Chemical Study and Larvicidal Activity against Aedes Aegypti of Essential Oil of Piper Aduncum L. (piperaceae)

Chemical study and larvicidal activity against Aedes aegypti of essential oil of Piper aduncum L. ABSTRACT Piper aduncum L. is used in folk medicine to treat respiratory and inflammatory diseases. The aim of this study was to analyze the essential oil from leaves of P. aduncum collected in the Brazilian Cerrado, North of Minas Gerais, as well as to evaluate the larvicidal activity of this oil and of its major constituent. The essential oil was analyzed by gas chromatography coupled to flame ionization detector and gas chromatography coupled to mass spectrometry that allowed characterizing 23 compounds (monoterpenes: 90.4%; sesquiterpenes: 7.0%). The major component was 1,8-cineole (53.9%). This oil showed to be very different from those obtained from the same species. Larvae of A. aegypti were exposed to different concentrations of the essential oil and 1,8-cineole. The mortality rate of 100% was obtained after 24h of treatment with the oil at concentrations of 500 and 1,000 ppm. After 48h of treatment, the mortality rate was 80% and 50% for concentrations of 250 and 100 ppm, respectively. The LC 50 obtained after 24h was estimated in 289.9 ppm and after 48h was 134.1 ppm. The major compound 1,8-cineole showed no larvicidal activity.


INTRODUCTION
Dengue is a tropical disease, characterized by acute infectious of short duration and can take severe and lethal forms than concern for medical and health authorities around the world.Dengue is transmitted by Culicidae of the genus Aedes, specifically, by the bite of female mosquito infected with dengue virus.The A. aegypti, widely distributed in tropical and GISELE L. OLIVEIRA et al. subtropical regions, is the most important arbovirus vector of dengue in the Americas and the main vector of dengue in the world (Costa et al. 2005).
In Brazil, the A. aegypti program controls are focused on environmental management associated with the use of biological products, such as Bacillus thuringiensis Berliner, or chemicals products, such as insecticides of the pyrethroid and organophosphate classes, that act at the mosquito in the larval stage.However, the use of insecticides associated with the educational actions and environmental management have not controlled the infestations of the vector and are making the insects increasingly resistant, besides being pollutants and toxic (Carvalho et al. 2004, Santiago et al. 2005).
Currently, there is a growing search for new insecticides using products derived from plants as an alternative to control disease-transmitting mosquitoes.In this context, substances found in essential oils are distinguishing themselves in control of insects.These insecticides are important and interesting because they are biodegradable and the near absence of toxicity (Franzios et al. 1997, Isman 2000, Pimenta et al. 2006).
Piper, the largest genus in the family Piperaceae, is known to be rich in essential oils and some species of this genus such as P. aduncum, P. brachystachyum, P. falconeri, P. guineense and P. hispidum stand out for having insecticidal activity or as an insect repellent (Parmar et al. 1997, Pessini et al. 2003).
Piper aduncum, popularly known as "apertaruão", "falso-jaborandi", "aduncum", "jaborandido-mato" and "pimenta-de-macaco", is a widely distributed species in tropical regions, and it is considered an opportunistic plant that invades cleared areas, high hardiness and resistance to climate change.P. aduncum occurs spontaneously in pastures and in the edge of forests of the Southeast of Brazil, where it is considered weed (Lorenzi andMatos 2002, Sousa et al. 2008).
Phytochemical studies carried out with P. aduncum led to the isolation and characterization of chromenes and flavonoids, with emphasis on the 2',6'-dihydroxy-4'-methoxychalcone which showed excellent leishmanicidal effect (Moreira et al. 1998, Torres-Santos et al. 1999a, b).This plant is used in folk medicine as anti-inflammatory, to treat respiratory diseases, gynecological and intestinal disorders, as diuretic, carminative, digestive stimulant, for treatment of liver ailments and of chronic ulcers (Berg 1993, Coimbra 1994, Guadalupe-Rojas et al. 1999).
The essential oil obtained from P. aduncum was active against cercariae intermediate form of schistosomiasis, and as insecticidal and larvicidal against phytophagous insects and mosquitoes that transmit dengue and malaria (Lorenzi and Matos 2002, Souto 2006, Almeida et al. 2009).According to literature records, the major substance of the essential oil of P. aduncum, the arylpropanoid derivative dillapiole, is responsible for fungicide, larvicidal, insecticide, and molluscicide activities (Maia et al. 1998, Almeida 2004).As there are only records of the volatile composition of P. aduncum from woodlands of the Southeast and Northern Brazil, this study aims to investigate the chemical composition of essential oil obtained from this species that occurs spontaneously in the Cerrado ecosystem (Savanna) of Northern Minas Gerais, as well as to evaluate the larvicidal activity of this oil and of its major constituent, against the larvae of A. aegypti.

MATERIALS AND METHODS
COLLECT AND  The obtained essential oil was packed in amber vial, lightless.A sample of the oil was diluted in dichloromethane and subjected to analysis by gas chromatography coupled to flame ionization detector (GC-FID) and by gas chromatography coupled to mass spectrometry (GC-MS), in Farmanguinhos, FIOCRUZ, Rio de Janeiro.
Analysis conditions by GC-FID were: column HP-5MS (30m x 0.32 mm x 0.25 µm film thickness), temperature programming from 60°C to 240°C, with increase of 3°C.min -1 , using hydrogen and synthetic air as carrier gases, with a flow rate of 1.0 mL.min -1 .The Retention Indices (RI) were determined from the retention time of a homologous series of hydrocarbons (C8-C28), obtained by GC-FID under the same conditions of analysis of essential oils.The conditions of analysis by GC-MS were: column HP-5MS (30m x 0.32 mm x 0.25 µm), temperature programming from 60°C to 240°C, with increase of 3°C.min -1 , using helium as carrier gas, with a flow rate of 1.0 mL.min -1 .
The relative percentage of substances in the essential oil was estimated by GC-FID.The quantification of the major substance was accomplished using GC-FID with external standard 1,8-cineole (99.0%), obtained from Aldrich (Lot 1398664V), from the calibration curve made with different concentrations of standard (500 to 100 μg.mL -1 , R 2 = 0.996283 ± 0.003855).
The substances present in the essential oil were identified by comparing their mass spectra with database registration (WILEy7n) and by comparison of Retention Index (RI) calculated with those from literature data (Adams 2001).

BIOLOGICAL ASSAyS
Aedes aegypti larvae were provided by Center of Zoonoses of Montes Claros, Minas Gerais.Larvicidal activity was conducted in the Laboratory of Zoology of Faculdade de Saúde Ibituruna (FASI), Montes Claros, Minas Gerais.Initially, the larvae of A. aegypti were placed in a container with water and were selected according to the larval stage.Larvae in the third final instar or fourth initial were collected and separated for the test.A stereoscopic microscope it was used for identification of larvae stages.The biological assays were performed in triplicate, according to the methodology described by Oliveira et al. (2002).Different concentrations of essential oil from leaves of P. aduncum and pure 1,8-cineole varying from 1,000, 500, 250, 100, 50 and 10 ppm were used.The results represent mean of larvae mortality.
The essential oil was dissolved in 0.4 mL of dimethylsulfoxide (DMSO), and after with 30 mL of water in containers of 50 mL.The test was performed in triplicate and in each container were added 10 larvae.As a blank control was used water with DMSO (2%).The same procedure was used for the 1,8-cineole.The result was assessed after 24h and 48h, counting the dead larvae and observing the occurrence of some morphological deformation.For this purpose, larvae of each treatment were placed separately in a container with water to observe the mortality.
The calculation of regression analysis was used to estimate the concentrations capable of causing 50% (LC 50 ), 90% (LC 90 ) and 100% (CL 100 ) mortality of the larvae.The standard-error was determined by Probit analysis with software PROBIT GW-Basic, considering a significance level of p<0.05 (StatPlus 2009).

CHEMICAL COMPOSITION
The analysis of the essential oil from fresh leaves of P. aduncum allowed characterizing 23 different substances (Table I), distributed in monoterpenes (90.4%) and sesquiterpenes (7.0%).The major compound was identified as 1,8-cineole (53.9%), an oxygenated monoterpene.The following monoterpenes were also identified in great amounts: α-pinene (12.7%), β-pinene (8.5%) and transocimene (5.7%), comprising almost 27% of the mixture.Considering these four monoterpenes that represents about 80% of the volatiles, biological activity of this oil can be due to these substances.Noteworthy is the great qualitative difference of the constituents of the volatile oil extracted from leaves of P. aduncum of the Savanna of North of Minas Gerais, in comparison with literature records.A study about the constituents of the essential oil from P. aduncum leaves, collected at different locations in the Amazon Region of Brazil, showed that the oil has excellent yield (2.5 to 3.5%) and that the major constituent is a phenylpropanoid, dillapiole, ranging from 31.5 to 97.3% in the samples (Maia et al. 1998).This substance was not identified in the analyzed sample and the monoterpene 1,8-cineole was not identified in any sample of the Amazon oil (Maia et al. 1998).The presence of dillapiole as the major substance of the P. aducnum essential oil was also confirmed in a study lead in Eastern of Ecuador, where this substance represents 46.0% of the sample (Guerrini et al. 2009).However, interestingly, the volatile composition of P. aduncum collected in Bolivia and Panama is quite similar to that found in this study (Vila et al. 2005).
Dillapiole is responsible for much of the biological activities attributed to P. aduncum (Almeida 2004).In the Bolivian sample, the monoterpene group was the main fraction (76.0%), with high amount of the oxygenated compound 1,8-cineole (40.5%).The sesquiterpene fraction did not reach 10.0% of the total sample and the phenylpropanoids accounted for only 13.6% of essential oil (Vila et al. 2005).
In the sample collected in Panama, the sesquiterpenes were identified as the majority (49.0%), being the main components characterized as E-caryophyllene (17.4%) and aromadendrene (13.2%).Monoterpernes represented about 33.0% of the volatile fraction.It is interesting to note the absence of phenylpropanoids in the sample collected in Panama.In addition, the dillapiole, considered the substance responsible for the biological activities of essential oil of P. aduncum, was not detected in the two samples investigated in Bolivia and Panama (Vila et al. 2005).Thus, the differences in the volatile composition of leaves from P. aduncum from Amazon Region, Bolivia, Ecuador, Panama and our data may suggest the existence of some chemical polymorphism that could be due to difference in altitude and climate, as well as genetic factors.

LARVICIDAL ACTIVITy
The essential oil of P. aduncum showed larvicidal activity against the larvae of A. aegypti.After 24h there was a mortality rate of 100% of the larvae to concentrations of 500 and 1,000 ppm of essential oil, and 40%, 30%, 20% and 10% for 250, 100, 50 and 10 ppm, respectively (Figure 1).In the concentrations of 500 and 1,000 ppm the Larvicidal activity was registered 10 minutes after the larvae come into contact with the essential oil.
The evaluating of the larvae after a period of 48h of exposure, it was observed that the essential oil remained active, being registered a mortality rate of 80%, 50%, 40% and 10% for concentrations of 250, 100, 50 and 10 ppm, respectively (Figure 1).exposure time, even considering the volatility of the oil.It was also shown that the activity is dose-dependent.
Studies published in the literature show that the essential oil of P. aduncum is very active as insecticidal and larvicidal against phytophagous insects and mosquitoes that transmit dengue and malaria (Souto 2006, Almeida et al. 2009).Pohlit et al. (2004), for example, tested the larvicidal activity of leaf extracts of P. aduncum, collected in the Amazonas State, against the larvae of A. aegypti.The extract showed larvicidal activity that was attributed to the major component of the extract, the dillapiole.The indication of this phenylpropanoid as responsible for the larvicidal activity of this species was initially proposed by Bernard et al. (1995) that compared the alcoholic extract of leaves of sixteen species of Piper, registering P. aduncum as the highest insecticidal activity against second instar larvae of Aedes atropalpus.In this essay, it was shown that the aqueous extract of fresh leaves of P. aduncum applied directly into the water at a concentration of 10 ppm, caused 50% mortality of second instar larvae of this Culicidae and the dillapiole isolated and purified presented, under the same experimental conditions, 92% efficiency in the control of larvae at a concentration of 1 ppm.
Still, an essential oil of P. aduncum containing 86.9% of dillapiole and a purified fraction, containing between 95.0 and 98.9% of dillapiole, were tested as larvicidal against the larvae of mosquistos Anopheles marajoara e A. aegypti (vectors of malaria and dengue, respectively) using five different concentrations (from 20 to 100 ppm) for 24h and 48h, separately.It was observed a mortality rate of 96% for the oil and 100% for the dillapiole, after the exposure time.For both larvae the LC 50 was calculated as 52.7 ppm and 42.9 ppm for 24h and 48h respectively (Almeida et al. 2009).
Tests for the larvicidal activity of the oxygenated monoterpene 1,8-cineole (eucalyptol) against the larvae of A. aegypti, in this study, it was not observed mortality for any of the concentrations, excluding, thus, the major compound (53.9%) of oil as responsible for Based on the results obtained after 24h, the LC 50 was calculated at 289.9 ppm, the LC 90 was estimated at 654.9 ppm and the LC 100 in 717.04 ppm.However, the lethal concentration values decreased after 48h: LC 50 = 134.1 ppm, LC 90 = 527.1 ppm, LC 100 = 594.1 ppm.These data suggest an increase of activity after a longer this activity.The other identified monoterpenes, in lower percentage, α-pinene (12.7%), β-pinene (8.5%) and trans-ocimene (5.7%), but in combination, may be responsible for this activity.In a study with essential oils from the leaves of Pinus caribaea and Pinus tropicalis, endemic species of Cuba, it was observed larvicidal activity against larvae of A. aegypti.Both essential oils showed monoterpenes in their chemical composition, mainly α-tuyen, α-pinene, sabinene, β-pinene and myrcene.The compound β-pinene represented 26.5% of Pinus caribaea and 23.6% of Pinus tropicalis essential oils (Leyva et al. 2009).In another search for larvicidal activity it was observed for pine resin (turpentine), rich in α-pinene and β-pinene, a great activity against A. aegypti.The isolated pinenes were tested and also showed larvicidal activity (Lucia et al. 2007).
Other studies have shown a potential larvicidal activity for species rich in monoterpenes.Leyva et al. (2009) studied the larvicidal potential of essential oil of Piper racemosa against the larvae of A. aegypti and observed a positive result, noting that this activity can be attributed to the presence of monoterpenes 4-terpineol and 1,8-cineole, which together represent 41.1% of the total composition of the oil.The monoterpene 4-terpineol was identified in the studied P. aduncum essential oil (Table I), but at low percentage (0.6%).It is reasonable that 4-terpineol can be responsible for the activity registered in the P. racemosa essential oil since we not registered any activity for 1,8-cineole.
In addition, in a survey performed with essential oils of species of other families, such as Hyptis martiusii (Lamiaceae) and Lippia sidoides (Verbenaceae), Costa et al. (2005) described the larvicidal activity of essential oils against larvae of A. aegypti and of Culex quinquefasciatus.It was observed in this study the presence of mono and sesquiterpenes in the chemical constitutions.The major constituents were monoterpenes 1,8-cineole (H.martiusii) and thymol (L.sidoides).Thus, the authors state that these constituents are, probably, responsible for this activity, in isolated forms or in synergistic action with other constituents.
The main difference between our results and others published is the fact that the specimen collected in the Savanna of Northern of Minas Gerais State does not exhibit dillapiole as constituent of the volatile fraction.The major constituent is an oxygenated monoterpene, 1,8-cineole (53.9%).Therefore, the larvicidal activity of essential oil for the studied specimen cannot be attributed to dillapiole nor to its major constituent, consequently, can not be attributed in a general way the biological activity of an essential oil to its major component, being the dillapiole, or any other substance.

MORPHOLOGICAL CHARACTERISTICS OF LARVAE
Besides mortality, there was a morphological change of the larvae exposed to concentrations of 250, 500 and 1,000 ppm of essential oil of P. aduncum, both 24h and 48h.Several dead larvae showed a blackish color on their edges and curved body (Figure 2A); characteristic not observed before exposure to oil (Figure 2B).
The darkening of the larvae and the curved body were also observed in other studies (Barreto et al. 2006, Abed et al. 2007, Aciole 2009).Abed et al. (2007) investigated the morphological changes of the larvae of A. aegypti caused by essential oils from Copaifera reticulata (Fabaceae).The authors described in this study that this darkening of the edges of the larvae, possibly, occur due to the overlap of cuticle of abdominal segments.
According with Aciole (2009), morphological and behavioral changes in larvae subjected to treatments with substances isolated from plants are very important, because it will probably lead to a full explanation of the toxic action of essential oils on larvae of Culicidae.These changes provide indications of the way of action of plant substances in the body of the larvae.Thus, through of more studies could potentiate their effects and produce a highly effective insecticide and that at the same In conclusion, the volatile chemical composition of this specimen was very different of the specimens from the Amazon region, and similar to specimens from Bolivia and Panama.These data suggest for P. aduncum the occurrence of chemotypes, a hypothesis that should be investigated.This essential oil showed great larvicidal activity against A. aegypty, unlike its major component, which showed no activity.The potential lethality of the essential oil against A. aegypti, even after 48h, suggesting a residual effect, coupled with excellent oil yield (0.8%), may represent a new possibility for obtaining an active natural product to combat the mosquito that transmits yellow fever and dengue.

Fig. 1 -
Fig.1 -A.aegypti larvae mortality rate after 24h and 48h of exposure to different concentrations of essential oil of P. aduncum.

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ESSENTIAL OIL AND ANTI-LARVAE EFFECT OF P. aduncum time do not interfere in other organisms nor to environment that are being exposed.

Fig. 2 -
Fig. 2 -Larvae dead of A. aegypti with blackened color and curved body (A); larvae alive and healthy, before the exposure to the essential oil (B).

TABLE I Substances identified in the essential oil of Piper aduncum leaves.
RI = Retention Index, RI lit = Retention Index from literature.*Amount obtained using calibration curve with pure standard.