Chemosphere 104 (2014) 37–43

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Larvicidal activity of Mentha x villosa Hudson essential oil, rotundifolone

and derivatives
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, Brazil
b
Department of Pharmaceutical Sciences, Federal University of Paraíba, CEP 58051-970 João Pessoa, Paraíba, Brazil
c
Center of Genetics, Molecular Biology and Phytochemistry, Agronomic Institute, CEP 13001-970 Campinas, São Paulo, Brazil
d
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 a b s t r a c t

Article history: The aim of this study was to evaluate the larvicidal activity of Mentha x villosa essential oil (MVEO) and its
Received 3 July 2013 major constituent, rotundifolone, against larvae of Aedes aegypti. Additionally, a set of 15 analogues of the
Received in revised form 6 October 2013 rotundifolone were evaluated to identify the molecular characteristics which contribute to the larvicidal
Accepted 8 October 2013
effect. The results from the present study showed that the MVEO exhibited outstanding toxic effects
Available online 22 November 2013
against 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-
Keywords:
pounds were less potent than rotundifolone, except ()-limonene. In general, replacement of C–C double
Larvicidal activity
Essential oil
bonds by epoxides groups decreases the larvicidal potency. The presence of a,b-unsaturated carbonyls
Aedes aegypti contributes to the larvicidal toxicity. The addition of hydroxyl groups in the chemical structure resulted
Terpene in less potent compounds. Furthermore, the enantioselectivity seems to play an important role for the
Dengue larvicidal toxicity.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction mosquitoes breeding sites and by the use of synthetic chemical

Dengue is an infectious disease caused by an arbovirus trans-
mitted to humans by the bite of female mosquitoes of the genus
Aedes. 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 by
arthropod vectors in terms of mortality and morbidity
(Rigau-Pérez et al., 1998; Rosen, 1999).
In the absence of effective and safe vaccines for dengue
prevention, the epidemics control is accomplished by monitoring
⇑ Corresponding author. Tel.: +55 7988329710.
E-mail address: damiao_desousa@yahoo.com.br (D.P. de Sousa).
insecticides to control the spreading of adult or larval forms of
Ae. aegypti (Tauil, 2001; Porto et al., 2008). Although the chemical
products are effective, the continuous use in large scale has con-
tributed to the emergence of many negative consequences, such
as, 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 problems
have 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 major
constituents have received much attention as potential bioactive
agents against mosquito vector (Cheng et al., 2009; Waliwitiya
et al., 2009; Zahran and Abdelgaleil, 2011).
The genus Mentha is represented by about 19 species and 13
natural hybrids and it is an important member of the family

0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2013.10.035
38 T.C. Lima et al. / Chemosphere 104 (2014) 37–43
Lamiaceae (Kumar et al., 2011a). Various essential oils of Mentha
have 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 specie
Mentha x villosa Hudson popularly known as ‘‘hortelã-da-folha-
miúda’’ or Cuban mint is an herb largely cultivated in northeastern
Brazil (Alencastro et al., 1965). The essential oil from M. x villosa
Hudson possess several biological properties (Arruda et al., 2006;
De Sousa et al., 2007, 2008), however, there are no studies about
its insecticidal activity against Ae. aegypti.
Therefore, the present study aims to evaluate the larvicidal
activity of M. x villosa Hudson (MVEO) leaves essential oil and its
main constituent, the monoterpene rotundifolone, against third-
instar larvae of Ae. aegypti. Additionally, the Structure-Activity
Relationships (SAR) involving a set of 15 analogues compounds
of 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 the
Federal University of Paraíba/Brazil in July 2008 (7°080 2900 S,
34°500 4800 W). A voucher specimen has been deposited at the
Herbarium 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 steam
distillation 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 was
calculated 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 spectrometer
model 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 at
3 °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 their
mass 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 plates
were developed three times using hexanes as eluent. When the
plates were exposed to UV light (254 nm), rotundifolone was visu-
alized as the major component of the essential oil. Rotundifolone
was removed from chromatographic plates and later recovered
by extraction with dichloromethane followed by filtration and
evaporation to obtain a yellowish oil. The structural identification
was made by infrared, 1H and 13C Nuclear Magnetic Resonance
(NMR) analysis and comparison with the literature data (Almeida
et al., 1996).
2.5. Rotundifolone analogues
The compounds pulegone epoxide (Katsuhara, 1967), (+)- and
()-carvone epoxide (Santos et al., 1997), (+)- and ()-limonene
epoxide (Thomas and Bessière, 1989), ()-perillaldehyde (Furniss
et 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 literature
and 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 the
evaluated compounds are shown in Fig. 1.
2.6. Larvicidal assay
Larval mortality bioassays were performed according to the
methodology recommended by WHO and adapted by Santos
et al. (2011). The larvicidal properties of the MVEO, rotundifolone
and its analogues were evaluated on third-instar larvae and in all
the experiments was used only the Rockefeller lineage. Eggs of
Ae. aegypti were obtained from the insectary of the Federal Univer-
sity of Sergipe attached to paper stips. Paper strips (1000 eggs L1)
were placed in a rectangular polyethylene container containing
natural mineral water and cat food (whiskas) to allow regular
development of the larvae. The container was kept in the insectary
for hatching and monitoring of larvae development for 3–4 days.
The concentration ranges were determined by a preliminary curve
concentration–response with 20 larvae. Standard solutions
(20,000 ppm) of the tested oil and compounds were prepared using
MVEO (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 prepared
ranging from 10 to 2500 ppm. Twenty larvae were collected with
a Pasteur pipette and placed on a 50 mL graduated cylinder. The
volume was completed to 20 mL with natural mineral water and
transferred to disposable cups containing variable concentrations
of 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 only
Tween-80 (0.1 mL) and mineral water (19.9 mL). Three replicates
were 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 for
larvae control, was used as positive control. The mortality was re-
corded after 24 h exposure to different concentration of testing
solutions. Larvae were considered dead when they did not respond
to stimulus.
2.7. Statistical analysis
The larvicidal assays mortality data were subjected to Probit
analysis (Finney, 1971) to estimate the lethal concentration for
50% mortality (LC50) and 95% confidence intervals (CI) values for
the respective oil and compounds (Table 1). In all cases where
deaths had occurred in the control experiment between 5% and
20%, correction was performed by applying the Abbott’s formula
(1925):
% test mortality  % control mortality  100
% Deaths ¼
100  % mortality
 T.C. Lima et al. / Chemosphere 104 (2014) 37–43 39

O
O

O O O
O

Rotundifolone, 1 (+)-Pulegone, 2 Pulegone epoxide, 3 (-)-Carvone, 4

O O O
O O

(+)-Carvone (-)-Carvone (-)-Limonene, 7 (+)-Limonene

epoxide, 5 epoxide, 6 epoxide, 8

H O H O
O O

(-)-Limonene (-)-Perillaldehyde, 10 Perillaldehyde Trans-isopulegone, 12

epoxide, 9 epoxide, 11

O
O O
O

O
OH OH
Trans- Hydroxycarvone, 14 Hydroxydihydrocarvone, 15
dihydrocarvone, 13 O
Acetoxycarvotanacetone, 16

Fig. 1. Structures of evaluated compounds.

When the control experiment mortality was over 20% the
test were discarded and repeated. Compounds activity was
considered significantly different if the 95% confidence limits did
not overlap.
3. Results and discussion
The MVEO was obtained by steam distillation with 0.1% yield.
Table 1 shows the identified chemical constituents, their retention
indices, and percentage composition, listed in order of elution in
the 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 oxygenated
monoterpene rotundifolone, also known as piperitenone oxide,
found in high percentage (70.96%). All the other constituents were
present in low amount (<8.76%). Similar composition results have
been observed in Sousa et al. (2009), in which M. x villosa leaves
essential oil exhibited rotundifolone in high concentration
(79.03%).
Although several studies have reported the larvicidal activity of
essential oils against Ae. aegypti larvae, few works reported the lar-
vicidal properties of their constituents. In this study, the MVEO, its
major metabolites (rotundifolone) and a set of 15 analogues of the
rotundifolone were subjected to laboratory bioassay studies to
evaluate 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 are
summarized in Table 2. The results of larvicidal assay revealed that
the MVEO and all test compounds exhibited larvicidal activity
against Ae. aegypti larvae and the rates of mortality were directly
proportional to concentration.
The MVEO exhibited high larvicidal effect when compared with
other essential oils, LC50 value of 45.0 ppm, inducing 100% mortal-
ity of the larvae in the concentration of 110 ppm. Several species of
the 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.,
40 T.C. Lima et al. / Chemosphere 104 (2014) 37–43

Table 1 Zoubiri and Baaliouamer (2011), as essential oils are complex

Composition of the essential oil from leaves of Mentha x villosa characterized by GC/ mixtures of various molecules, their biological effects may reflect
MS.
only the activity of the main constituent, or could result of a syn-
RI Constituents % ergism between all molecules, expressing the importance of the
972 Sabinene 0.23 compositional complexity in the bioactivity of natural mixtures
976 b-Pinene 0.81 of terpenes.
990 Myrcene 3.10 The comparative study of the rotundifolone and its analogues
1029 Limonene 8.75
1035 cis-b-Ocimene 0.85
showed that all tested analogues (Fig. 1) were found to have a lar-
1363 Rotundifolone 70.96 vicidal effect less potent than rotundifolone itself (Table 2), except
1386 b-Bourbonene 0.09 for ()-limonene. The ()-limonene (7), a monoterpene unsatured
1393 b-Elemene 0.24 cyclic hydrocarbon was significantly the more toxic compound
1420 trans-Caryophyllene 1.46
(LC50 = 33.9 ppm), followed by rotundifolone (1) (LC50 = 62.5 ppm)
1454 a-Humulene 0.21
1457 trans-b-Farnesene 0.39 and ()-perillaldehyde (10) (LC50 = 115.8 ppm). Acetoxycarvota-
1481 Germacrene-D 3.81 nacetone (6) (LC50 = 230.7 ppm), (+)-carvone epoxide (5)
1521 cis-Calamenene 0.22 (LC50 = 254.6 ppm) and ()-carvone epoxide (6) (LC50 = 217.5 ppm)
1523 d-Elemene 0.31 exhibited intermediate larvicidal activity, whereas hydroxydihy-
1653 a-Muurolol 0.49
drocarvone exhibited the lowest overall larvicidal potency, induc-
– Monoterpenes 84.70
– Sesquiterpenes 7.22 ing 100% mortality only at 2000 ppm.
– Total 91.92 The addition of oxygen function to the structure of ()-limo-
nene (7) resulted in decrease in potency, and this reduction varied
RI, Retention indices determined on the OV-5 column.
%, Constituents percentage. according 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-
2011a, 2011b). Govindarajan et al. (2012)evaluated the larvicidal pared the other monoterpenes tested is an important property for
activity of essential oil from M. spicata (Linn.) against three mos- the larvicidal activity. Some studies have shown the importance of
quito species, Ae. aegypti, Anopheles stephensi and Cx. quinquefasci- the relationship between the chemical structure and the biological
atus. The oil of M. spicata exhibited a significant toxic effect against activity of natural products. Simas et al. (2004) demonstrated the
larvae of three species under study, with LC50 values of 49.71 ppm importance of the lipophilicity of terpenes for the larvicidal activity
for An. stephensi, 56.08 ppm for Ae. aegypti and 62.62 ppm for Cx. in Ae. aegypti, when comparing mono- and sesquiterpenes of re-
quinquefasciatus. lated structures. These authors concluded that the sesquiterpene
Rotundifolone, the most abundant component of MVEO, also farnesol, an isomeric form of nerolidol, exhibited a good larvicidal
exhibited strong toxic effect against the larvae of Ae. aegypti activity, with an LC50 value of 13.0 ppm. In contrast, its biosyn-
(LC50 = 62.5 ppm). Thus, rotundifolone probably is the main com- thetic precursor, the monoterpene geraniol, was less bioactive
ponent responsible for the activity of this oil. This monoterpene ke- (LC50 = 81.6 ppm).
tone has also been described in the literature due to its toxic effect To investigate if the position of the ketone of carbonyl group in
against the larvae of other mosquito species. Tripathi et al. (2004) the molecule affects the larvicidal activity, ()-carvone (4) was
reported the larvicidal activity the rotundifolone against malarial compared to (+)-pulegone (2) and trans-isopulegone (12) was com-
vector, An. stephensi, with LD50 value of 61.64 lg/mL. Koliopoulos pared to trans-dihydrocarvone (13). Compounds 2 and 4 have a
et al. (2010) recorded the efficacy of the rotundifolone against carbonyl in different positions on the ring resulting on different
the West Nile virus mosquito Cx. pipiens, with LC50 value of larvicidal effects between these monoterpenes with LC50 values
9.95 mg/L. of 129.1 ppm to the ()-carvone (4) and 188.1 ppm to the (+)-pule-
Although rotundifolone (1) has shown a good larvicidal effect gone (2). Similarly, 12 and 13 have a ring carbonyl group differing
against the Ae. aegypti, it was less potent than its essential oil of from each other in the position of this group on the cyclohexane
origin. The higher toxicity of the MVEO compared with its major ring. Trans-dihydrocarvone (13) exhibited more potent larvicidal
component may be possibly justified by the existence of a syner- activity than trans-isopulegone (12) (LC50 = 361.3 ppm and
gistic interaction between rotundifolone and other constituents, 538.8 ppm, respectively). These results indicate that the disposi-
present in minor proportion or due to the presence of other con- tion of ketone of carbonyl group on the cyclohexane skeleton alters
stituents also be responsible for activity of this essential oil. The the biological activity. In addition, ()-carvone (4) and (+)-pule-
plants frequently produce essential oils as a mixture of many gone (2) showed variation in the position of the conjugated C–C
ingredients with strong interactions among them. According to double bond, endocyclic in the compound 4 and exocyclic in the

Table 2
Larvicidal activity (LC50) and 95% confidence interval (CI) of evaluated oil and compounds on third-instar larvae of Ae. aegypti.

Compound LC50 ppm (CI 95%) Compound LC50 ppm (CI 95%)
MVEO 45.0 ppm (35.4–53.4) 9 522.5 ppm (515.9–529.2)
1 62.5 ppm (56.2–68.3) 10 115.8 ppm (114.9–116.7)
2 188.1 ppm (186.4–189.8) 11 715.1 ppm (708.2–721.9)
3 1116.2 ppm (1107.6–1124.8) 12 538.8 ppm (532.7–544.9)
4 129.1 ppm (128.2–130.0) 13 361.3 ppm (358.4–364.3)
5 254.6 ppm (252.5–256.8) 14 1470.9 ppm (1462.6–1479.3)
6 217.5 ppm (215.8–219.2) 15 1628.2 ppm (1622.9–1633.6)
7 33.9 ppm (29.9–38.8) 16 230.7 ppm (229.0–233.1)
8 525.0 ppm (521.1–528.8) Temephos 0.043 ppm (0.036–0.051)

The mortality data of the larvicidal assays were subjected to Probit analysis after 24 h treatment to estimate the lethal concentration for 50% mortality (LC50) and 95%
confidence intervals (CI) values for the respective compounds and Temephos. Compounds and oil activity was considered significantly different if the 95% confidence limits
did not overlap.
 T.C. Lima et al. / Chemosphere 104 (2014) 37–43
compound 2. This difference may also be contributing to the
observed difference in potency between these two monoterpenes
ketones. Michaelakis et al. (2011) demonstrated the importance
of the position of a ketone to the larvicidal activity in Culicidae.
Carvone and piperitone exhibited different larvicidal activity
against Cx. pipiens, probably due to the position of the ketone on
the ring. Furthermore, these authors also reported the importance
of the location of the C–C double bond (endocyclic or exocyclic) to
the larvicide toxicity, since pulegone, with an exocyclic double
bond, was 5-fold more active than its isomer piperitone, with an
endocyclic double bond. Our results corroborate with the later
findings. Additionally, the presence of an a,b-unsaturated ketone
appears to be important for biological activity, once (+)-pulegone
(2) (LC50 = 188.1 ppm), ()-carvone (4) (LC50 = 129.1 ppm), and
hydroxycarvone (14) (LC50 = 1470.9 ppm) containing this func-
tional group were more bioactive than a,b-saturated ketones
trans-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 by
García 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) reported
that 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. than
dihydrocarvone (saturated ketone).
The presence of an exocyclic carbonyl group and conjugated the
C–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 reported
that isolated aldehydes are not important for the larvicidal effect;
however, when it is a,b-unsaturated or conjugated to an aromatic
system 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 aromatic
aldehyde, was more bioactive (LC50 = 136 ppm) against the larvae
of Ae. aegypti than the phenol (absence of the aldehyde of carbonyl
group) (LC50 = 194 ppm).
In general, the replacing of C–C double bond by an epoxide
group 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 ()-limonene
epoxide (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 pulegone
epoxide (3) (LC50 = 1116.2 ppm). These data corroborate with the
results obtained by Santos et al. (2011), in which these authors re-
ported that replacing a double bond by a more reactive epoxide
group decreased the larvicidal potency. However, according to
our results rotundifolone (1) (have an epoxide group) behaved as
an exception. When compared with (+)-pulegone (2) (absence of
epoxide group) (LC50 = 188.1 ppm), the monoterpene 1 was 3-fold
more potent than compound 2 (LC50 = 62.5 ppm).
In order to ascertain whether the position of the epoxide group
related 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 epoxide
and a a,b-saturated ketone) have the carbonyl group in different
positions 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 the
ring affects the larvicidal activity. In addition, the carbonyl group of
41
rotundifolone is a,b-unsaturated, while that of (+)-carvone is
saturated, 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 in
the p-menthane skeleton: endo- and exocyclic epoxide groups,
respectively. This difference has resulted in a severe decrease in
the 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 data
suggest that exocyclic epoxide groups result in reduction of larvi-
cidal potency, while endocyclic epoxide groups result in increase
of 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 different
positions in the p-menthane skeleton: a,b-exo- and endocyclic
epoxide groups, respectively. Additionally, the carbonyl group is
located at different positions in the ring. Compound 5
(LC50 = 254.6 ppm) showed a significant increase of the larvicidal
activity when compared with compound 3 (LC50 = 1116.2 ppm),
suggesting that the position of the epoxide and ketone of carbonyl
functional groups in the p-menthane skeleton affects the larvicidal
activity.
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 potent
than compound 14 (LC50 = 1470.9 ppm). Furthermore, compound
13 (LC50 = 361.3 ppm) was 4.5-fold more bioactive than the com-
pound 15 (LC50 = 1628.2 ppm), suggesting that the presence of a
hydroxyl group in the chemical structure decreases the larvicidal
action. Some authors have reported that replacing a carbonyl group
by a hydroxyl group, resulting in an alcohol, promotes a decrease
in the fumigant and larvicidal toxicity (Rice and Coats, 1994; Lee
et al., 2003). García et al. (2005), for example, showed that the
monoterpene alcohol pulegol was less toxic against the adults of
T. castaneum than the monoterpene ketone pulegone. According
to Lopez et al. (2005), a plausible explanation for this result may
be the presence of polar hydroxyl groups on the chemical struc-
ture, which may prevent the penetration of these substances in
the larvae cuticle and as a consequence, such compounds cannot
achieve their aims and interact with an active site.
To determine the importance of the ester group in the larvicidal
activity, we compared hydroxycarvone (14) with acetoxycarvota-
nacetone (16). The replacement of a hydroxyl group by an acetate
group 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 than
hydroxycarvone (14, LC50 = 1470.9 ppm). Other authors have pre-
viously evaluated the influence of an ester group in the larvicidal
activity when compared to the hydroxyl group, obtaining similar
results. 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 less
toxic effect (LC50 > 500 mg/L) against the larvae of Ae. aegypti than
borneol (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 ()-carvone
epoxide (6), as well as, (+)-limonene epoxide (8) and ()-limonene
epoxide (9) were compared to investigate the role of enantioselec-
tivity in the larvicidal activity against mosquitoes. (+)- and ()-
carvone epoxide showed different larvicides activities, LC50 values
of 254.6 ppm and 217.5 ppm, respectively. These data indicate that
the enantioselectivity may play an important role in the larvicidal
activity. However, there is no difference in the larvicidal potency
when 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 authors
reported 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 the
enantioselectivity seems to play an important action for the toxic-
ity of essential oils.
Some of the LC50 values expressed in this report are different
from 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 of
different 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 the
MVEO, and its main constituent, the monoterpene rotundifolone,
against larvae of Ae. aegypti. Additionally, we have attempted to
learn 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 results
of comparing rotundifolone and its analogues confirmed that the
different functional groups and their positions in the p-menthane
skeleton influence the larvicidal activity, suggesting that appropri-
ate structural modifications in the monoterpenes may be possible
to develop new larvicides agents.
Acknowledgements
The authors are thankful to the CAPES, CNPq, FAPITEC, and
Federal University of Sergipe for financial support.
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