Journal of Ethnopharmacology 97 (2005) 199206

A study of the larvicidal and molluscicidal activities of some medicinal plants from northeast Brazil
J. de S. Lunaa , A.F. dos Santosb , M.R.F. de Limab , M.C. de Omenab , F.A.C. de Mendonc ab , L.W. Biebera , A.E.G. SantAnab,
a

Departamento de Qu mica Fundamental-CCEN, Universidade Federal de Pernambuco, 50740-901 Recife PE, Brazil b Departamento de Qu mica-CCEN, Universidade Federal de Alagoas, 57072-970 Maceio AL, Brazil Received 9 August 2004; received in revised form 6 October 2004; accepted 7 October 2004 Available online 4 January 2005

Abstract In a search for natural products that could be used to control the vectors of tropical diseases, 23 extracts of medicinal plants from the northeast of Brazil have been tested for molluscicidal activity against egg masses and adults of the snail Biomphalaria glabrata, for larvicidal activity against the larvae of the mosquito Aedes aegypti, and for general toxicity against the larvae of the brine shrimp Artemia salina. Of these extracts, two were active against the adult snail, one against snail egg masses, eight against the larvae of the mosquito, and 16 showed toxicity towards the brine shrimp. Chemical tests indicated that a wide variety of natural product classes were present in those extracts that showed signicant activities in the bioassays. 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Biomphalaria glabrata; Artemia salina; Aedes aegypti

1. Introduction Brazil possesses the largest genetic diversity of plant species in the world, but less than 10% have been evaluated with respect to their biological characteristics, and fewer than 5% have been subjected to detailed phytochemical studies. Despite a recent increase in research activity in this area, plants still constitute a relatively under-utilised, and potentially very valuable, source for the further discovery of biologically active substances. Two of the most important health problems facing much of tropical Latin America, including large parts of northern Brazil, are the diseases of schistosomiasis and dengue fever. Since a large proportion of the population living in these areas suffer from varying degrees of poverty, the discovery of plant-derived compounds that could help with the control or eradication of these diseases would of great value, particularly if the plants concerned were readily available to those

Corresponding author. Tel.: +55 82 214 1388; fax: +55 82 214 1700. E-mail address: aegs@telurio.qui.ufal.br (A.E.G. SantAna).

who most need to use them. In this context, our continuing project has the aim of screening plants grown locally in northeast Brazil for biological activities that could be of value in the ght against local diseases. Schistosomiasis, also known as bilharzia, is a disease caused by infestation of the host by the parasite Schistosoma, a atworm that lives in blood during one stage of its life cycle. Schistosoma mansoni is the most common human schistosome in Brazil, and the disease is transmitted to humans by skin contact with fresh water containing cercariae. One plausible method of attacking and breaking down the transmission cycle (worm-egg-miracidium-sporocyst-cercariae-worm) of Schistosoma mansoni is through control, using natural molluscicides, of the snail (Biomphalaria glabrata), the intermediate host in which the transformation from miracidium to cercariae occurs. The discovery of new methods for the control of the mosquito Aedes aegypti is of paramount importance because of its role as a vector for arboviruses responsible for dengue fever and yellow fever, both of which are endemic to Central and South America, Asia and Africa. Currently, the situa-

0378-8741/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.10.004

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J. de S. Luna et al. / Journal of Ethnopharmacology 97 (2005) 199206

tion with respect to dengue is critical in all American countries and particularly in Brazil (FUNASA, 2003): in many endemic regions, contraction of the disease is considered almost inevitable (Gibbons and Vaughn, 2002). However, there are no specic therapeutic agents for dengue: steroids, antiviral compounds and carbazochrome (which decrease capillary permeability) have no proven role (Gibbons and Vaughn, 2002). The only way to prevent the occurrence of the dengue arbovirus is through control of the proliferation of Aedes aegypti (Marcondes, 2001), but this is not an easy task because the mosquito shows large genetic plasticity. In order for insecticides to remain efcient at killing the mosquito, it is necessary continuously to increase the dosage, but eventually the level of insecticide required will give rise to toxicity problems for man and his domesticated animals. Clearly new drugs and novel methods, which can be employed with greater safety, for the control of the mosquito must be developed (Neves, 2000). A number of studies have recently been concerned with the larvicidal properties of plants, and interest in this area is now growing rapidly. Artemia salina, commonly known as the brine shrimp, is a small crustacean, which has been the subject of many physiological studies. The brine shrimp lethality assay is considered to be one of the most useful tools for the preliminary assessment of general toxicity, and the bioassay has shown good correlation with cytotoxic activity against some human solid tumours and with pesticidal activity (McLaughlin et al., 1991). The specic objective of the present study was to examine crude extracts of medicinal plants from the northeast of Brazil for their molluscicidal activities against the snail Biomphalaria glabrata, their larvicidal activities against the mosquito Aedes aegypti, and their general toxicity towards Artemia salina.

ground in a Nogueira (Itapira, SP, Brazil) laboratory mill to a moderately-ne powder (mesh size 2.0 mm). Powdered material (500 g) was extracted with 95% ethanol (1.5 L) at room temperature (2527 C) for 3 days and ltered: the residue was extracted twice more in a similar manner. The extracts were evaporated under reduced pressure in a rotary evaporator and bulked: if necessary, concentrated extracts were further dried in a freeze-drier. Crude extracts were stored in a freezer at 20 C until required. A stock solution containing 2000 g mL1 of each crude extract was prepared by suspending 200 mg of extract in 1 mL of pure dimethylsulphoxide (DMSO: Aldrich, Milwaukee, WI, USA), mixing by sonication in a Cole Parmer (Vernon Hills, IL, USA) model 8892E-MT ultrasonic bath (47 kHz; 120 W) for 20 min, and completing to volume (100 mL) with dechlorinated water. Stock solutions were diluted with an appropriate volume of dechlorinated water in order to provide assay solutions containing 1000, 500 and 100 ppm as required. 2.3. Assay for molluscicidal activity The molluscicidal activities of extracts against adults and egg masses of Biomphalaria glabrata were determined (four replicates for each sample) according to previously described techniques (dos Santos and SantAna, 1999). Both positive (cupric carbonate at 50 g mL1 or niclosamide at 3 g mL1 ) and negative (dechlorinated water containing 1% DMSO) control assays were carried out in order to verify the susceptibility of the snails under the assay conditions employed. For assays involving mollusc eggs, pentachlorophenol was used as the positive control. 2.4. Assay for larvicidal activity The larvicidal activities of extracts against 4th instar larvae of Aedes aegypti were assayed (four replicates for each sample) according to a previously published method (WHO, 1975). Both positive (temephos at 3 g mL1 ) and negative (distilled water containing 1% DMSO) control assays were carried out. 2.5. Assay for cytotoxic activity Eggs of the brine shrimp (Artemia salina Leach) were hatched in seawater and used after 48 h. Homogeneous 2nd instar larvae, obtained by two successive incubations for 24 h, were employed in the assay using the method of Lima et al. (2002), which was carried out in quadruplicate for each sample. Both positive (thymol at 10 g mL1 ) and negative (sea water containing 1% DMSO) control assays were carried out in order to verify the susceptibility of Artemia under assay conditions employed.

2. Material and methods 2.1. Plant material Fresh materials (at least 500 g) of each of the plant species, shown in Table 1, were collected in the States of Bahia, Sergipe, Alagoas and Pernambuco in Brazil (see Table 1), and the material classied systematically by Prof. Jos e Elias de Paula (Universidade de Bras lia, Brasilia, DF, Brazil), Prof. Ros ario Rocha (Universidade Federal de Alagoas, Macei o, AL, Brazil), and Prof. Suzene Iz dio da Silva (Universidade Federal Rural de Pernambuco, Recife PE, Brazil). Voucher specimens of all plant species collected are deposited in the herbaria of the respective institutions (Table 1). 2.2. Preparation of extracts and samples Selected parts (see Table 1) of freshly collected plant material were separated, immediately air dried and nally

Table 1 Plants employed in this study and the activities of their extracts against Aedes aegypti, Biomphalaria glabrata and Artemia salina Family Species [locality: date of collect], voucher number Anacardiaceae Spondias mombin L. [Penedo-AL: 03/98] 17578 Common name Ethnobotanical use Reference Plant part extracted Aedes aegyptia Activity (expressed as % mortality) against Biomphalaria glabratab Adults Eggs 0 96 J. de S. Luna et al. / Journal of Ethnopharmacology 97 (2005) 199206 Artemia salinac

Caj a

Used to treat erysipelas and wounds; shows anti-spasmodic, anti-diarrhoeal, anti-bacterial, anti-viral, oxytocic, abortifacient, molluscicidal and astringent properties Used to treat diabetes and loss of weight; shows anti-diarrhoeal, anti-viral, anti-leishmania, sedative, tranquillising, ansiolitic, insecticidal, molluscicidal and parasiticidal properties Some species of this genus are used to treat gonorrhoea and show anti-asthmatic, anti-fertility, and anti-cancer properties Used to treat hypoglycaemia and as a tonic Used to treat chest and bladder diseases

Ofah and Anyanwu (1989), Corthout et al. (1991, 1992, 1994), Abo et al. (1999), Mello (2000)

Seeds

85

Annonaceae Annona muricata L. [Macei o-AL: 08/00] 8530

, Araticum Graviola, Cruac a do Grande, Jaca do Par a

Martins (1989), Bories et al. (1991), Hasrat et al. (1997), Padma et al. (1998), Jaramillo et al. (2000), dos Santos and SantAna (2001)

Leaves

100

100

100

Asclepiadaceae Marsdenia altissima (Jacq.) Dugand [Barra de S ao Miguel-AL: 09/00] JEP 3652 (UB) Burseraceae Bauhinia cheilantha (Bong.) Steud. [Macei o-AL: 04/99] 23567 Bursera leptophloeos Mart. [Santa Luzia-AL: 12/99] 00789

Cip o-Seda

Chowdhury et al. (1994), Ma et al. (1997), Kumar et al. (1999)

Wood bark

40

80

62

Moror o

da Silva and Cechinel Filho (2002) Balbach (1966)

Imburana-de-Camb ao

Wood Leaves Roots Wood

100 0 0 10

0 0 24 9

50 61 100 57

201

202

Table 1 (Continued ) Family Species [locality: date of collect], voucher number Caesalpiniaceae Caesalpinia echinata Lam. [Macei o-AL: 12/98] 20045 Common name Ethnobotanical use Reference Plant part extracted Activity (expressed as % mortality) against Aedes aegyptia Biomphalaria glabratab Adults Eggs 7 100 78 57 Artemia salinac

Pau-Brasil

Caesalpinia ferrea Mart. [Macei o-AL: 02/99] 43256 Caesalpinia pyramidalis Tul. [Marechal Deodoro-AL: 11/97] 21754 Convolvulaceae Operculina macrocarpa (Linn) Urb. [Rio Largo-AL: 09/96] 16876 Fabaceae Anadenanthera macrocarpa (Benth.) Brenan [Bet ania-PE: 11/01] Dioclea virgata (Rich.) Amshoff [Camac ari-BA: 09/00] JEP 3654 (UB) Lauraceae Ocotea glomerata (Nees.) Mez [Murici-AL: 09/00] JEP 3635 (UB)

Pau-Ferro Catingueira

Used as a tonic, astringent and for toothache. Many species of this genus show anti-bacterial, anti-fungal, anti-viral and anti-tubercular activities Used to treat diabetes, and bronchial and lung diseases Used to treat catarrh, diarrhoea and dysentery

Corr ea (1978), Ragasa et al. (2002, 2003), Chiang et al. (2003), Promsawan et al. (2003)

Leaves Wood

80 50

J. de S. Luna et al. / Journal of Ethnopharmacology 97 (2005) 199206

Balbach (1966) Braga (1960)

Leaves Leaves Wood

10 10 20 100

0 3 14 7

68 0 100 100

Batata-de-Purga

Used in utero stimulation; shows low level molluscicidal activity Shows anti-inammatory properties Some species of this genus used to treat fever and malaria; some show toxic properties Some species of this genus used to treat pain, rheumatism, fever, dyspepsia, dermatitis and tumours

Barros et al. (1970), Sousa and Rouquayrol (1974)

Tubers

Angico preto

Desmarchelier et al. (1999)

Wood bark

80

Cip o-pixuma

Braga (1960), Milliken (1997)

Leaves

88

Louro-pinho

Van den Berg (1982), Vieira (1992)

Wood bark Wood Leaves

15 0 35

0 0 0

29 0 0

Table 1 (Continued ) Family Species [locality: date of collect], voucher number Mirtaceae Eugenia uniora L. [Macei o-AL: 05/97] 29879 Common name Ethnobotanical use Reference Plant part extracted Activity (expressed as % mortality) against Aedes aegyptia Biomphalaria glabratab Adults Eggs 0 5 45 0 Artemia salinac

Pitanga

Used to treat fever and diarrhoea; shows anti-oxidant, anti-fungal, anti-microbial, hypotensive, trypanocidal and diuretic properties

Olacaceae Ximenia americana L. [Juazeiro do Piau -PI: 04/02} 14580 Piperaceae Piper arboreum Aublet. [S ao Sebasti ao-AL: 09/00] JEP 3647 (UB) Rhamnaceae Ziziphus joazeiro Mart. [Propri a-SE: 09/00] JEP 3637 (UB) Control samples Assay for molluscicidal activity Assay for cytotoxic activity Assay for mollusc eggs mass Assay for larvicidal activity

Sim oes (1989), Lima et al. Leaves (1993), Almeida et al. (1995), Wood Consolini et al. (1999), Adewunmi et al. (2001), Consolini and Sarubbio (2002), Holetz et al. (2002), Souza et al. (2002), Velazquez et al. (2003) Braga (1960), Benoit et al. Wood bark (1996)

65 10

J. de S. Luna et al. / Journal of Ethnopharmacology 97 (2005) 199206

Ameixa, Ameixa de Espinho, Ameixa do Brasil

Used to treat ulcers and malaria and as an astringent

Alecrim de Angola, Pau de Angola

Used to treat rheumatism and as a Van den Berg (1982) carminative and an emollient

Leaves

15

83

Ju a

Used to treat stomach problems and intermittent fevers and as a soap and tonic Cupric carbonate (50 g mL1 ) Niclosamide (3 g mL1 ) Dechlorinated water containing 1% DMSO Temephos (3 g mL1 ) Distilled water containing 1% DMSO Thymol (10 g mL1 ) Sea water containing 1% DMSO Pentachlorophenol (LD90 = 0.49 g mL1 )

Braga (1960), Delorme and Miolla (1979), Matos (1994)

Wood

15

83

100 0 100

100 100 0

0 90

0 0

a b c

Extract tested at a concentration of 500 ppm. Extract tested at a concentration of 100 ppm. Extract tested at a concentration of 1000 ppm.

203

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2.6. Chemical tests of the plant extracts The chemical tests were performed following the protocols described by Matos (1988).

3. Results A total of 23 ethanolic extracts (Table 1) of various parts of 16 medicinal plants were assayed for biological activity. The assays were carried out using concentrations of extracts selected according to the susceptibility of the test organism employed in the assay. The concentrations of extracts used were 1000 ppm for the toxicity test against Artemia salina, 500 ppm for the larvicidal assay against Aedes aegypti, and 100 ppm for the molluscicidal assay against Biomphalaria glabrata. Eight of the samples assayed showed signicant activity (40% mortality) against Aedes aegypti. The most active extracts, showing 100% mortality at 500 ppm, were from the leaves of Annona muricata, the wood of Bauhinia cheilantha, and the tubers of Operculina macrocarpa: extracts from seeds of Spondias mombin, leaves of Caesalpinia echinata and of Eugenia uniora, wood of Caesalpinia echinata, and wood bark of Marsdenia altissima exhibited, in the order shown, decreasing levels of mortality against the mosquito. Only two of the extracts screened in the present study, those from leaves of Annona muricata and from wood bark of Marsdenia altissima, showed activity at 100 ppm against adult snails, whilst an extract from the wood of Caesalpinia echinata was unique in showing very strong activity against the egg masses of Biomphalaria glabrata. Sixteen of the extracts assayed in our study showed significant toxicity (40% mortality) at 100 ppm against the larvae of the brine shrimp. The strongest activities (ca. 100% mortality) were observed with seed extracts of Spondias mombin, leaf extracts of Annona muricata, root extracts of Bauhinia cheilantha, wood extracts of Caesalpinia pyramidalis and tuber extracts of Operculina macrocarpa. The only extract (from wood of Caesalpinia echinata) that showed 100% mortality (at 100 ppm) against the egg masses of the snail was also partially active (ca. 50% mortality) against mosquito larvae and the brine shrimp. On the other hand, of the two extracts that showed strong activity (80% mortality at 100 ppm) against adult snails, one (from leaves of Annona muricata) was very toxic to both the mosquito larvae and the brine shrimp, whilst the other (from wood bark of Marsdenia altissima) was only partially active in the mosquito and brine shrimp bioassays.

4. Discussion and conclusions The data, shown in Table 1, indicate that extracts from medicinal plants from the northeast of Brazil show very promising activities against organisms that act as interme-

diates or carriers of human diseases. Table 2 presents a summary of the classes of compounds that we have shown by chemical tests to be present in the plants used in this survey. Among the 23 plant extracts that were tested in this study, two showed signicant activity against adult snails, one was active against the snail egg mass, and eight were active against mosquito larvae. The activities detected did not appear to be limited to any morphologically distinct part of the plant or to any particular class of secondary metabolite. There was, however, a correlation between larvicidal activity against Aedes aegypti and toxicity to Artemia salina, and this may indicate a general toxicity of the active components, which will require further investigation. The activities of the extracts described in Table 1 against mosquito larvae are comparable with those previously determined (Ciccia et al., 2000) for extracts from Xanthium spinosum, Eupatorium hecatanthum, Baccharis coridifolia and Aristolochia triangularis, all of which caused 50% mortality to mosquitoes at concentrations in the region of 300 ppm. Although extracts from the leaves of Annona muricata were by far the most active (90% mortality at 73 ppm) and could have direct application, extracts from the other plants studied in the present work will certainly be of value in the search for new lead compounds with larvicidal activity. With respect to molluscicidal activity, extracts from the leaves of Kigelia africana (Adewunmi and Sofowora, 1980) and Dysoxylum lenticellare (Aladesanmi and Adewunmi, 1995) possess activities similar to that reported here for the wood bark extract from Marsdenia altissima. On the other hand, extracts from the roots of Jatropha gossypiifolia (LC90 35 ppm; Adewunmi and Adesogen, 1986) and Annona squamosa (LC90 8.6 ppm; dos Santos and SantAna, 2001) and from the latex of Euphorbia milii (LC50 0.12 ppm; Oliveira-Filho and Paumgartten, 2000) have reported activities that are much higher than that of the most active extract described in the present study (i.e. leaves of Annona muricata; LC90 50 ppm). WHO, 1993 recommends that plant extracts showing LC50 values <40 ppm may be employed directly against mollusc populations, whilst less active extracts may very well provide sources of new lead compounds with molluscicidal activities. Any consideration, however, of the future commercial exploration of the plants found to show signicant activities in this study must take into account the growing habits, ease of cultivation and availability of the plants to the local population. Some of the species of interest, e.g. Annona muricata, Spondias mombin and Eugenia uniora, are already cultivated in Brazil as sources of edible fruits, thus providing a good possibility for the commercial exploitation of the other readily-available plant parts such as stems, seeds, leaves, etc. It is of interest to note that, in northeast Brazil, farmers already use a slurry prepared from plant material of Annona muricata and Annona squamosa to control nematodes, bacteria and fungi in the soil, and they also believe that such treatment protects plants from insect attack. The chromatographic fractionation, guided by larvicidal and mol-

J. de S. Luna et al. / Journal of Ethnopharmacology 97 (2005) 199206 Table 2 Classes of compounds detected in the plant extracts employed in this study Species (part ex- Phenols Flavonones Flavonoids, tracted) avanonols, avanonois, free xanthones, heterosides Spondias mombin (seeds) Annona muricata (leaves) Marsdenia altissima (wood bark) Bauhinia cheilantha (wood) Bauhinia cheilantha(leaves) Bauhinia cheilantha(roots) Caesalpinia echinata (leaves) Caesalpinia echinata(wood) Operculina macrocarpa (tubers) Anadenanthera macrocarpa(wood bark) Eugenia uniora (leaves) Eugenia uniora(wood)
a

205

Flavones, avonoids, xanthones

Free Pentacyclic Leucoantho- Anthra-quinones Anthrones Alkaloids Steroids triterpenes cyanidins

+a

a +

+ + +

+ +

+ +

+ +

+ + +

+ + +

+ + +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+: detected; : not detected. Barros, G.S.G., Matos, F.J.A., Vieira, J.E.V., 1970. Pharmacological screening of some Brazilian plants. Journal of Pharmacy and Pharmacology 22, 116122. Benoit, F., Valentin, A., Pelissier, Y., Diafouka, F., Marion, C., KoneBamba, D., Kone, M., Mallie, M., Yapo, A., Bastide, J.M., 1996. Anti-bacterial activity of plant extractsa comparison of agar dilution and microtiter in in vitro anti-malarial activity of vegetal extracts used in West African traditional medicine. American Journal of Tropical Medicine and Hygiene 54, 6771. Bories, C., Loiseau, P., Cortes, D., Myint, S.H., Hocquemiller, R., Gayral, P., Cav e, A., Laurens, A., 1991. Anti-parasitic activity of Annona muricata and Annona cherimolia seeds. Planta Medica 57, 434436. Braga, R., 1960. Plantas do Nordeste, Especialmente do Cear a, fourth ed. Editora Universit aria, Natal. Chiang, L.C., Chiang, W., Liu, M.C., Lin, C.C., 2003. In vitro antiviral activities of Caesalpinia pulcherrima and its related avonoids. Journal of Anti-microbial Chemotherapy 52, 194198. Chowdhury, A.K.A., Hashim, M.F., Sen, B.C., Khan, F., Ahmed, M., 1994. Anti-fertility principles from M. tinctoria. Pharmacological and photochemical studies. Pure and Applied Chemistry 66, 23432346. Ciccia, G., Coussio, J., Mongelli, E., 2000. Insecticidal activity against Aedes aegypti larvae of some medicinal South American plants. Journal of Ethnopharmacology 72, 185189. Consolini, A.E., Sarubbio, M.G., 2002. Pharmacological effects of Eugenia uniora (Myrtaceae) aqueous crude extract on rats heart. Journal of Ethnopharmacology 81, 5763.

luscicidal activity, of extracts of these plants is currently in progress.

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