Chemosphere 104 (2014) 37–43

Contents lists available at ScienceDirect

Chemosphere

journal homepage: www.elsevier .com/locate /chemosphere

Larvicidal activity of Mentha x villosa Hudson essential oil, rotundifoloneand derivatives

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.10.035

⇑ Corresponding author. Tel.: +55 7988329710.E-mail address: damiao_desousa@yahoo.com.br (D.P. de Sousa).

Tamires Cardoso Lima a, Tayane Kayne Mariano da Silva a, Fabiana Lima Silva b, José Maria Barbosa-Filho b,Márcia Ortiz Mayo Marques c, Roseli La Corte Santos d, Sócrates Cabral de Holanda Cavalcanti a,Damião Pergentino de Sousa b,⇑a Department of Pharmacy, Federal University of Sergipe, CEP 49100-000 São Cristóvão, Sergipe, Brazilb Department of Pharmaceutical Sciences, Federal University of Paraíba, CEP 58051-970 João Pessoa, Paraíba, Brazilc Center of Genetics, Molecular Biology and Phytochemistry, Agronomic Institute, CEP 13001-970 Campinas, São Paulo, Brazild Department of Morphology, Federal University of Sergipe, CEP 49100-000 São Cristóvão, Sergipe, Brazil

h i g h l i g h t s

� The Mentha x villosa Hudson essential oil exhibited toxic effects against Aedes aegypti mosquitoes larvae.� Rotundifolone, the major constituent of the Mentha x villosa Hudson essential oil, exhibited larvicidal activity.� The study showed the structural characteristics which may contribute to the larvicidal activity of rotundifolone and its analogues.

a r t i c l e i n f o

Article history:Received 3 July 2013Received in revised form 6 October 2013Accepted 8 October 2013Available online 22 November 2013

Keywords:Larvicidal activityEssential oilAedes aegyptiTerpeneDengue

a b s t r a c t

The aim of this study was to evaluate the larvicidal activity of Mentha x villosa essential oil (MVEO) and itsmajor constituent, rotundifolone, against larvae of Aedes aegypti. Additionally, a set of 15 analogues of therotundifolone were evaluated to identify the molecular characteristics which contribute to the larvicidaleffect. The results from the present study showed that the MVEO exhibited outstanding toxic effectsagainst Ae. aegypti larvae (LC50 = 45.0 ppm). Rotundifolone exhibited reasonable larvicidal activity(LC50 = 62.5 ppm). With respect to comparative study of rotundifolone and its analogues, all tested com-pounds were less potent than rotundifolone, except (�)-limonene. In general, replacement of C–C doublebonds by epoxides groups decreases the larvicidal potency. The presence of a,b-unsaturated carbonylscontributes to the larvicidal toxicity. The addition of hydroxyl groups in the chemical structure resultedin less potent compounds. Furthermore, the enantioselectivity seems to play an important role for thelarvicidal toxicity.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Dengue is an infectious disease caused by an arbovirus trans-mitted to humans by the bite of female mosquitoes of the genusAedes. Among many vectors, Aedes aegypti L. (Diptera: Culicidae)is the one of utmost epidemiological importance (Ligon, 2005;Coelho et al., 2009). Over the past 60 years the incidence, distribu-tion, and clinical severity of dengue have increased dramatically;resulting in the most important viral disease transmitted byarthropod vectors in terms of mortality and morbidity(Rigau-Pérez et al., 1998; Rosen, 1999).

In the absence of effective and safe vaccines for dengueprevention, the epidemics control is accomplished by monitoring

mosquitoes breeding sites and by the use of synthetic chemicalinsecticides to control the spreading of adult or larval forms ofAe. aegypti (Tauil, 2001; Porto et al., 2008). Although the chemicalproducts are effective, the continuous use in large scale has con-tributed to the emergence of many negative consequences, suchas, the appearance of insecticide resistance in dengue vectors lead-ing to decreased efficacy and adverse effects on non-target species,including humans (Zahran and Abdelgaleil, 2011). These problemshave persuaded numerous researches in order to develop alterna-tive strategies using natural products to control Ae. aegypti prolif-eration. In this direction, the plant essential oils and their majorconstituents have received much attention as potential bioactiveagents against mosquito vector (Cheng et al., 2009; Waliwitiyaet al., 2009; Zahran and Abdelgaleil, 2011).

The genus Mentha is represented by about 19 species and 13natural hybrids and it is an important member of the family

38 T.C. Lima et al. / Chemosphere 104 (2014) 37–43

Lamiaceae (Kumar et al., 2011a). Various essential oils of Menthahave been described in the literature for its insecticidal properties,such as the essential oil of Mentha pulegium (Franzios et al., 1997),Mentha piperita (Ansari et al., 2000), Mentha spicata, Mentha longi-folia, and Mentha suaveolens (Koliopoulos et al., 2010). The specieMentha x villosa Hudson popularly known as ‘‘hortelã-da-folha-miúda’’ or Cuban mint is an herb largely cultivated in northeasternBrazil (Alencastro et al., 1965). The essential oil from M. x villosaHudson possess several biological properties (Arruda et al., 2006;De Sousa et al., 2007, 2008), however, there are no studies aboutits insecticidal activity against Ae. aegypti.

Therefore, the present study aims to evaluate the larvicidalactivity of M. x villosa Hudson (MVEO) leaves essential oil and itsmain constituent, the monoterpene rotundifolone, against third-instar larvae of Ae. aegypti. Additionally, the Structure-ActivityRelationships (SAR) involving a set of 15 analogues compoundsof rotundifolone was investigated.

2. Material and methods

2.1. Plant material

M. x villosa leaves were collected in the Medicinal Plants’Garden of the Pharmaceutical Technology Laboratory of theFederal University of Paraíba/Brazil in July 2008 (7�080290 0S,34�500480 0W). A voucher specimen has been deposited at theHerbarium Prisco Bezerra of the Federal University of Ceará/Brazil,with the number 14996.

2.2. Extraction of the essential oil

Fresh leaves of M. x villosa (10 kg) were subjected to a steamdistillation for 8 h and an oil of yellowish coloration and character-istic odor was obtained. Subsequently, the oil was dried over anhy-drous sodium sulfate, filtered, and stored at 4 �C. The yield wascalculated from weight of fresh material (Matos et al., 1999).

2.3. Analysis by GC/MS

The steam-distilled oil was analyzed by gas chromatography-mass spectrometry (GC/MS) using a Hewlett Packard equipment,chromatograph model 5890 equipped with a mass spectrometermodel 5988A and an OV-5 capillary column(30 m � 0.25 mm � 0.25 lm) using the following analytical condi-tions: electron impact, 70 eV; carrier gas, Helium; flow rate,1.0 mL/min; oven temperature programmed from 60 to 240 �C at3 �C/min; injector temperature, 240 �C; detector temperature,230 �C; split ratio 1/20. The injected volume was 1.0 ll of a solu-tion containing ca. 0.1 ll of oil in 1.0 mL of ethyl acetate. The iden-tification of each component was performed by comparing theirmass spectra with the database of the GC/MS (Nist 62 lib.) and Ko-vats retention indices (Adams, 2001).

2.4. Isolation of rotundifolone from the essential oil of M. x villosa

Rotundifolone was isolated of MVEO using a procedure previ-ously described by Almeida et al. (1996). The essential oil was sub-mitted to preparative thin layer chromatography (PTLC). The plateswere developed three times using hexanes as eluent. When theplates were exposed to UV light (254 nm), rotundifolone was visu-alized as the major component of the essential oil. Rotundifolonewas removed from chromatographic plates and later recoveredby extraction with dichloromethane followed by filtration andevaporation to obtain a yellowish oil. The structural identificationwas made by infrared, 1H and 13C Nuclear Magnetic Resonance

(NMR) analysis and comparison with the literature data (Almeidaet al., 1996).

2.5. Rotundifolone analogues

The compounds pulegone epoxide (Katsuhara, 1967), (+)- and(�)-carvone epoxide (Santos et al., 1997), (+)- and (�)-limoneneepoxide (Thomas and Bessière, 1989), (�)-perillaldehyde (Furnisset al., 1998), perillaldehyde epoxide (Kido et al., 1992), trans-isopulegone (Moreira and Corrêa, 2003), hydroxycarvone, hydrox-ydihydrocarvone (Büchi and Wuest, 1979), trans-dihydrocarvone(Faria et al., 2000) and acetoxycarvotanacetone (Andrade et al.,2011) were prepared in our laboratory according to the literatureand analyzed by infrared, 1H and 13C NMR. (+)-Pulegone, (�)-carv-one, (�)-limonene and Temephos was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Chemical structures of theevaluated compounds are shown in Fig. 1.

2.6. Larvicidal assay

Larval mortality bioassays were performed according to themethodology recommended by WHO and adapted by Santoset al. (2011). The larvicidal properties of the MVEO, rotundifoloneand its analogues were evaluated on third-instar larvae and in allthe experiments was used only the Rockefeller lineage. Eggs ofAe. aegypti were obtained from the insectary of the Federal Univer-sity of Sergipe attached to paper stips. Paper strips (1000 eggs L�1)were placed in a rectangular polyethylene container containingnatural mineral water and cat food (whiskas) to allow regulardevelopment of the larvae. The container was kept in the insectaryfor hatching and monitoring of larvae development for 3–4 days.The concentration ranges were determined by a preliminary curveconcentration–response with 20 larvae. Standard solutions(20,000 ppm) of the tested oil and compounds were prepared usingMVEO (20 mg), Tween-80 (10% v/v) and natural mineral water(90% v/v) or each compound (20 mg), Tween-80 (10% v/v), di-methyl sulfoxide (30% v/v) and natural mineral water (60% v/v).From the standard solution, a series of dilutions was preparedranging from 10 to 2500 ppm. Twenty larvae were collected witha Pasteur pipette and placed on a 50 mL graduated cylinder. Thevolume was completed to 20 mL with natural mineral water andtransferred to disposable cups containing variable concentrationsof the standard solution. For each test, negative control was con-ducted using the same number of larvae in Tween-80 (0.1 mL), di-methyl sulfoxide (0.3 mL), and mineral water (19.6 mL) or onlyTween-80 (0.1 mL) and mineral water (19.9 mL). Three replicateswere used for each concentration and the control. The organ-ophosphorate Temephos (O,O0-(thio-di-4,1-phenylene)bis(O,O-dimethylphpsphorothiotate)), a commonly used insecticide forlarvae control, was used as positive control. The mortality was re-corded after 24 h exposure to different concentration of testingsolutions. Larvae were considered dead when they did not respondto stimulus.

2.7. Statistical analysis

The larvicidal assays mortality data were subjected to Probitanalysis (Finney, 1971) to estimate the lethal concentration for50% mortality (LC50) and 95% confidence intervals (CI) values forthe respective oil and compounds (Table 1). In all cases wheredeaths had occurred in the control experiment between 5% and20%, correction was performed by applying the Abbott’s formula(1925):

% Deaths ¼ % test mortality�% control mortality� 100100�% mortality

O

O

OO

OO

O

(+)-Pulegone, 2 Pulegone epoxide, 3 (-)-Carvone, 4

(+)-Carvone epoxide, 5

(+)-Limonene epoxide, 8

O

OH

OO

OH

OO

(-)-Carvone epoxide, 6

O

(-)-Limonene, 7

O

Trans-isopulegone, 12

OH

(-)-Perillaldehyde, 10

Hydroxycarvone, 14 Trans-dihydrocarvone, 13

O

O

O

O

O

Rotundifolone, 1

(-)-Limonene epoxide, 9

OHO

Perillaldehyde epoxide, 11

Hydroxydihydrocarvone, 15 Acetoxycarvotanacetone, 16

Fig. 1. Structures of evaluated compounds.

T.C. Lima et al. / Chemosphere 104 (2014) 37–43 39

When the control experiment mortality was over 20% thetest were discarded and repeated. Compounds activity wasconsidered significantly different if the 95% confidence limits didnot overlap.

3. Results and discussion

The MVEO was obtained by steam distillation with 0.1% yield.Table 1 shows the identified chemical constituents, their retentionindices, and percentage composition, listed in order of elution inthe OV-5 column. A total of 15 compounds, representing 91.92%of the essential oil, were characterized using GC/MS analyses.Among these 84.70% were monoterpenes and 7.22% sesquiter-penes. The major component was identified as the oxygenatedmonoterpene rotundifolone, also known as piperitenone oxide,found in high percentage (70.96%). All the other constituents werepresent in low amount (<8.76%). Similar composition results havebeen observed in Sousa et al. (2009), in which M. x villosa leavesessential oil exhibited rotundifolone in high concentration(79.03%).

Although several studies have reported the larvicidal activity ofessential oils against Ae. aegypti larvae, few works reported the lar-vicidal properties of their constituents. In this study, the MVEO, itsmajor metabolites (rotundifolone) and a set of 15 analogues of therotundifolone were subjected to laboratory bioassay studies toevaluate their relative toxicities against mosquito larvae of Ae. ae-gypti, as well as, their biological activity and SAR.

The results on the larvicidal activity were evaluated by compar-ing the LC50 values, expressed as ppm. LC50 values and 95% confi-dence limits of the MVEO, rotundifolone and its analogues aresummarized in Table 2. The results of larvicidal assay revealed thatthe MVEO and all test compounds exhibited larvicidal activityagainst Ae. aegypti larvae and the rates of mortality were directlyproportional to concentration.

The MVEO exhibited high larvicidal effect when compared withother essential oils, LC50 value of 45.0 ppm, inducing 100% mortal-ity of the larvae in the concentration of 110 ppm. Several species ofthe genus Mentha have been cited in the literature due to the insec-ticidal properties of its essential oils against different organisms(Mohamed and Abdelgaleil, 2008; Leite et al., 2009; Kumar et al.,

Table 1Composition of the essential oil from leaves of Mentha x villosa characterized by GC/MS.

RI Constituents %

972 Sabinene 0.23976 b-Pinene 0.81990 Myrcene 3.10

1029 Limonene 8.751035 cis-b-Ocimene 0.851363 Rotundifolone 70.961386 b-Bourbonene 0.091393 b-Elemene 0.241420 trans-Caryophyllene 1.461454 a-Humulene 0.211457 trans-b-Farnesene 0.391481 Germacrene-D 3.811521 cis-Calamenene 0.221523 d-Elemene 0.311653 a-Muurolol 0.49

– Monoterpenes 84.70– Sesquiterpenes 7.22– Total 91.92

RI, Retention indices determined on the OV-5 column.%, Constituents percentage.

40 T.C. Lima et al. / Chemosphere 104 (2014) 37–43

2011a, 2011b). Govindarajan et al. (2012)evaluated the larvicidalactivity of essential oil from M. spicata (Linn.) against three mos-quito species, Ae. aegypti, Anopheles stephensi and Cx. quinquefasci-atus. The oil of M. spicata exhibited a significant toxic effect againstlarvae of three species under study, with LC50 values of 49.71 ppmfor An. stephensi, 56.08 ppm for Ae. aegypti and 62.62 ppm for Cx.quinquefasciatus.

Rotundifolone, the most abundant component of MVEO, alsoexhibited strong toxic effect against the larvae of Ae. aegypti(LC50 = 62.5 ppm). Thus, rotundifolone probably is the main com-ponent responsible for the activity of this oil. This monoterpene ke-tone has also been described in the literature due to its toxic effectagainst the larvae of other mosquito species. Tripathi et al. (2004)reported the larvicidal activity the rotundifolone against malarialvector, An. stephensi, with LD50 value of 61.64 lg/mL. Koliopouloset al. (2010) recorded the efficacy of the rotundifolone againstthe West Nile virus mosquito Cx. pipiens, with LC50 value of9.95 mg/L.

Although rotundifolone (1) has shown a good larvicidal effectagainst the Ae. aegypti, it was less potent than its essential oil oforigin. The higher toxicity of the MVEO compared with its majorcomponent may be possibly justified by the existence of a syner-gistic interaction between rotundifolone and other constituents,present in minor proportion or due to the presence of other con-stituents also be responsible for activity of this essential oil. Theplants frequently produce essential oils as a mixture of manyingredients with strong interactions among them. According to

Table 2Larvicidal activity (LC50) and 95% confidence interval (CI) of evaluated oil and compounds

Compound LC50 ppm (CI 95%)

MVEO 45.0 ppm (35.4–53.4)1 62.5 ppm (56.2–68.3)2 188.1 ppm (186.4–189.8)3 1116.2 ppm (1107.6–1124.8)4 129.1 ppm (128.2–130.0)5 254.6 ppm (252.5–256.8)6 217.5 ppm (215.8–219.2)7 33.9 ppm (29.9–38.8)8 525.0 ppm (521.1–528.8)

The mortality data of the larvicidal assays were subjected to Probit analysis after 24 hconfidence intervals (CI) values for the respective compounds and Temephos. Compounddid not overlap.

Zoubiri and Baaliouamer (2011), as essential oils are complexmixtures of various molecules, their biological effects may reflectonly the activity of the main constituent, or could result of a syn-ergism between all molecules, expressing the importance of thecompositional complexity in the bioactivity of natural mixturesof terpenes.

The comparative study of the rotundifolone and its analoguesshowed that all tested analogues (Fig. 1) were found to have a lar-vicidal effect less potent than rotundifolone itself (Table 2), exceptfor (�)-limonene. The (�)-limonene (7), a monoterpene unsaturedcyclic hydrocarbon was significantly the more toxic compound(LC50 = 33.9 ppm), followed by rotundifolone (1) (LC50 = 62.5 ppm)and (�)-perillaldehyde (10) (LC50 = 115.8 ppm). Acetoxycarvota-nacetone (6) (LC50 = 230.7 ppm), (+)-carvone epoxide (5)(LC50 = 254.6 ppm) and (�)-carvone epoxide (6) (LC50 = 217.5 ppm)exhibited intermediate larvicidal activity, whereas hydroxydihy-drocarvone exhibited the lowest overall larvicidal potency, induc-ing 100% mortality only at 2000 ppm.

The addition of oxygen function to the structure of (�)-limo-nene (7) resulted in decrease in potency, and this reduction variedaccording to the amount and the functional group added (epoxide,ketone of carbonyl, ester, aldehyde of carbonyl or alcohol group).This result suggests the higher lipophilicity of compound (7), com-pared the other monoterpenes tested is an important property forthe larvicidal activity. Some studies have shown the importance ofthe relationship between the chemical structure and the biologicalactivity of natural products. Simas et al. (2004) demonstrated theimportance of the lipophilicity of terpenes for the larvicidal activityin Ae. aegypti, when comparing mono- and sesquiterpenes of re-lated structures. These authors concluded that the sesquiterpenefarnesol, an isomeric form of nerolidol, exhibited a good larvicidalactivity, with an LC50 value of 13.0 ppm. In contrast, its biosyn-thetic precursor, the monoterpene geraniol, was less bioactive(LC50 = 81.6 ppm).

To investigate if the position of the ketone of carbonyl group inthe molecule affects the larvicidal activity, (�)-carvone (4) wascompared to (+)-pulegone (2) and trans-isopulegone (12) was com-pared to trans-dihydrocarvone (13). Compounds 2 and 4 have acarbonyl in different positions on the ring resulting on differentlarvicidal effects between these monoterpenes with LC50 valuesof 129.1 ppm to the (�)-carvone (4) and 188.1 ppm to the (+)-pule-gone (2). Similarly, 12 and 13 have a ring carbonyl group differingfrom each other in the position of this group on the cyclohexanering. Trans-dihydrocarvone (13) exhibited more potent larvicidalactivity than trans-isopulegone (12) (LC50 = 361.3 ppm and538.8 ppm, respectively). These results indicate that the disposi-tion of ketone of carbonyl group on the cyclohexane skeleton altersthe biological activity. In addition, (�)-carvone (4) and (+)-pule-gone (2) showed variation in the position of the conjugated C–Cdouble bond, endocyclic in the compound 4 and exocyclic in the

on third-instar larvae of Ae. aegypti.

Compound LC50 ppm (CI 95%)

9 522.5 ppm (515.9–529.2)10 115.8 ppm (114.9–116.7)11 715.1 ppm (708.2–721.9)12 538.8 ppm (532.7–544.9)13 361.3 ppm (358.4–364.3)14 1470.9 ppm (1462.6–1479.3)15 1628.2 ppm (1622.9–1633.6)16 230.7 ppm (229.0–233.1)Temephos 0.043 ppm (0.036–0.051)

treatment to estimate the lethal concentration for 50% mortality (LC50) and 95%s and oil activity was considered significantly different if the 95% confidence limits

T.C. Lima et al. / Chemosphere 104 (2014) 37–43 41

compound 2. This difference may also be contributing to theobserved difference in potency between these two monoterpenesketones. Michaelakis et al. (2011) demonstrated the importanceof the position of a ketone to the larvicidal activity in Culicidae.Carvone and piperitone exhibited different larvicidal activityagainst Cx. pipiens, probably due to the position of the ketone onthe ring. Furthermore, these authors also reported the importanceof the location of the C–C double bond (endocyclic or exocyclic) tothe larvicide toxicity, since pulegone, with an exocyclic doublebond, was 5-fold more active than its isomer piperitone, with anendocyclic double bond. Our results corroborate with the laterfindings. Additionally, the presence of an a,b-unsaturated ketoneappears to be important for biological activity, once (+)-pulegone(2) (LC50 = 188.1 ppm), (�)-carvone (4) (LC50 = 129.1 ppm), andhydroxycarvone (14) (LC50 = 1470.9 ppm) containing this func-tional group were more bioactive than a,b-saturated ketonestrans-isopulegone (12) (LC50 = 538.8 ppm), trans-dihydrocarvone(13) (LC50 = 361.3 ppm) and hydroxydihydrocarvone (15)(LC50 = 1628.2 ppm), respectively. Similar results were found byGarcía et al. (2005), which pulegone (a,b-unsaturated ketone)exhibited a strong repellent activity against adults of Tribolium cas-taneum, while dihydropulegone (saturated ketone) was inactive.Corroborating with these results, Tripathi et al. (2003) reportedthat the L-carvone and D-carvone (a,b-unsaturated ketones) exhib-ited a higher fumigant and contact toxicities against adults of Sito-philus oryzae L., T. castaneum and Rhyzopertha dominica F. thandihydrocarvone (saturated ketone).

The presence of an exocyclic carbonyl group and conjugated theC–C double bond appears to contribute to the larvicidal activity,since the (�)-perillaldehyde (perillyl aldehyde) (10) exhibited rea-sonable activity (LC50 = 115.8 ppm). Some authors have reportedthat isolated aldehydes are not important for the larvicidal effect;however, when it is a,b-unsaturated or conjugated to an aromaticsystem contributes positively to biological activity (Simas et al.,2004; Abdelgaleil et al., 2009). Santos et al. (2010), for example, re-ported that the salicylic aldehyde (salicylaldehyde), an aromaticaldehyde, was more bioactive (LC50 = 136 ppm) against the larvaeof Ae. aegypti than the phenol (absence of the aldehyde of carbonylgroup) (LC50 = 194 ppm).

In general, the replacing of C–C double bond by an epoxidegroup decreases larvicidal activity. Reduction of the biological po-tency was observed by comparison of (�)-carvone (4)(LC50 = 129.1 ppm) with (+)-carvone epoxide (5) (LC50 = 254.6 -ppm), (�)-limonene (7) (LC50 = 33.9 ppm) with (�)-limoneneepoxide (9) (LC50 = 522.5 ppm), (�)-perillaldehyde (10)(LC50 = 115.8 ppm) with perillaldehyde epoxide (11) (LC50 = 715.1 -ppm), and (+)-pulegone (2) (LC50 = 188.1 ppm) with pulegoneepoxide (3) (LC50 = 1116.2 ppm). These data corroborate with theresults obtained by Santos et al. (2011), in which these authors re-ported that replacing a double bond by a more reactive epoxidegroup decreased the larvicidal potency. However, according toour results rotundifolone (1) (have an epoxide group) behaved asan exception. When compared with (+)-pulegone (2) (absence ofepoxide group) (LC50 = 188.1 ppm), the monoterpene 1 was 3-foldmore potent than compound 2 (LC50 = 62.5 ppm).

In order to ascertain whether the position of the epoxide grouprelated to the carbonyl group influence the larvicidal activity, rot-undifolone (1), (+)-carvone epoxide (5), and pugelone epoxide (2)were compared. Rotundifolone (1) (have an epoxide and a a,b-unsaturated ketone) and (+)-carvone epoxide (5) (have an epoxideand a a,b-saturated ketone) have the carbonyl group in differentpositions on the cyclohexane skeleton and resulted in different bio-logical effects. Rotundifolone (1) was 4-fold more bioactive(LC50 = 62.5 ppm) than (+)-carvone epoxide (5) (LC50 = 254.6 ppm),suggesting that the position of the ketone of carbonyl group on thering affects the larvicidal activity. In addition, the carbonyl group of

rotundifolone is a,b-unsaturated, while that of (+)-carvone issaturated, which also may be contributing to the difference of po-tency between these two compounds. Rotundifolone (1) and pule-gone epoxide (3) have an epoxide group in different positions inthe p-menthane skeleton: endo- and exocyclic epoxide groups,respectively. This difference has resulted in a severe decrease inthe biological activity of pugelone epoxide (3) (LC50 = 1116.2 ppm)compared with rotundifolone (1) (LC50 = 62.5 ppm), since the com-pound 1 was 18-fold more potent than compound 3. These datasuggest that exocyclic epoxide groups result in reduction of larvi-cidal potency, while endocyclic epoxide groups result in increaseof this potency. Both, pugelone epoxide (3) and (+)-carvone epox-ide (5) have carbonyl and an epoxide group in their chemical struc-ture. However, these functional groups are located in differentpositions in the p-menthane skeleton: a,b-exo- and endocyclicepoxide groups, respectively. Additionally, the carbonyl group islocated at different positions in the ring. Compound 5(LC50 = 254.6 ppm) showed a significant increase of the larvicidalactivity when compared with compound 3 (LC50 = 1116.2 ppm),suggesting that the position of the epoxide and ketone of carbonylfunctional groups in the p-menthane skeleton affects the larvicidalactivity.

To investigate the influence of the hydroxyl group in the larvi-cidal activity, we compared (�)-carvone (4) with hydroxycarvone(14) and trans-dihydrocarvone (13) with the hydroxydihydrocarv-one (15). Compound 4 (LC50 = 129.1 ppm) was 11-fold more potentthan compound 14 (LC50 = 1470.9 ppm). Furthermore, compound13 (LC50 = 361.3 ppm) was 4.5-fold more bioactive than the com-pound 15 (LC50 = 1628.2 ppm), suggesting that the presence of ahydroxyl group in the chemical structure decreases the larvicidalaction. Some authors have reported that replacing a carbonyl groupby a hydroxyl group, resulting in an alcohol, promotes a decreasein the fumigant and larvicidal toxicity (Rice and Coats, 1994; Leeet al., 2003). García et al. (2005), for example, showed that themonoterpene alcohol pulegol was less toxic against the adults ofT. castaneum than the monoterpene ketone pulegone. Accordingto Lopez et al. (2005), a plausible explanation for this result maybe the presence of polar hydroxyl groups on the chemical struc-ture, which may prevent the penetration of these substances inthe larvae cuticle and as a consequence, such compounds cannotachieve their aims and interact with an active site.

To determine the importance of the ester group in the larvicidalactivity, we compared hydroxycarvone (14) with acetoxycarvota-nacetone (16). The replacement of a hydroxyl group by an acetategroup promoted a significant increase of the larvicidal activity,which can be explained by the increased lipophilicity. Acetoxycar-votanacetone (16, LC50 = 230.7 ppm) was 6-fold more potent thanhydroxycarvone (14, LC50 = 1470.9 ppm). Other authors have pre-viously evaluated the influence of an ester group in the larvicidalactivity when compared to the hydroxyl group, obtaining similarresults. Wang et al. (2008) showed that the menthyl acetate exhib-ited more potent repellent activity than its corresponding alcohol,menthol. Waliwitiya et al. (2009), however, found different results.These authors showed that bornyl acetate (ester) exhibited a lesstoxic effect (LC50 > 500 mg/L) against the larvae of Ae. aegypti thanborneol (alcohol) (LC50 = 183.1 mg/L).

In view of the importance of chirality in the biological activity,the pairs of enantiomers (+)-carvone epoxide (5) and (�)-carvoneepoxide (6), as well as, (+)-limonene epoxide (8) and (�)-limoneneepoxide (9) were compared to investigate the role of enantioselec-tivity in the larvicidal activity against mosquitoes. (+)- and (�)-carvone epoxide showed different larvicides activities, LC50 valuesof 254.6 ppm and 217.5 ppm, respectively. These data indicate thatthe enantioselectivity may play an important role in the larvicidalactivity. However, there is no difference in the larvicidal potencywhen comparing compounds 8 and 9, exhibiting LC50 values of

42 T.C. Lima et al. / Chemosphere 104 (2014) 37–43

525.0 ppm and 522.5 ppm, respectively. Michaelakis et al. (2009)evaluated the larvicidal activity and selectivity of four enantio-meric pinenes against the mosquito Cx. pipiens. The authorsreported that the enantiomers (1R)-(+)-a-pinene and (1S)-(�)-a-pinene showed similar activity, while the enantiomers (1R)-(+)-b-pinene and (1S)-(�)-b-pinene exhibit different larvicidal effects.Additionally, the (�)-b enantiomer was the most toxic among pin-enes, with LC50 value of 36.53 mg/L. Michaelakis et al. (2011) re-vealed that menthone was more toxic against the larvae of Cx.pipiens than its enantiomer isomenthone, indicating that theenantioselectivity seems to play an important action for the toxic-ity of essential oils.

Some of the LC50 values expressed in this report are differentfrom published data in the literature (Cheng et al., 2009;Waliwitiya et al., 2009) which may be the result of different anal-ysis and methodologies (Santos et al., 2011). Moreover, larvae ofdifferent species or populations and that occupy different ecologi-cal niches may have different susceptibility level to specific com-pounds (Waliwitiya et al., 2009).

4. Conclusion

This paper presented a study about the larvicidal activity of theMVEO, and its main constituent, the monoterpene rotundifolone,against larvae of Ae. aegypti. Additionally, we have attempted tolearn the SAR involving a set of 15 analogues of rotundifolone.The MVEO showed strong toxic effect against Ae. aegypti larvae,suggesting a potential use to control the dengue vector. The resultsof comparing rotundifolone and its analogues confirmed that thedifferent functional groups and their positions in the p-menthaneskeleton influence the larvicidal activity, suggesting that appropri-ate structural modifications in the monoterpenes may be possibleto develop new larvicides agents.

Acknowledgements

The authors are thankful to the CAPES, CNPq, FAPITEC, andFederal University of Sergipe for financial support.

References

Abbott, W.S., 1925. A method for computing the effectiveness of insecticides. J.Econ. Entomol. 18, 265–267.

Abdelgaleil, S.A.M., Mohamed, M.I.E., Badawy, M.E.I., El-Arami, S.A.A., 2009.Fumigant and contact toxicities of monoterpenes to Sitophilus oryzae (L.)and Tribolium castaneum (Herbst) and their inhibitory effects onacetylcholinesterase activity. J. Chem. Ecol. 35, 518–525.

Adams, R.P., 2001. Identification of Essential Oil Components by GasChromatography/mass Spectroscopy. Allured Publishing Corporation, CarolStream, Illinois (USA), p. 456.

Andrade, L.N., Batista, J.S., De Sousa, D.P., 2011. Spasmolytic activity of p-menthaneesters. J. Med. Plant Res. 5, 6995–6999.

Alencastro, F.M.M.R., Scatone, Z., Prisco, J.T., Laboriau, L.F.G., 1965. Contribuiçãopara uma bibliografia do gênero Mentha L. Associação Brasileira de Pesquisa dePlantas Aromáticas. Óleos Essenciais, Campinas, SP, Brazil, p. 354.

Almeida, R.N., Hiruma, C.A., Barbosa-Filho, J.M., 1996. Analgesic effect ofrotundifolone in rodents. Fitoterapia 67, 334–338.

Ansari, M.A., Vasudevan, P., Tandon, M., Razdan, R.K., 2000. Larvicidal and mosquitorepellent action of peppermint (Mentha piperita) oil. Bioresour. Technol. 71,267–271.

Arruda, T.A., Antunes, R.M.P., Catão, R.M.R., Lima, E.O., Sousa, D.P., Nunes, X.P.,Pereira, M.S.V., Barbosa-Filho, J.M., Da Cunha, E.V.L., 2006. Preliminary study ofthe antimicrobial activity of Mentha x villosa Hudson essential oil, rotundifoloneand its analogues. Rev. Bras. Farmacog. 16 (3), 307–311.

Büchi, G., Wuest, H.J., 1979. New synthesis of b-agarofuran and of dihydroagarofu-ran. J. Org. Chem. 44, 546–549.

Cheng, S.S., Chang, H.T., Lin, C.Y., Chen, P.S., Huang, C.G., Chen, W.J., Chang, S.T.,2009. Insecticidal activities of leaf and twig essential oils from Clausena excavataagainst Aedes aegypti and Aedes albopictus larvae. Pest Manage. Sci. 65, 339–343.

Coelho, A.A.M., De Paula, J.E., Espíndola, L.S., 2009. Atividade larvicida de extratosvegetais sobre Aedes aegypti (L.) (Diptera: Culicidae), em condições delaboratório. Bioassay 4, 1–6.

De Sousa, D.P., Júnior, E.V.M., Oliveira, F.S., Almeida, R.N., Nunes, X.P., Barbosa-Filho,J.M., 2007. Antinociceptive activity of structural analogues of rotundifolone:structure-activity relationship. Z. Naturforsch. 62c, 39–42.

De Sousa, D.P., Júnior, G.A.S., Andrade, L.N., Calasans, F.R., Nunes, X.P., Barbosa-Filho,J.M., Batista, J.S., 2008. Structural relationships and spasmolytic activity ofanalogues of rotundifolone, monoterpenes found in many aromatic plants. Z.Naturforsch. 16c, 808–812.

Faria, M.L., Magalhães, R.A., Silva, F.C., Matias, L.G.O., Ceschi, M.A., Brocksom, U.,Brocksom, T.J., 2000. Enantiodivergent synthesis of cycloheptenoneintermediates for guaiane sesquiterpenes. Tetrahedron Asymmetry 11, 4093–4103.

Finney, D.J., 1971. Probit Analysis, third ed. University Press, Cambridge.Franzios, G., Mirotsou, M., Hatziapostolou, E., Kral, J., Scouras, Z.G., Mavragani-

Tsipidou, P.J., 1997. Insecticidal and genotoxic activities of mint essential oils.Agric. Food Chem. 45, 2690–2694.

Furniss, B.S., Hannaford, A.J., Smith, P.W.G., Tatchell, A.R., 1998. Vogel’s Text Book ofPractical Organic Chemistry, fifth ed. Addition Wesley Longman, England.

García, M., Donadel, O.J., Ardanaz, C.E., Tonn, C.E., Sosa, M.E., 2005. Toxic andrepellent effects of Baccharis salicifolia essential oil on Tribolium castaneum. PestManage. Sci. 61, 612–618.

Govindarajan, M., Sivakumar, R., Rajeswari, M., Yogalakshmi, K., 2012. Chemicalcomposition and larvicidal activity of essential oil from Mentha spicata (Linn.)against three mosquito species. Parasitol. Res. 110, 2023–2032.

Katsuhara, J., 1967. Absolute configuration of pulegone oxide and piperitenonedioxide. J. Org. Chem. 32, 797–799.

Kido, F., Abiko, T., Kato, M., 1992. Spiroannulation by the [2,3]sigmatropicrearrangement via the cyclic allylsulfonium ylide. A stereoselective synthesisof (+)-acorenone B. J. Chem. Soc. Perkin Trans. 2, 229–233.

Koliopoulos, G., Pitarokili, D., Kioulos, E., Michaelakis, A., Tzakou, O., 2010. Chemicalcomposition and larvicidal evaluation of Mentha, Salvia, and Melissa essentialoils against the West Nile virus mosquito Culex pipiens. Parasitol. Res. 107, 327–335.

Kumar, P., Mishra, S., Malik, A., Satya, S., 2011a. Insecticidal properties of Menthaspecies: a review. Ind. Crops Prod. 34, 802–817.

Kumar, S., Wahab, N., Warikoo, R., 2011b. Bioefficacy of Mentha piperita essential oilagainst dengue fever mosquito Aedes aegypti L. Asian Pac. J. Trop. Med., 85–88.

Lee, S., Peterson, C.J., Coats, J.R., 2003. Fumigation toxicity of monoterpenoids toseveral stored product insects. J. Stored Prod. Res. 39, 77–85.

Leite, A.M., Lima, E.O., Souza, E.L., Diniz, M.F.F.M., Leite, S.P., Xavier, A.L., Medeiros,I.A., 2009. Preliminary study of the molluscicidal and larvicidal properties ofsome essential oils and phytochemicals from medicinal plants. Rev. Bras.Farmacogn. 19, 842–846.

Ligon, B.L., 2005. Dengue fever and dengue hemorrhagic fever: a review of thehistory, transmission, treatment, and prevention. Semin. Pediatr. Infect. Dis. 16(1), 60–65.

Lopez, O., Fernandez-Bolanos, J.G., Gil, M.V., 2005. New trends in pest control: thesearch for greener insecticides. Green Chem. 7, 431–442.

Matos, F.J.A., Machado, M.I., Craveiro, A.A., Barbosa-Filho, J.M., Alencar, J.W.,Barbosa-Filho, J.M., Cunha, E.V.L., Hiruma, C.A., 1999. Essential oil of Mentha xvillosa huds. An antiparasitic medicinal herb from Nordestern Brazil. J. Essent.Oil Res. 11, 41–44.

Michaelakis, A., Papachristos, D., Kimbaris, A., Koliopoulos, G., Giatropoulos, A.,Polissiou, M.G., 2009. Citrus essential oils and four enantiomeric pinenesagainst Culex pipiens (Diptera: Culicidae). Parasitol. Res. 105, 769–773.

Michaelakis, A., Papachristos, D., Kimbaris, A., Polissiou, M., 2011. Larvicidalevaluation of three Mentha species essential oils and their isolated majorcomponents against the West Nile virus mosquito. Hellenic Plant Prot. J. 4, 35–43.

Mohamed, M.I.E., Abdelgaleil, S.A.M., 2008. Chemical composition and insecticidalpotential of essential oils from Egyptian plants against Sitophilus oryzae (L.)(Coleoptera: Curculionidae) and Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae). Appl. Entomol. Zool. 43, 599–607.

Moreira, J.A., Corrêa, A.G., 2003. Enantioselective synthesis of three stereoisomers of5,9-dimethylpentadecane, sex pheromone component of Leucoptera coffeella,from (�)-isopulegol. Tetrahedron Asymmetry 14, 3787–3795.

Porto, K.R.A., Roel, A.R., Silva, M.M., Coelho, R.M., Scheleder, E.J.D., Haroldo, J.A.H.,2008. Atividade larvicida do óleo de Anacardium humile Saint Hill sobre Aedesaegypti (Linnaeus, 1762) (Diptera: Culicidae). Rev. Soc. Bras. Med. Trop. 41 (6),586–589.

Rice, P.J., Coats, J.R., 1994. Insecticidal properties of several monoterpenoids to thehouse fly (Diptera: Muscidae), red flour beetle (Coleoptera: Tenebrionidae), andsouthern maize rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 87,1172–1179.

Rigau-Pérez, J.G., Clark, G.G., Gubler, D.J., Reiter, P., Sanders, E.J., Vorndam, A.V.,1998. Dengue and dengue haemorrhagic fever. Lancet 352, 971–977.

Rosen, L., 1999. Comments on the epidemiology, pathogenesis and control ofdengue. Med. Trop. 59, 495–498.

Santos, R.B.T., Brocksom, J., Brocksom, U., 1997. A convenient deoxygenation of a, b-epoxy ketones to enones. Tetrahedron Lett. 38, 745–748.

Santos, S.R.L., Silva, V.B., Melo, M.A., Barbosa, J.D.F., Santos, R.L.C., De Sousa, D.P.,Cavalcanti, S.C.H., 2010. Toxic effects on and structure-toxicity relationships ofphenylpropanoids, terpenes, and related compounds in Aedes aegypti larvae.Vector-Borne Zoon Dis. 10, 1049–1054.

Santos, S.R.L., Melo, M.A., Cardoso, A.V., Santos, R.L.C., De Sousa, D.P., Cavalcanti,S.C.H., 2011. Structure-activity relationships of larvicidal monoterpenes andderivatives against Aedes aegypti linn. Chemosphere 84 (1), 150–153.

T.C. Lima et al. / Chemosphere 104 (2014) 37–43 43

Simas, N.K., Lima, E.C., Conceição, S.R., Kuster, R.M., Oliveira Filho, A.M., 2004.Natural products for dengue transmission control-larvicidal activity ofMyroxylon balsamum (red oil) and of terpenoids and phenylpropanoids. Quim.Nova 27, 46–49.

Sousa, P.J.C., Linard, C.F.B.M., Azevedo-Batista, D., Oliveira, A.C., Coelho-de-Souza,A.N., Leal-Cardoso, J.H., 2009. Antinociceptive effects of the essential oil ofMentha x villosa leaf and its major constituent piperitenone oxide in mice. Braz.J. Med. Biol. Res. 42, 655–659.

Tauil, P.L., 2001. Urbanização e ecologia do dengue. Cad. Saúde Pública. 17, pp. 99–102.

Thomas, A.F., Bessière, Y., 1989. Limonene. Nat. Prod. Rep. 6, 291–309.Tripathi, A.K., Prajapati, V., Ahmad, A., Aggarwal, K.K., Khanuja, S.P.S., 2004.

Piperitenone oxide as toxic, repellent, and reproduction retardant towardmalarial Anopheles stephensi (Diptera: Anophelinae). J. Med. Entomol. 41, 691–698.

Tripathi, A.K., Prajapati, V., Kumar, S., 2003. Bioactivities of l-carvone, d-carvone,and dihydrocarvone toward three stored product beetles. J. Econ. Entomol. 96,1594–1601.

Waliwitiya, R., Kennedy, C.J., Lowenberger, C.A., 2009. Larvicidal and oviposition-altering activity of monoterpenoids, transanethole and rosemary oil to theyellow fever mosquito Aedes aegypti (Diptera: Culicidae). Pest Manage. Sci. 65,241–248.

Wang, Z., Song, J., Chen, J., Song, Z., Shang, S., Jiang, Z., Han, Z., 2008. QSAR study ofmosquito repellents from terpenoid with a six-member-ring. Bioorg. Med.Chem. Lett. 18, 2854–2859.

Zahran, H.E.M., Abdelgaleil, S.A.M., 2011. Insecticidal and developmental inhibitoryproperties of monoterpenes on Culex pipiens L. (Diptera: Culicidae). J. Asia Pac.Entomol. 14, 46–51.

Zoubiri, S., Baaliouamer, A., 2011. Potentiality of plants as source of insecticideprinciples. J. Saudi Chem. Soc. http://dx.doi.org/10.1016/j.jscs.2011.11.015.