against Aedes aegypti of Foeniculum vulgare Essential Oils from Portugal and Cape Verde

Diara Kady Rochaa,b*, Olivia Matosc,b, Maria Teresa Novoa,b, Ana Cristina Figueiredod, Manuel Delgadoe and Cristina Moiteirof aInstituto de Higiene e Medicina Tropical, UNL, Rua Junqueira, 100, 1349-008 Lisboa, Portugal

bUnidade de Parasitologia e Microbiologia Médicas, IHMT, Rua Junqueira, 100, 1349-008 Lisboa, Portugal

cInstituto Nacional de Investigação Agrária e Veterinária, INIAV. Quinta do Marquês, Av. República, 2784-505 Oeiras, Portugal

dUniversidade de Lisboa, Faculdade de Ciências de Lisboa, Departamento de Biologia Vegetal, IBB, Centro de Biotecnologia Vegetal, C2, Piso 1, Campo Grande, 1749-016 Lisboa, Portugal

eDelegação do Ministério do Desenvolvimento Rural, MDR. Porto Novo, Santo Antão, Cabo Verde

fUniversidade de Lisboa, Faculdade de Ciências, Departamento de Química e Bioquímica; Centro de Química e Bioquímica, Campo Grande, 1749-016 Lisboa, Portugal dkrocha@fc.ul.pt

Received: November 7th, 2014; Accepted: February 24th, 2015

Dengue is a potentially fatal mosquito-borne infection with 50 million cases per year and 2.5 billion people vulnerable to the disease. This major public health problem has recurrent epidemics in Latin America and occurred recently in Cape Verde and Madeira Island. The lack of anti-viral treatment or vaccine makes the control of mosquito vectors a high option to prevent virus transmission. Essential oil (EO) constituents can affect insect’s behaviour, being potentially effective in pest control. The present study evaluated the potential use of Foeniculum vulgare (fennel) EO in the control of the dengue vector Aedes aegypti. EOs isolated from fennel aerial parts collected in Cape Verde and from a commercial fennel EO of Portugal were analysed by NMR, GC and GC-MS. trans-Anethole (32 and 30%, respectively), limonene (28 and 18%, respectively) and fenchone (10% in both cases) were the main compounds identified in the EOs isolated from fennel from Cape Verde and Portugal, respectively. The larvicidal activity of the EOs and its major constituents were evaluated, using WHO procedures, against third instar larvae of Ae. aegypti for 24 h. Pure compounds, such as limonene isomers, were also assayed. The lethal concentrations LC50, LC90 and LC99 were determined by probit analysis using mortality rates of bioassays. A 99% mortality of Ae. aegypti larvae was estimated at 37.1 and 52.4 µL L-l of fennel EOs from Cape Verde and Portugal, respectively. Bioassays showed that fennel EOs from both countries displayed strong larvicidal effect against Ae. aegypti, the Cape Verde EO being as active as one of its major constituents, (-)-limonene. Keywords: Aedes aegypti, Vector control, Larvicidal activity, Aromatic plants, Foeniculum vulgare, Essential oils. Mosquitoes are vectors of diseases like malaria, lymphatic filariasis, dengue and yellow fever, among other arboviruses [1]. These diseases cause high levels of mortality and/or morbidity, especially in countries from subtropical and tropical regions, where they are more prevalent, with consequent social and economic impacts [2]. Aedes aegypti (Linnaeus 1762) is the main vector of dengue and yellow fever (via an urban cycle of transmission). It is an invasive species, adapted to human environments, promptly feeding on humans, and which breeds mainly in domestic or peridomestic areas. A rapid rise in urban populations, without proper urbanization, is placing a growing number of people in contact with the vector. Consequently, the number of dengue infections has increased worldwide over the last decades, with the occurrence of serious outbreaks with haemorrhagic fever [3]. In the African archipelago of Cape Verde, the first dengue epidemic, with 21,000 cases in 2009, demonstrated that dengue virus is still expanding into new territories [4-5]. In 2012, an outbreak of dengue fever in Madeira Island was reported [6], which proves that this infection is not only confined to the tropical region. Vector control remains an important component of the strategies to fight vector-borne diseases. Mosquito control has been based on continued applications of organophosphates and insect growth regulators, such as diflubenzuron and methoprene. Vector control

relies mainly on the use of insecticides applied to mosquito larvae habitats or, indoors, against adult mosquitoes. However, insecticide resistance has evolved in many Ae. aegypti mosquito populations worldwide [7-8]. Environmentally friendly compounds with high larval mortality effect are currently needed. Products of plant origin have been assessed for the detection of biocide substances that might reduce, or replace, the use of organophosphate and pyrethroid insecticides for disease vector control. Essential oils (EOs) have a broad spectrum of bioactivity due to the presence of several active components [9]. It is known that several plant EOs or plant extracts have toxic effects on mosquito species. In particular, fennel extracts have been reported as having insecticidal activity against different mites and insects [10], including disease mosquito vectors [11-15]. Their lipophilic nature facilitates them to interfere with basic metabolic, biochemical, physiological and behavioural functions of insects. Foeniculum vulgare Mill. (fennel) is cultivated in every country surrounding the Mediterranean Sea, where it is used extensively in cooking. Several fennel parts are edible (bulbs, leaves, stalks and fruits), and the plant is medicinally used as secretomotor, secretolytic and antiseptic expectorant, spasmolytic, carminative, diuretic, anti-inflammatory and as a constituent of cosmetic products [16]. Recently, it was reported that fennel EOs possessed

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678 Natural Product Communications Vol. 10 (4) 2015 Rocha et al.

emmenagogue and galactagogue properties for lactating women [17]. There is a great diversity in the composition of commercial fennel EOs. The Committee on Herbal Medicinal Products (HMPC) of the European Medicines Agency includes the monographs for the two varieties of fennel, namely F. vulgare Miller subsp. var. vulgare (bitter fennel) and F. vulgare Miller subsp. vulgare var. dulce (Miller) Thellung (sweet fennel), and their chemical profiles [16]. According to the European Pharmacopeia bitter fennel EO is characterized by a minimum of 60% trans-anethole, 15% fenchone, 1-10% pinene, ≤6% estragole (methyl chavicol) and 1-5% limonene. Sweet fennel EO contains a minimum of 80% anethole and a maximum of 10% estragole and 8% fenchone [18]. However, the International Organization for Standardization defines two bitter fennel EO types, the trans-anethole and the phellandrene type [19]. Commercial fennel fruit EOs from pharmacies in different countries were characterized by a large variation of trans-anethole (34.8-82.0%), fenchone (1.6-22.8%), estragole (2.4-17.0%), limonene (0.8-16.5%), and cis-anethole (0.1-8.6%) [20]. Portuguese fennel EOs showed the existence of the chemotypes trans-anethole, trans-anethole/fenchone and trans-anethole/methyl chavicol [21]. A study of the morphological data and chemical characterization of the EOs of wild Portuguese fennel fruits showed that this species presents high hybridization rates and a wide number of intermediate forms with highly variable characters. However, despite such difference, it is evident that Portuguese fennel EO displays the presence of methyl chavicol and trans-anethole chemovarieties [22]. The main goal of this study was to evaluate, for the first time, the larvicidal properties of EOs from F. vulgare of different geographical origins against the mosquito vector Ae. aegypti. The chemical profiles of these EOs were analysed through 13C NMR, GC and GC-MS techniques, which permitted the identification of characteristic fingerprinting markers of fennel EOs. Additionally, the LC50, LC90 and LC99 values for major constituents of EOs were also determined. Hydrodistillation of F. vulgare aerial parts collected in Cape Verde produced a yellow EO with a yield of 0.6%, v/w. The application of 13C NMR spectroscopy to the field of EOs analysis is a very fast and reliable methodology, which allows analysing samples at room temperature without degradation of unstable components. 13C APT NMR analysis of EOs isolated from fennel aerial parts collected in Cape Verde and Portugal showed similar chemical profiles, with trans-anethole (1) and limonene (2) (Figure 1) as their main constituents (Table 1). 13C APT NMR analysis allowed attribution of the carbons of the aromatic ring at δ 114.0 (C-2 and C-6) and 126.9 (C-3 and 5), an exocyclic double bound at δ 130.5 (C-1´) and 123.4 (C-2´), methyl groups at δ 55.2 (C-7) and 18.5 (C-3´), and quaternary carbons at δ 158.7 and 130.9 (C-1 and C-4), which are characteristic of trans-anethole. Moreover, in the APT 13C spectra were also well-distinguished resonances for limonene (2), at δ 150.2, 120.7 and 108.4 assigned to unsaturated carbons (C-1´, C-2 and C-2´), a quaternary carbons at δ 133.7 and 41.7 (C-1 and C-4), methylene groups at 30.8, 30.6 and 28.0 ppm (C-6, C-3 and C-5) and methyl groups of double bounds at δ 23.5 and 20.9 (C-7 and C-3´). This preliminary study evidenced that the EOs from both plant origins exhibited similar chemical profiles, with trans-anethole (1) and limonene (2) as their main constituents (Table 1). The data obtained are in agreement with that present in the literature [23]. This approach allowed a prompt identification and evaluation of both EOs and its main constituents, simultaneously.

Table 1: Assignment of main peaks of 13C NMR spectra of F. vulgare EOs.

Peak Compounds (ppm) Assignment 1 trans-Anethole 1 18.5 CH3 (C-3´) 2 Limonene 2 20.9 CH3 (C-3´) 3 Limonene 2 23.5 CH3 (C-7) 4 Limonene 2 30.6 CH2 (C-5) 5 Limonene 2 30.8 CH2 (C-6) 6 Limonene 2 28.0 CH2 (C-3) 7 Limonene 2 41.2 CH (C-4) 8 trans-Anethole 1 55.4 CH3 (C-7) 9 Limonene 2 108.4 CH2 (C-2´) 10 trans-Anethole 1 114.0 CH (C-2 and C-6) 11 Limonene 2 120.7 CH= (C-2) 12 trans-Anethole 1 123.4 CH (C-2´) 13 trans-Anethole 1 126.9 CH (C-3 and C-5) 14 trans-Anethole 1 130.5 CH= (C-1´) 15 trans-Anethole 1 130.9 Cq (C-4) 16 Limonene 2 150.2 Cq=C (C-1´) 17 trans-Anethole 1 158.7 Cq (C-1)

Figure 1: Chemical structures of main constituents of F. vulgare EOs. GC and GC-MS analysis of Cape Verde and Portugal fennel EOs led to identification of 55 and 36 compounds representing 98 and 99% of the total volatiles, respectively (Table 2). Both EOs were characterized by high contents of monoterpene hydrocarbons (47 and 53%, respectively) and phenylpropanoids (34% in both cases), followed by oxygen-containing monoterpenes (16 and 13%, respectively). The EOs of F. vulgare from Cape Verde and Portugal showed similar major constituents, trans-anethole (32 and 30%, respectively), limonene (28 and 18%, respectively) and fenchone (10% in both cases) (Table 2). Other minor compounds such as -pinene (7 and 5%, respectively), -pinene (5 and 12%, respectively), p-cymene (2 and 7%, respectively), -phellandrene (1 and 4%, respectively) and estragole (1 and 3%, respectively), were also identified in both EOs. The range of the main components found in the present work for the commercial fennel EO from Portugal is in agreement with previous data on the essentials oil isolated from dried aerial parts of F. vulgare collected in Portugal, namely high in trans-anethole (31-36%), -pinene (14-20%) and limonene (11-13%) [24], and low in estragole, which is dominant in the fruit EOs [22-24]. The chemical composition of the presently studied EOs was also quite similar to those obtained from fruits of Egyptian fennel cultivar var. dulce [16]. To consider the fennel EO from Cape Verde as a chemotype, due to the high levels of trans-anethole (1) and limonene (2), a morphological and chemical study for better characterization is still needed. The high content of limonene in the samples does not allow the characterization of the fennel EOs studied as sweet or bitter based on the European Pharmacopeia classification [25]. However, the presence of limonene undoubtedly contributed to the relevant larvicidal effects obtained and evaluation of both EOs and its main constituents, simultaneously. EOs obtained from both Cape Verde and Portuguese fennel exhibited significantly high larvicidal activity against Ae. aegypti third instar larvae (Table 3). Portuguese and Cape Verde fennel

Larvicidal activity against Aedes aegypti of Foeniculum vulgare essential oils Natural Product Communications Vol. 10 (4) 2015 679

Table 2: Chemical constituents of essential oils isolated from F. vulgare aerial parts collected in Cape Verde and a commercial sample from Portugal.

Foeniculum vulgare Components RIa Cape Verde Portugal Tricyclene 921 t t α-Thujene 924 t 0.1 α-Pinene 930 4.9 11.9 Camphene 938 0.4 0.9 Sabinene 958 t 0.3 β-Pinene 963 7.3 5.2 2-Pentyl furan 973 t t β-Myrcene 975 1.8 3.7 α-Phellandrene 995 0.7 4.4 δ-3-Carene 1000 0.3 α-Terpinene 1002 t 0.1 p-Cymene 1003 1.9 6.9 1,8-Cineole 1005 t 2.5 β-Phellandrene 1005 t t Limonene 1009 28.4 17.5 cis-β-Ocimene 1017 0.4 0.7 trans-β-Ocimene 1027 t 0.2 γ-Terpinene 1035 1.1 0.3 Fenchone 1050 9.7 9.6 Terpinolene 1064 0.1 0.1 Linalool 1074 t 0.1 Isopentyl isovalerate 1084 t t cis-Limonene oxide 1095 0.1 t Camphor 1102 0.1 t trans-Limonene oxide 1112 1.0 t cis-Verbenol 1113 t t trans-Verbenol 1114 t t Menthone 1120 0.8 Pinocarvone 1121 0.1 iso-Menthone 1126 t Menthol 1148 t Terpinen-4-ol 1148 0.3 0.1 Myrtenal 1153 t cis-Dihydro carvone 1159 t α-Terpineol 1159 t 0.2 Methyl chavicol (= Estragole) 1163 1.3 3.1 Myrtenol 1168 0.1 trans-Carveol 1189 0.5 cis-Carveol 1202 0.3 t Pulegone 1210 0.8 p-Anisaldehyde * 1210 0.8 0.5 Carvone 1210 t β-Fenchyl acetate 1212 1.7 cis-Anethole 1220 t 0.3 trans-Anethole 1254 31.8 29.8 Bornyl acetate 1265 t Carvacrol 1286 0.2 t α-Copaene 1375 0.2 β-Caryophyllene 1414 t t trans-α-Bergamotene 1434 t α-Humulene 1447 t trans-Methyl iso-eugenol 1471 0.4 ar- curcumene 1474 0.1 Germacrene D 1474 0.1 δ-Cadinene 1505 0.1 Elemicin 1525 t % Identification 97.6 98.9 Grouped components Monoterpene hydrocarbon 47.0 52.6 Oxygen-containing monoterpenes 15.8 12.6 Sesquiterpene hydrocarbon 0.4 t Phenylpropanoids 34.3 33.7 Others t t

a Retention index relative to C9-C16 n-alkanes on a DB-1 column; t - trace (<0.05); * Identification based on mass spectra only.

EOs, at 52.4 µL L-1 and 37.1 µL L-1 respectively, showed 99% larval mortality. By applying ANOVA and Mann-Whitney tests, no significant difference was found between the variances of mosquito mortality observed after exposure to the different concentrations of each oil. However, the highest toxicity against third instar larvae was shown by fennel EOs from Cape Verde. Larvicidal activity of standard compounds, (-)-limonene, (+)-limonene, trans-anethole and estragole was also evaluated against the 3rd instar mosquito larvae (Table 3). trans-Anethole (LC50= 29.3 and LC90= 45.2 l l-1), revealed a larvicidal effect similar to that of the F. vulgare crude EO. However, the strong larvicidal results obtained seem to be related to the presence of (-)-

limonene (LC50= 13.0 and LC90= 23.2 l l-1). The results were submitted to Pearson regression coefficient analysis (sigma 2 tailed=0.01) showing high correlation between the larvicidal activity of the fennel EO from Cape Verde and (-)-limonene, the most active compound against this mosquito larvae. The bioassays for larvicidal activity revealed that 3rd instar larvae of Ae. aegypti are susceptible to EO of F. vulgare from Cape Verde and Portugal, with LC50 = 23.3; 28.2 L L-1 and LC90 = 30.1; 39.6 L L-1, respectively. The larvicidal effects of the fennel EOs are high and comparable with one of the major compounds, limonene, as confirmed by the Pearson correlation test (two-tailed). Larvicidal effects observed with both Cape Verde and Portuguese fennel EOs are relevant, being stronger against Ae. aegypti than those of lemon grass (Cymbopogon flexuosus), eucalyptus (Eucalyptus globulus), palmarosa (Cymbopogon martinii), and orange (Citrus sinensis), which are referred to as having, LC50 = 138.4, 106.2, 87.9 and 85.9 ppm and LC90 = 759.7, 198.8, 156.1 and 153.3 ppm, respectively [15]. Reports on the effectiveness of fennel EO against larvae of Ae. aegypti were found, but the plants from Portugal and Cape Verde were screened for the first time. At 40 mg L-1, fennel EO induced 50% mortality on second instar larvae 2 h after exposure, and at 60 mg L-1 ensured 90% mortality of fourth instar larvae after 4 h exposure [26]. The larvicidal activity of trans-anethole from other EOs against several mosquito species has already been reported [26; 27; 28]. The LC50 and LC90 obtained in this work are in accordance with reports on the larvicidal activity of EO from fennel flowers against Culex pipiens larvae, despite its slightly lower level of trans-anethole and the significantly higher concentration of limonene [29]. Although the World Health Organization (WHO) does not specify any criteria for classifying the larvicidal potential of new products, some authors use the values of the minimum lethal concentration that eliminates 50% of the population (LC50) as a criterion for activity. Specifically, if LC50<50 mg L-1 the product is considered very active, if 50 < LC50 < 100 mg L-1 the product is considered active, and when LC50 > 750 mg L-1 the product is considered inactive [30-32]. Based on this norm the fennel EOs from Cape Verde and Portugal are undoubtedly in the class of very active. The same is true for the main compounds when separately assayed. However, the highest larvicidal activity corresponds to the EO from Cape Verde fennel (LC50= 23.3 μl L-1, which is equivalent to 4.4 mg L-1).

Microscopic analysis showed morphological changes in the anal papillae of Ae. aegypti larvae after contact with essential oils and standards of the F. vulgare major compounds, (+)-limonene and (-)-limonene (Figure 2). An accumulation of dark pigmentation was observed throughout the chest and on the base of the anal papillae insertion after contact with these two compounds. Larvae exposed to (-)-limonene showed major structural damage such as destruction of the gut and extrusion of hemolymphatic content. Larvae exposed to (+)-limonene exhibited darkening at the base of the anal papillae extended to the apex region. The larvae of Ae. aegypti exposed to either fennel EOs or (-)-limonene and (+)-limonene exhibited morphological changes, such as dark pigmentation through the chest and at the base of the anal papillae insertion. Such modification was not observed in the control group (water and Tween 20). Earlier study, regarding the evaluation of Azadirachta indica extracts against Culex quinquefasciatus (= fatigans) larvae, revealed similar morphological changes, including modifications in

680 Natural Product Communications Vol. 10 (4) 2015 Rocha et al.

Table 3: EOs and standards larvicidal activity on 3rd instar Ae. aegypti larvae, 24 h after exposure.

Lethal Concentration (µL L-1)* LC50 LC90 LC99 Equation line Correlation confidence

Fennel EOs

Cape Verde 23.3a (22.6-24.0) 30.1 (28.7-32.3) 37.1 (34.2-41.8) 11.1x-15.1 0.999

Portugal 28.2b (27.2-29.3) 39.6 (37.5-42.6) 52.4 (48.0-58.7) 9.4x-12.5 0.998

Standards

(-)-Limonene 13.0 (10.5-16.7) 23.2 (18.8-36.0) 35.7 (26.1-77.3) 5.3x-6.1 0.972

(+)-Limonene 17.8 (12.8-23.2) 41.0 (25.5-69.7) 59.1 (38.2-197.7) 4.1x-5.2 0.968

trans-Anethole 29.3 (28.0-30.6) 45.2 (42.5-48.8) 64.3 (58.2-73.3) 7.6x-10.4 0.981

Estragole 46.4 (42.5-50.0) 59.1 (54.2-69.6) 72.1 (63.3-95.3) 11.1x-18.5 0.972

* Confidence interval 95%, a Equivalent 4.4 mg L-1 and 23.3 ppm, b Equivalent 5.3 mg L-1 and 28.2 ppm.

Figure 2: Morphological alteration of Ae. aegypti larvae observed under stereomicroscope (A) and light microscope (B); Ap = anal papillae, cont = control; (-)-lim = (-)-limonene; (+)-lim = (+)-limonene.

head and abdomen pigmentation, but not the darkening of anal papillae [33]. In the current study, we observed discoloration of anal papillae, which are involved in the osmoregulation process, so we recommend further ultrastructural studies, to better understand the role of this organ and the fennel EO’s mode of action. However, according to these above mentioned criteria, it is clear that the EO of the aerial parts of F. vulgare has high larvicidal properties and that these results are very promising for further development of a larvicidal formulation. According to our findings, the larvicidal potential of F. vulgare EO was confirmed and it could be an alternative to synthetic insecticides being useful as an integrated strategy to prevent dengue by controlling the mosquito vector. In conclusion, the EOs of F. vulgare aerial parts and their main constituents exhibited strong activity against Ae. aegypti larvae. Biological assays displayed that one of the isomers of limonene has a strong and similar larvicidal effect to the fennel crude oil, indicating that enantioselectivity seems to play an important action in the activity of essential oils. These results suggest the potential application of fennel EO as a possible natural larvicide for the control of mosquitoes. However, additional studies of human toxicity and effects of the EO on non-target organisms are recommended before formulation development.

Experimental

Plant material: Aerial parts of wild F. vulgare Mill. from Cape Verde were collected during flowering in the Islands of Santo Antão. Five Kg of the material was collected from random plants over a large area. Voucher specimens were identified and deposited in the Herbarium of the Instituto de Investigação Científica Tropical, in Lisbon, under voucher number 3. Aerial plant parts were dried in a ventilated room at 40-45ºC, in continuous dark, for about 8 days, until reaching a constant dry weight, and then the essential oil extraction occurred immediately after the plant material was coarsely ground. Essential oil extraction: The EO was obtained by hydrodistillation for 3 h, using a modified Clevenger apparatus in a ratio of 100 g of dry aerial parts to 700 mL of deionized water according to the European Pharmacopoeia method [34]. The EO thus obtained was stored at 4ºC until further analysis. The EO of F. vulgare aerial parts from Portugal was commercially supplied by Terra Pura®; this was processed in a similar manner to the Cape Verde sample. Standard trans-anethole, estragole, (+)-limonene and (-)-limonene were purchased from Merck. NMR spectroscopic analysis: 13C NMR spectra were recorded on a Bruker Avance 400 spectrometer operating at 100.61 MHz, equipped with a 5 mm QNP probe at room temperature. The EO (100 mg) was dissolved in deuterochloroform (0.5 mL) and all

Larvicidal activity against Aedes aegypti of Foeniculum vulgare essential oils Natural Product Communications Vol. 10 (4) 2015 681

chemical shift values given in ppm ( tetramethylsilane (TMS) was the internal standard. 13C NMR spectra were recorded with the following parameters: spectral width 23.98 kHz under low-power proton decoupling and 13C pulse width of 9.0 µs, 2.0 s relaxation delay (D1) between scans, acquisition time 1.366 s, line broadening 1.0 Hz, for 64 k data points. An APT experiment from the Bruker library was used with 1 k accumulated scans and the plot limits were +219 to -19 ppm. Chemical shifts and peak attributions of 13C NMR spectra were made according to literature data [35], and trans-anethole, and limonene standard spectra. GC and GC-MS analysis: Gas chromatographic (GC) analyses were performed using a Perkin Elmer Clarus 400 gas chromatograph equipped with two flame ionization detectors (FIDs), a data handling system and a vaporizing injector port into which two columns of different polarities were installed: a DB-1 fused-silica column (polydimethylsiloxane, 30 m x 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific Inc., Rancho Cordova, CA, USA) and a DB-17HT fused-silica column [(50% phenyl)-methylpolysiloxane, 30 m x 0.25 mm i.d., film thickness 0.15 µm; J & W Scientific Inc.]. Oven temperature was programmed for 45-175°C, at 3°C/min, subsequently at 15°C/min up to 300°C, and then held isothermal for 10 min; injector and detector temperatures, 280°C and 300°C, respectively; carrier gas, hydrogen, adjusted to a linear velocity of 30 cm s-1. The samples were injected using a split sampling technique, ratio 1:50. The volume of injection was 0.1 µL of a n-pentane-oil solution (1:1). The percentage composition of the essential oil was computed by the normalization method from the GC peak areas, calculated as mean values of 2 injections from each essential oil, without using correction factors. The Gas Chromatography-Mass Spectrometry (GC-MS) unit consisted of a Perkin Elmer Clarus 600 gas chromatograph, equipped with DB-1 fused-silica column (30 m x 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific, Inc.), and interfaced with a Perkin-Elmer Clarus 600T mass spectrometer (software version 4.1, Perkin Elmer, Shelton, CT, USA). Injector and oven temperatures were as above; transfer line temperature, 280°C; ion source temperature, 220°C; carrier gas, helium, adjusted to a linear velocity of 30 cm/s; split ratio, 1:40; ionization energy, 70 eV; scan range, 40-300 u; scan time, 1 s. The identity of the components was assigned by comparison of their retention indices, relative to C9-C16 n-alkane indices and GC-MS from a home-made library, constructed based on the analyses of reference oils, laboratory-synthesised components and commercial available standards. Biological assays

Target mosquito colony: Immature stages of Ae. aegypti were collected in Santiago Island, Cape Verde. From these larvae, a colony has been installed and maintained for 6 generations at 26±2ºC and 70-80% relative humidity, under a photoperiod of 12:12h (light/dark) in a secure insectarium, free of exposure to pathogens, insecticides and repellents. Larvae were fed on triturated fish food. The adults were provided with continuous access to 10% glucose water solution and anaesthetized Rattus rattus was used as a blood meal source for the females.

Assay procedures: Different EO concentrations were tested on the third larval stage of Ae. aegypti. Sets of 25 third instar larvae per concentration were exposed to F. vulgare EO and its major compounds, trans-anethole and (-)- and (+)-limonene stereoisomers. Although the amount of estragole in both Cape Verdean and Portuguese EOs was low, this compound was also included in biological assays considering the relevance of the Portuguese fennel trans-anethole and methyl chavicol (=estragole) chemovarieties, associated with the knowledge that these two monoterpenes are isomers, both being referred to as active against insects [36]. After preliminary bioassays to optimize the concentrations, the larvae were exposed to 5.0, 6.0, 7.0 or 10.0 µL of Cape Verde fennel EO and to 6.0, 7.0, 9.0 or 12.0 µL of Portugal fennel EO. For each concentration assayed, either the essential oils or compounds were prepared in Eppendorf vials containing 0.04% of Tween 20® (Sigma, 99% purity) in 1 mL of distilled water and then transferred to glass containers with distilled water at a final volume of 250 mL. For all the experiments, 4 replicates of each essential oil or compound concentration were prepared, including negative controls with water and Tween® 20 and only with distilled water. Mortality of larvae exposed to each treatment was determined 24 h after contact. Larvae unable to reach the water surface when touched were considered dead [37]. Morphological analysis of larvae after EO contact: A preliminary evaluation of the mode of action of F. vulgare EOs and their major compounds was carried out through stereomicroscope and light microscope observation. For this purpose, larvae exposed to treatments with different concentrations and negative controls were preserved in 2 % glycerine alcohol. Groups of 5 larvae were mounted between slide and coverslip in Hoyer medium for further observation and comparison between groups. Statistical analysis: Mortality results from bioassays were recorded 24 h after treatment. Data from each treatment were subjected to probit analysis [38] in which probit-transformed mortality was regressed against the log10-transformed dose allowing, when statistically significant, LC50, LC90, and LC99 values and slope estimation (SPSS). To evaluate the hypothesis of equality or differences between two means and ANOVA, parametric tests were applied, including Student's t test for independent samples, which allows comparison of the variability of the average of all samples with the intrinsic variability of the samples. For the sake of accuracy, without disregarding the results of the Kolmogorov-Smirnov test, samples where normality was not confirmed (p <α) were selected, by considering the Mann-Whitney test, which is an alternative nonparametric method used for comparison of ordinal or quantitative variables between two independent groups, when the distribution is not symmetric (normal).

Acknowledgements - The authors thank to Doctor Cândida Liberato for the identification of the plant specimens and Doctor Ana Amaro for her help in the stereomicroscopic analysis. This work was supported by Diara Rocha PhD Grant, Fundação Calouste Gulbenkian (Neglected Tropical Diseases for PALOP students) by UPMM/IHMT funds and National Funds through the FCT under Pest-OE/EQB/LA0023/2011 and Pest-OE/QUI/UI0612/2013.

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Volume 10, Number 4

Contents

Original Paper

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Bioactive Oleanane Glycosides from Polyscias duplicata from the Madagascar Dry Forest Alexander L. Eaton, Peggy J. Brodie, Martin W. Callmander, Roland Rakotondrajaona, Etienne Rakotobe, Vincent E. Rasamison and David G. I. Kingston 567

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Continued inside backcover