Fitoterapia 76 (2005) 629–636

www.elsevier.com/locate/fitote

Activities of some Brazilian plants against larvae of

the mosquito Aedes aegypti

Fernando A.C. de Mendonca a,b,*, K.F.S. da Silva b,

K.K. dos Santos b, K.A.L. Ribeiro Junior b, A.E.G. Sant’Ana b

aDepartamento de Fitotecnia e Fitossanidade, CECA, Universidade Federal de Alagoas,

57.100-000, Rio Largo, AL, BrazilbDepartamento de Quımica, CCEN, Universidade Federal de Alagoas, 57.072-970, Maceio, AL, Brazil

Received 17 October 2004; accepted 24 June 2005

Available online 25 October 2005

Abstract

The insecticidal activities of extracts and oils of seventeen medicinal plants of Brazil have been

determined using an Aedes aegypti larvicidal bioassay. Oils from Anacardium occidentalis,

Copaifera langsdorffii, Carapa guianensis, Cymbopogon winterianus and Ageratum conyzoides

showed high activities with LC50 values of 14.5, 41, 57, 98 and 148 Ag/l, respectively. The most

active ethanolic extract tested was that from the stem of Annona glabra which presented an LC50

value of 27 Ag/l. The potential application of cashew nut oil, an industrial by-product with low

commercial value, in the control of the vector of dengue and yellow fever, may be proposed.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Larvicidal activity; Aedes aegypti; Dengue; Yellow fever

1. Introduction

In Brazil, the rates of incidence of dengue and yellow fever have attained levels that are of

considerable concern to local authorities. Themosquito that carries the arbovirus responsible

for these diseases is Aedes aegypti [1]. This vector is now to be found in all parts of Brazil, its

0367-326X/$

doi:10.1016/j.

* Correspon

57.100-000, R

E-mail add

- see front matter D 2005 Elsevier B.V. All rights reserved.

fitote.2005.06.013

ding author. Departamento de Fitotecnia e Fitossanidade, CECA, Universidade Federal de Alagoas,

io Largo, AL, Brazil. Tel.: +55 82 261 1688.

ress: facm_al@ibest.com.br (F.A.C. de Mendonca).

F.A.C. de Mendonca et al. / Fitoterapia 76 (2005) 629–636630

distribution and abundance being strongly influenced by the presence of man and by the

level of poverty of the population [2]. Increasing deforestation, which permits greater

agricultural utilisation, decreases the rural habitats of the mosquito leading to its migration to

urban centres [3], thus following a similar migration pattern to that of the poorer section of

the human population.

For dengue, at least, there are currently no effective methods by which to control the

advance of the epidemic within the country. Despite the immense resource presented by

the natural flora of Brazil, control of A. aegypti still depends basically on the use of

synthetic pesticides. This strategy is, however, becoming inefficient because of the genetic

plasticity of the mosquito. Each year, larger doses of synthetic insecticides are required

leading to increased dangers for man and progressive contamination of the ecosystem.

The development of techniques that would provide more efficient insect control without

serious effects on the environment is clearly required in the fight against the spread of this

disease.

As part of our continued search of the biodiversity resource available in Brazil for natural

products with utilisable bioactivity, we have assayed larvicidal activity towards A. aegypti of

extracts and oils derived from nineteen Brazilian plants. The species, distributed between ten

botanical families, were selected based on ethnobotanical and chemosystematic information,

a summary of which is presented in Table 1.

2. Experimental

2.1. Chemicals

Solvents were of commercial grade and were distilled prior to use. Anhydrous sodium

sulphate and dimethylsulphoxide (DMSO) (Merck, Darmstadt, Germany) were of

analytical grade.

2.2. Plant material

Plant specimens (minimum of 500 g fresh weight for each species) were collected from

their natural habitats in the northeast of Brazil, mainly in the states of Alagoas,

Pernambuco and Bahia. Plants were identified by botanist Dr. Elias de Paula of the

Universidade Federal de Brasilia (UnB, Brasilia, Brazil); voucher specimens are deposited

in the Herbarium at UnB.

Copaıba oil (Copaifera langsdorffii) and Andiroba oil (Carapa guianensis) were

purchased from a local healer in Belem (Para, Brazil): cashew nut oil (Anacardium

occidentalis) was obtained commercially from EMBRAPA–CNPAT (Fortaleza, Ceara,

Brazil).

2.3. Preparation of extracts

The ethanol extracts were prepared by the use of a Soxhlet apparatus. The essential oils

from Ageratum conyzoides and Cymbopogon winterianus were prepared by steam

Table 1

Ethnobotanical data of the reported plants

Family/species Part used Popular use References

Anacardiaceae

Anacardium occidentalis L. Oil Treatment of malaria and yellow fever [23]

Anacardium occidentalis L. Stem Treatment of malaria and yellow fever [24]

Schinus terebinthifolius Raddi. Stem Wound healing; antiseptic,

anti-inflammatory

[25]

Annonaceae

Annona crassiflora Mart. Roots Treatment of snake bites: extracts show in

vitro cytotoxicity to human

lung carcinoma

[10]

Annona glabra L. Stem Anti-parasitic, anti-rheumatic,

emollient

[26]

Asteraceae

Ageratum conyzoides L. Leaves Treatment of malaria and yellow fever [16]

Vernonia brasiliana L. Branches Treatment of malaria and yellow fever [23]

Celastraceae

Maytenus rigida Mart. Leaves Insecticide [27]

Esterculiaceae

Guazuma ulmifolia Lam. Leaves Treatment of dermatosis; astringent,

depurative, detoxicant,

[26]

Fabaceae

Andira inermis (W. Wright) Kunth Stem Wound healing; vermifuge [26]

Caesalpinia pyramidalis Tul. Wood bark Anti-inflammatory, anti-diarrhoeal [28]

Copaifera langsdorffii Desf. Oil Wound healing and treatment

of malaria and yellow fever;

antibiotic, anti-inflammatory

[26,23]

Meliaceae

Carapa guianensis Aubl. Oil Wound healing and treatment of

insect bites, malaria and yellow fever

[23,26,29]

Cedrela fissilis Vell. Stem Astringent, febrifuge [26]

Swietenia macrophylla King. Leaves Treatment of malaria and yellow fever [23]

Poaceae

Cymbopogon winterianus Jowitt Leaves Mosquito repellent [15]

Simaroubaceae

Simarouba amara Aubl. Leaves Wound healing and treatment of colic,

malaria and yellow fever; vermifuge,

anti-diarrhoeal

[26,30]

Verbenaceae

Aegiphilai lhotskiana Cham. Roots Treatment of malaria and yellow fever [23]

F.A.C. de Mendonca et al. / Fitoterapia 76 (2005) 629–636 631

distillation. Samples were prepared at initial concentrations of 500 Ag/l for the preliminary

activity tests by solubilising an appropriate aliquot in water containing 1% DMSO (with

sonication if required).

2.4. Determination of larvicidal activity

The bioassays were conducted in the Department of Chemistry, Universidade Federal

de Alagoas (Maceio, Brazil) employing a colony of A. aegypti maintained for this purpose.

F.A.C. de Mendonca et al. / Fitoterapia 76 (2005) 629–636632

Standard methods for assaying larvicidal activity as recommended by the World Health

Organisation [4] were followed in all experiments. Preliminary bioassays were performed

with 4th instar larvae of A. aegypti and were carried out in duplicate using 10 larvae for

each replicate assay. The larvae were placed into 200-ml disposable plastic cups

containing 25 ml of the test solution and incubated at 27 8C. Larvae were considered

dead when they were unable to reach the surface of the solution when the cups were

shaken. The number of dead larvae was determined at the start of the experiment (0 h) and

24 and 48 h thereafter. An aqueous solution of DMSO (1%) was employed as the negative

control whilst rotenone served as the positive control.

Treatments that showed at least 50% mortality within 48 h were followed-up by further

bioassays of the same sample at different concentrations in order to determine the

concentration required to kill 10% (LC10), 50% (LC50) and 90% (LC90) of the larvae

present. The analysis of the follow-up assays was carried out according to the Finney

Probit method [5].

3. Results and discussion

The results obtained in the preliminary assays of eighteen extracts or oils of

seventeen Brazilian plants against larvae of A. aegypti (Table 2) showed that only six

were active according to our accepted norm (i.e. N50% lethality at 500 Ag/l). Of the

active samples, all of which caused 100% lethality of larvae at 500 Ag/l, five were

oils and one was an ethanolic extract of stem material. The most active sample was

cashew nut (A. occidentalis) oil which showed an LC50 value of 14.5 Ag/l (Table 3).

Although this oil sample was less active than the positive control (rotenone), the result

is particularly noteworthy because the crude material is obtained as a by-product of

the processing of cashew nuts and, as an industrial residue, is of very low commercial

value.

The demonstration of considerable larvicidal activity (LC50 26.9 Ag/l) against A.

aegypti by stem extracts of Annona glabra is, perhaps, not unexpected since it has been

previously demonstrated that extracts of the seeds are strongly larvicidal [6]. The plant is

used in traditional medicine and shows strong insecticidal activity [7,8]: furthermore, two

new Annonaceous acetogenins possessing insecticide and anti-tumour activity have

recently been isolated from the wood and leaves of this species [9]. The flora of Brazil is

rich in Annonaceous plants, and extracts from many (Annona muricata, Annona

crassiflora and Annona squamosa amongst others) show strong larvicidal activities

[6,9,10]. The major constituents identified in members of the Annonaceae are, typically,

acetogenins, and this class of compound exhibits numerous biological properties including

antibiotic, anti-tumour, anti-malarial, anti-parasitic and insecticidal activities [11].

Oils from C. langsdorffii (Copaıba oil), C. guianensis, C. winterianus (citronella grass

oil), and A. conyzoides showed significant activities (in decreasing order) in the larvicidal

assay against A. aegypti. In an earlier study, Kumar and Dutta [12] assayed the oils from

ten plants against larvae of Anophelis stephensis and concluded that those from Cedrus

deodora, Lavendula officinalis and Mentha arvensis were the most active with LC50

values (in the range 63–84 Ag/l) that are very similar to those reported in the present work.

Table 2

Toxicity of the Brazilian plants ethanol extracts and oils on A. aegypti larvae

Plants Voucher

specimen localityaSample

(500 ppm)

Yield of

extract or oilbLarvae

mortality (%)c

Anacardiaceae

Anacardium occidentalis L. EMBRAPA–CE Oil 100

Anacardium occidentalis L. Ethanol extract 15.0 0

Schinus terebinthifolius Raddi. JEP 3643 (UB) Ethanol extract 18.0 35

Annonaceae

Annona crassiflora Mart. JEP 3369 (UB) Ethanol extract 15.0 10

Annona glabra L. JEP 3649 (UB) Ethanol extract 17.0 100

Asteraceae

Ageratum conyzoides L. Oil 0.3 100

Vernonia brasiliana JEP 3607 (UB) Ethanol extract 8.0 5

Celastraceae

Maytenus rigida Mart. 00767 UFS Ethanol extract 19.0 15

Esterculiaceae

Guazuma ulmifolia Lam. JEP 3644 (UB) Ethanol extract 18.0 35

Fabaceae

Andira inermis (W. Wright) Kunth JEP 3642 (UB) Ethanol extract 18.0 35

Caesalpinia pyramidalis Tul. JEP 3592 (UB) Ethanol extract 16.0 5

Copaifera langsdorffii Desf. Commercial oil Oil 100

Meliaceae

Carapa guianensis Aubl. JEP 3959 (UB) Oil 100

Cedrela fissilis Vell. JEP 3656 (UB) Ethanol extract 15.0 20

Swietenia macrophylla King. Ethanol extract 16.5 0

Poaceae

Cymbopogon winterianus Jowitt Oil 1.3 100

Simaroubaceae

Simarouba amara Aubl. JEP 3640 (UB) Ethanol extract 1.6 0

Verbenaceae

Aegiphila lhotskiana Cham. JEP 3540 (UB) Ethanol extract 8.0 30

a Voucher specimens are deposited in the Herbarium of Universidade Federal de Brasilia, Brasilia, Brazil.b Yield of extract (w/w) in terms of original starting material.c After 48 h of treatment.

F.A.C. de Mendonca et al. / Fitoterapia 76 (2005) 629–636 633

Copaıba oil (together with the related Andiroba oil) has been used for centuries by

indigenous populations in the Amazon to cure many maladies ranging from a simple

headache to serious infections including malaria [13]. In 1972, the USA Food and Drug

Administration approved the use of Copaıba oil following the demonstration of negative

reactions to irritation and sensitivity tests performed on 25 volunteers [14]. In the present

work we demonstrate that Copaıba oil could also be used as a potent larvicide against A.

aegypti.

Also reported for the first time in this study is the larvicidal activity of citronella grass

oil (C. winterianus) against A. aegypti: the oil is considered to possess insect repellent

properties and is currently employed widely for this purpose [15].

The essential oil from A. conyzoides showed an LC50 value of 148 Ag/l which,

although somewhat high, is nevertheless of interest because this oil is known to

induce morphogenetic abnormalities in mosquito larvae [16]. Precocenes I and II,

Table 3

LC10, LC50 and LC90 index of A. aegypti larvae exposed to plants ethanol extracts and oils

Plants Sample LC10a LC50

a LC90a

Meliaceae

Carapa guianensis Aubl. Oil 9.9 (6.1, 14.2) 57 (47, 68) 330 (253, 473)

Asteraceae

Ageratum conyzoides Oil 86 (76, 97) 148 (138, 159) 256 (230, 298)

Annonaceae

Annona glabra Ethanol extract 11.4 (3.1, 18.1) 27 (16, 35) 64 (48, 120)

Poaceae

Cymbopogon winterianus Jowitt Oil 56 (48, 62) 98 (89, 107) 172 (151, 206)

Fabaceae

Copaifera langsdorffii Desf. Oil 21 (14, 26) 41 (34, 47) 79 (65, 109)

Anacardiaceae

Anacardium occidentalis Oil 4.4 (1.5, 7.7) 14.5 (8.7 , 21.0) 48 (32, 91)

Rotenone – 1.5 (0.9, 2.2) 6.2 (4.7 , 7.8) 25 (19, 38)

a Mean values (16 Ag/l) with lower and upper 95% confidence limits, respectively, shown in parenthesis.

F.A.C. de Mendonca et al. / Fitoterapia 76 (2005) 629–636634

which are present in the oil, possess anti-juvenile hormone activity [17] and this can

give rise, in some insects, to precocious metamorphosis by eliminating the control of

gene expression normally exerted by the juvenile hormones [18]. In the present study,

it was observed (data not shown) that, following treatment with different concentra-

tions of A. conyzoides oil, the larvae of A. aegypti that were not killed showed

problems in completing their development: pupae were pigmented or incompletely

emergent, adults showed intense rings of pigmentation on the tarsus and abdomen, and

some developed very stunted wings and low production of eggs. Thus, the oil of A.

conyzoides may represent an excellent candidate for use in the fight against A. aegypti

and other Culicid vectors since, together with its larvicidal activity, its effects on

insect metamorphosis would give rise to a decrease in the reproductive efficiency of

the adult insect further reducing the population.

All six of the active samples identified in the present study showed larvicidal activities

superior to that previously reported [19] for an extract of Quercus lusitania var. infectoria

galls (Oliv.), fractions of which, it was proposed, could be used to control Culex pipiens.

Furthermore, whilst use in the control of A. aegypti and C. pipiens of a commercial

saponin extract from Quillaja saponaria has been advocated [20], this extract produced

100% mortality of larvae only at concentrations N800 Ag/l: in our study, all six active

samples produced 100% mortality at concentrations b500 Ag/l, with individual LC50

values being well below 200 Ag/l.Neem oil (Azadirachta indica) is a widely used insecticide and, recently, LC50 values

for two new triterpenoids and nimocinol (all components of the oil) of 21, 83, and 100 Ag/l have been reported [21]. In our experiments, the oils of A. occidentalis, C. langsdorffii,

C. guianensis and C. winterianus, and the stem extract of A. glabra, all showed lower

LC50 values than that of nimocinol. The results relating to the oil of A. occidentalis are

similar to those reported in a previous study [22] in which the commercial insecticide h-asarone was used as the positive control (LC50=16 Ag/l). Furthermore, it is important to

note that our results relate to crude plant preparations and not to the purified active

F.A.C. de Mendonca et al. / Fitoterapia 76 (2005) 629–636 635

components, which, when isolated, would be expected to show much lower LC50 values

than those reported here for the extracts and oils.

The present study has identified extracts or oils from six Brazilian plants which show

potential for use in the control of A. aegypti. Identification of the components present in

the active samples that might be responsible for the larvicidal activity against A. aegypti

will be an important next step in the development of a verifiable application of these

materials for the field control of the insect vector.

Acknowledgements

The authors wish to thank Conselho Nacional de Pesquisa e Desenvolvimento and

Fundacao de Amparo a Pesquisa do Estado de Alagoas for their financial support.

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