Essential oils of leaves of Piper species display larvicidal activity against the dengue vector, Aedes aegypti (Diptera: Culicidae)

Óleo essencial da folha de espécies de Piper exibem atividade larvicida contra o vetor da dengue Aedes aegypti (Diptera: Culicidae)

Abstracts

The mosquito Aedes aegypti is the vector of the dengue virus, an endemic arbovirus from tropical and subtropical regions of the world. The increasing resistance of mosquitoes to commercial insecticides impairs regular control programs; therefore, chemical prospecting originating from the Amazonian flora is promising for potential new insecticides. Several Piper species are, notably, rich in phenylpropanoids and terpenoids, substances with proven insecticidal activity. The composition and the larvicidal activity of three Piper species against A. aegypti were evaluated. Essential oils were extracted by hydrodistillation in a modified Clevenger apparatus and analyzed by GC/MS. The major components found in Piper arboreum were germacrene D (31.83%) and bicyclogermacrene (21.40%); in Piper marginatum: (E)-methyl isoeugenol (27.08%), (E)-anethole (23.98%) and (Z)-methyl isoeugenol (12.01%); and in Piper aduncum: (E)-isocroweacin (29.52%) and apiole (28.62%) and elemicin (7.82%). Essential oils from the Piperaceae species studied resulted in Lethal Concentrations (LC50) of 34-55 ppm, while LC90 was higher than 100 ppm, except for P. marginatum (85 ppm).

Vector control; Essential oil; Piperaceae; Aedes aegypti

O mosquito Aedes aegypti é o vetor do vírus da dengue, um arbovírus endêmico em regiões tropicais e subtropicais do mundo. A crescente resistência dos mosquitos aos inseticidas comerciais prejudica programas regulares de controle, portanto, a prospecção química proveniente da flora amazônica surge como alternativa promissora para novos inseticidas. Várias espécies de Piper são notavelmente ricas em fenilpropanóides e terpenóides, substâncias com atividade inseticida comprovada. A composição e atividade larvicida de três espécies de Piper sobre A. aegypti foi avaliada. Os óleos essenciais foram extraídos por hidrodestilação em Clevenger modificado e analisado por GC/MS. Os principais componentes encontrados em Piper arboreum foram germacreno D (31,83%) e biciclogermacreno (21,40%); Piper marginatum: (E)-metilo de isoeugenol (27,08%), (E)-anetole (23,98%) e (Z)- methyl isoeugenol (12,01%), e Piper aduncum: (E)-Isocroweacin (29,52%), apiol (28,62%) e elemicin (7,82%). Os óleos essenciais a partir das espécies de Piperaceae estudadas resultou em concentrações letais (CL50), 34-55 ppm, enquanto que CL90 foi superior a 100 ppm, com excepção para P. marginatum (85 ppm).

Controle Vetorial; Óleo Essencial; Piperaceae; Aedes aegypti

INTRODUCTION

Mosquitoes are important vectors of diseases, such as dengue, that stands out as reemerging disease and public health concern worldwide, affecting mostly poor, urban populations; and also a leading cause of hospital admissions in several countries. Dengue incidence has increased about 30 times over the last 50 years and 50-100 million infections annually are estimated in more than 100 endemic countries, placing almost half the world's population at risk (Who, 2005).

Although, most patients are asymptomatic, subsequent infections with different viruses serotypes may result in hemorrhagic fever with high mortality. In the Latin America, Brazil stands up with 98.5% of cases, and the highest mortality rate (Who, 2009 and 2010).

Dengue is considered the most important viral disease transmitted and disseminated by arthropods worldwide. The mosquito vector, Aedes aegypti, can be found in all Brazilian states, and dengue outbreaks are often reported (Who, 1997; Who, 2002; MS, 2009).

Chemical control of A. aegypti is a challenging issue and dengue outbreaks have repeatedly occurred in Brazil over the last 10 years since this vector species is resistant to insecticides such as organochlorines, organophosphates and onset of resistance to pyrethroids (Tauil, 2006). Besides toxicity, resistance reported in several areas under continuous application of insecticides is also an important issue and alternatives to conventional chemical control based on natural products from plants, including essential oils, stands as a potential source for new molecules for mosquito control. The genus Piper stands out as a source of active principles derived from plants has been described as antimicrobial and insecticidal (Bergo, et al. 2005).

Essential oils of many Brazilian plants display larvicidal activity, e.g., Ocimum americanum, Ocimum gratissimum (Cavalcanti, et al. 2004), Croton zehntneri (Morais, et al. 2006), including Piper species (Morais, et al. 2007), therefore, in the present study, essential oils of three Piper species commonly found in Amazon region, Brazil were tested as larvicides against the dengue vector, A. aegypti.

MATERIAL AND METHODS

Plant material

Leaves of three different species of Piperaceae: P. marginatum Jacq. (pimenta-do-mato), P. arboretum Aubl. (jaborandi-pimenta) and P. aduncum Vell. (pimenta-de-macaco) were collected at different areas of the State of Rondonia, Western Amazon, Brazil. Piper species were identified by Dr. J. Gomes from INPA herbarium (Instituto Nacional de Pesquisa da Amazônia) and voucher specimens were deposited under the identification numbers: 216630 for P. marginatum, 226856 for P. arboretum and 211711 for P. aduncum.

GC and GC-MS

Fresh leaves of each plant were steam distilled in a Clevenger type apparatus and the oils (1 mL) analyzed by GC using a Varian CP-3800 gas chromatograph coupled to a computer equipped with a STAR WORKSTATION. The instrument operates at the following conditions: equipped with a fused silica 30 m (CP-Sil 8CB, Varian) capillary column with an internal diameter of 0.25 mm and a film thickness of 0.25 mm; the hydrogen carrier gas had a delivery rate of 1.5 ml/min (controlled constant flow); a capillary injector operating at 250 0C in the split mode (1:100); a flame ionization detector (FID) running at 250 0C; the oven temperature programming was 35 0C during injection, and then increased from 35 to 180 0C at the rate of 4 0C/min, increased again until a final temperature of 280 0C at a rate of 17 0C/min, and at 280 0C for 10 min.

GC-MS was performed on a Hewlett Packard 5971 instrument employing the following conditions: column: dimethylpolysiloxane DB-1 coated fused silica capillary column (30 m x 0.25 mm); carrier gas: He (1 mL/min); injector temperature was 2500C and the detector temperature 2000C. The column temperature programming was 35-180 0C at 4 0C/min then 180-250 0C at 10 0C/min; mass spectra: electron impact 70 eV (Adams 2001). Compounds were identified by their GC retention time relative to known compounds and by comparison of mass spectra with those present in the computer data bank (National Institute for Standard Technology e NIST and 62,235 compounds) and published spectra (Stenhagen, 1974).

Mosquito collection and breeding

The eggs of Aedes aegypti were collected using ovitraps, i.e., black plastic pots filled with tap water and a wooden paddle, placed in houses found with adult A. aegypti in the municipality of Porto Velho - RO (08º 44' 11,40"S, 63º 53'45,34"W). The ovitraps were removed three days after installation and wooden paddles were analyzed in the laboratory for the presence of A. aegypti eggs and placed in plastic trays filled with 1 L distillated water. After hatching, the larvae were kept under laboratory conditions (28 ºC, 80% RU and 12 h photoperiod) and fed with grinded dog food pellets. Pupae were transferred to plastic cages and mosquitoes were fed with sucrose 10% and blood fed on rabbits for egg production. Eggs were collected introducing beaker containing filter paper and distilled water. Third to fouth instar larvae were used in the larvicidal assays.

Larvicidal bioassay

Five concentrations (10, 50, 100, 250 and 500 ppm) of the essential oils were used based on (Morais, et al. 2007). Batches of 25 larvae of 3º- 4º instar for each concentration were tested with four replicates and included a control (1ml of DMSO diluted in 100 ml of water). Larval mortality in intervals of 24 h and 48h was recorded for LC (Lethal Concentration) calculation using Probit Analysis (Minitab 14.1; Minitab Inc). All experiments were repeated three times in different periods (Who, 2005).

The effect of different essential oil of Piper and concentration on larval mortality was analyzed using Two Way Anova using SigmaStat 2.0 (SPSS, Inc).

RESULTS AND DISCUSSION

A total of 40 volatile constituents were identified, accounting for 98.31- 99.79% of the chemical composition of the correspondent oils. The essential oils of P. marginatum and P. aduncum had similar percentages of the main terpenoid classes, but differed significantly from a qualitative point of view. The essential oils of P. marginatum and P. aduncum were characterized mostly by phenylpropanoids: 75.05 and 75.19%, respectively, while sesquiterpene hydrocarbons composed up to 95.21 % of P. arboreum oil (Table 1).

TABLE 1.
Composition (%) of the leaf oil of Piper marginatum, P. arboreum and P. aduncum collected in Rondonia State, Brazil.

Compound	K.I.*	P. marginatum	P. arboreum	P. aduncum
a-pinene	939	2.46	-	1.81
canfene	954	1.02	-	?
b-pinene	979	2.27	-	1.70
d-carene	1002	4.27	-	-
limonene	1029	?	-	1.43
cis-ocimene	1037	?	?	3.28
trans-ocimene	1050	-	-	7.70
(Z)-anethol	1253	7.90	-	-
(E)-anethol	1285	23.98	-	-
safrole	1287	-	-	3.31
d-elemene	1338	?	3.44	-
a-copaene	1377	1.85	1.13	0.40
b-bourbonene	1388	-	2.22	-
b-cubebene	1389	-	0.67	-
b-elemene	1391	0.95	3.63	-
b-caryophyllene	1409	2.28	10.88	2.51
b-curjunene	1434	-	1.73	-
(Z)-methyl-isoeugenol	1454	12.01	-	-
a-humulene	1455	-	1.49	0.64
aromadendrene- dehydro	1463	4.06	-	-
allo-aromadendrene	1460	-	1.72	-
b-chamigrene	1478	1.59	-	-
germacreneD	1485	?	31.83	2.28
b-selinene	1490	-	4.23	-
(E)-methyl-isoeugenol	1492	27.08	-	-
biciclogermacrene	1499	?	21.40	0.99
germacreneA	1509	?	1.41	?
miristicin	1519	-	-	5.92
elemol	1550	2.51	-	-
d-candinene	1523	?	1.36	?
(E)-isocroweacin	1555	-	-	29.52
elemicin	1557	2.41	-	7.82
germacreneB	1561	?	8.07	?
isoelemicin	1570	1.67	-	-
caryophyllene oxide	1583	-	1.44	-
globulol	1585	-	0.46	-
viridifloral	1593	-	0.54	-
himachalol	1650	-	1.50	-
a-cadinol	1654	-	0.64	-
apiole	1678	-	-	28.62
Terpenoid class composition
Monoterpene hydrocarbons		10.02	-	15.98
Sesquiterpene hydrocarbons		10.73	95.21	6.82
Oxygenated sesquiterpenes		2.51	4.58	-
Phenylpropanoids		75.05	-	75.19
Total		98.31	99.49	98.93

• *Retention index. The identified constituents are listed in their order of elution from a nonpolar Column.

The major phenylpropanoids in P. marginatum were (E)-methyl-isoeugenol (27.08%), (E)-anethol (23.98%), (Z)-methyl-isoeugenol (1.01%), and (Z)-anethol (7.9%); P. aduncum: (E)-isocroweacin (29.52%), apiole (28.62%) and elemicin (7.82%), and major sesquiterpenes in P. arboreum were germacreneD (31.83%), biciclogermacrene (21.40%) and (E)-caryophyllene (10.88%) (Table 1).

The phenylpropanoid anethol, a major component of P. marginatum, was also found in high percentage in P. marginatum chemotype V (Craveiro, et al. 1976) essential oil, Pimpinella anisum (Erler, et al. 2006), Illicium verum (Lima, et al. 2008), Croton zenhtneri (Santos, et al. 2001).

Santos et al. (2001) analyzed essential oils of 10 Piperaceae species from the Brazilian Atlantic Forest and found that the most frequently identified compounds were sesquiterpenes, differently from P. marginatum and P. aduncum in the present work, but similar to P. arboreum, except for biciclogermacrene. These authors also related (E)-caryophyllene and germacrene D as the most common sesquiterpenes identified, also found in lower percentages in the essential oils from leaves of Piper lanceaefolium from Costa Rica (Mundina, et al. 2001).

Cruz et al. (2011) found great diversity of the chemical composition of the essential oils from 15 Piper species from Guatemala and identified β-caryophyllene in all species, germacreneD in 11 and biciclogermacrene only in Piper sempervirens.

Besides the major components found here in the oil of P. aduncum, Maia et al. (1998) and Fazolin et al. (2007) also related other major components for the same plant species. Differences in oil components probably resulted from different environmental conditions for plant development (Simas, et al. 2004).

Aedes aegypti larvae were exposed to the essential oil of the studied plants all died after a few hours exposure only when exposed to 500 and 250 ppm. But larval mortality decreased significantly (F=312.66; P<0.001) in lower oil concentrations. The oil of Piper marginatum, usually, was the most effective against larvae (Table 2).

TABLE 2.
Larval mortality (%) of Aedes aegypti after 96 hours exposure to the essential oils of different Piper species (P. aduncum, P. arboreum and P. marginatum)

Concentrations (ppm)
Species	10	50	100	250	500	Mean (±1,9)
P. aduncum	19.0b4	49.0b3	71.3b2	99.31	1001	56b
P. arboreum	23.6a4	38.3b3	59.3b2	98.61	1001	53b
P. marginatum	13.0b3	78.0a2	99.3a1	1001	1001	65a
Mean (±2,35)	184	553	762	991	1001

• Two Way Anova (species and concentration) and Student-Newman-Keuls (comparisons). Different letters and number indicate significant differences (P<0,05) in the same columm and row, respectively. No mortality was detected in control groups.

Piper marginatum oil had the lowest CL50 and CL90 values and significantly (F=26.09; P<0.001) caused higher larval mortality at 50 and 100 ppm related to the other species (Table 2). Piper aduncum and P. arboreum had higher CL50 values (Table 3) and did not differed significantly in the mean mortality caused to A. aegypti larvae (Table 2).

TABLE 3.
Lethal Concentrations (LC₅₀ and LC₉₀) of the essential oils of Piper species on 3°- 4° instar larvae of the mosquito Aedes aegypti (Diptera: Culicidae).

Lethal Concentration (ppm)
Species	LC50	CI	SE	LC90	CI	SE
Piper aduncum	46	43.01- 48.99	2.99	156	148.83- 163.17	7.17
Piper arboreum	55	51.48- 58.52	3.52	204	198.45- 212.55	8.55
Piper marginatum	34	32.64- 35.36	1.36	85	82.83- 87.17	2.17

• PPM= parts per million. Calculated from data mortality from 24-48 hours
• CI 95%= Confidence Interval. Calculated from data used for lethal concentrations
• SE= Standard Error. Calculated from mean from data mortality from 24-48 hours

Interestingly, Autran et al. (2008) evaluated the larvicidal properties of the essential oils of different parts of P. marginatum against A. aegypti and reported a similar larvicidal activity for the essential oil extracted from leaves. But, majoritarian oil components, i.e, (Z)-azarone and patchouli alcohol, were not detected in the present work. Andrade et al. (2008) described seven chemotypes of P. marginatum based on the oil components. The present sample from Rondonia State was similar to chemotype V, i.e., high content of (E)-anethole, while Autran's was similar to chemotype VII, indicating that, despite of chemical characteristics, larvicidal activity is retained in different P. marginatum chemotypes due to other insecticidal molecules.

The presence of anethol in Piper marginatum composition could be responsible to the larvicidal activity because Cheng et al (2004) related a LC50 = 42 ppm of this compound to A. aegypti, very close the data informed in the present study. Also this phenylpropanoid was highly effective in the control mosquito larvae in other studies, e.g., Aedes aegypti (Chantraine, et al. 1998; Morais, et al. 2006), Ochlerotatus caspius (Knio, et al. 2008) and other insects, e.g., the cockroach Blattella germanica (Chang, et al. 2002), the beetle Zabrotes subfaciatus (Silva and Câmara, 2007). Besides, anethol also repelled Culex pipiens mosquitoes (Lima, et al. 2008) and the aphid Brevicoryne brassicae (Santos, et al. 2001).

Other components present in the oil of P. marginatum, e.g., methyl-isoeugenol, possible contributed to the larvicidal activity found in the present work. Park et al. (2007) related that this molecule had nematicidal activity against the pine wood nematode, Bursaphelenchus xylophilus. A similar molecule, methyl-eugenol, also presented contact toxicity against the cockroach Periplaneta americana and also larvicidal activity against A. aegypti (Morais, et al. 2006).

Piper aduncum also displayed similar insecticidal activity against workers of the fire ant, Solenopsis saevissima (Ngoh, et al. 1998), but the majoritarian compound, dillapiole, has a different methoxy group position in the benzene ring compared to apiole, one of the main compounds found in P. aduncum in the present work.

The main component of P. arboreum oil, the sesquiterpene germacrene D, displayed the highest mosquitocidal activity against Culex quinquefasciatus, Anopheles gambiae and A. aegypti (Souto, et al. 2012) and may be related to larvicidal activity of this species to A. aegypti since its percentage in P. arboreum was 3 times higher than found by Kiran and Devi (2006) in the plant Chloroxylon swietenia.

Interestingly, Piper humaytanum had similar percentage of b-caryophyllene, one of the main compounds of the essential oil from Piper arboreum, but germacrene D was not detected. This species displayed LC50 to A. aegypti three times higher than Piper arboreum in the present study (Table 3).

Hummelbrunner and Isman (2001) related significant synergistic effects of several essential oil compounds on the tobacco cutworm, Spodoptera litura. Therefore, future analysis of single components, e.g., anethol, methyl-isoeugenol and germacrene D from the Piper species studied and potential synergistic or addictive effects among them in the larvicidal activity on A. aegypti and also other important mosquito species, e.g, the malaria vector, Anopheles darlingi, may provide new affordable and efficient molecules for chemical control of these insects.

Concluding, the main components found in Piper arboreum were germacrene D (31.83%) and bicyclogermacrene (21.40%); Piper marginatum: (E)-methyl isoeugenol (27.08%), (E)-anethole (23.98%) and (Z) - methyl isoeugenol (12.01%) and Piper aduncum (E) - Isocroweacin (29.52%), apiol (28.62%) and elemicin (7.82%). Essential oils from the Piperaceae species studied resulted in lethal concentrations (LC50) 34-55 ppm, whereas LC90 was greater than 100 ppm, except for P. marginatum (85 ppm), the essential oil of this plant that showed the highest larvicidal activity against Aedes aegypti.

CONCLUSIONS

The main components found in Piper arboreum were germacrene D (31.83%) and bicyclogermacrene (21.40%); Piper marginatum: (E)-methyl isoeugenol (27.08%), (E)-anethole (23.98%) and (Z) - methyl isoeugenol (12.01%) and Piper aduncum (E) - Isocroweacin (29.52%), apiol (28.62%) and elemicin (7.82%). Essential oils from the Piperaceae species studied resulted in lethal concentrations (LC50) 34-55 ppm, whereas LC90 was greater than 100 ppm, except for P. marginatum (85 ppm), the essential oil of this plant that showed the highest larvicidal activity against Aedes aegypti.

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Publication Dates

• Publication in this collection
  Jan-Mar 2015

History

• Received
  01 May 2013
• Accepted
  16 June 2014