Microsoft Word - 14251_revisadoOSW.doc 255 Original Article Biosci. J., Uberlândia, v. 29, n. 1, p. 255-263, Jan./Feb. 2013 BRINE SHRIMP LETHALITY TEST AS A BIOLOGICAL MODEL FOR PRELIMINARY SELECTION OF PEDICULICIDAL COMPONENTS FROM NATURAL SOURCE TESTE DE TOXICIDADE FRENTE ARTEMIA COMO MODELO PRELIMINAR DE BUSCA DE SUBSTÂNCIAS PEDICULICIDAS A PARTIR DE FONTES NATURAIS Camila VIDOTTO1; Denise Brentan da SILVA2; Rodolfo PATUSSI1 ; Luis Fabrício Galdino BRANDÃO1; Jacqueline Domingues TIBÚRCIO3; Stênio Nunes ALVES3; João Máximo de SIQUEIRA3 1. Centro de Ciências Biológicas e da Saúde, CCBS, Universidade Federal de Mato Grosso do Sul, MS, Brasil; 2. Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP, Ribeirão Preto, SP, Brasil; 3. Campus CCO-Dona Lindu, Universidade Federal de São João Del Rei, Divinopolis, MG. jmaximo@ufsj.edu.br ABSTRACT: Brine shrimp lethality test as a biological model for the preliminary selection of pediculicidal components from a natural source. In order to achieve a good correlation between pediculicidal activity and brine shrimp lethality (BSL) test, several pediculicidal substances and active essential oils were tested in BSL test, with the purpose to use the latter as convenient preliminary protocol for pediculicidal activity. Benzyl benzoate, deltametrine and essential oil of Eucalyptus were purchased and clove essential oil obtained by hydrodistillation, besides essential oils, chloroform extracts from Duguetia furfuracea were also submitted to BSL test. All of them were carried out with same protocol described to pediculicidal assay found in the literature, i.e, flask tests were examined every five minutes in the first half hour and then every ten minutes until all the naupli were dead or no movements were observed (knockdown). During the BSL test, it was possible to observe the effect of a particular lethal dose or only a knockdown in the arthropod, as occurred in the test with lice. The results of the BSL test for essential oils and other active substances are essentially in agreement with those described in literature for pediculicidal activity. Extracts and essential oil obtained from aerial parts of D. furfuracea did not present activity, but the essential oil from underground stem bark was active. α-asarone has already been isolated from the underground stem and it has been previously described to possess insecticidal activity. KEYWORDS: Pediculicidal activity. Bioassay model. Essential oil. Duguetia furfuracea. INTRODUCTION Infestation with Pediculus capitis is a common disease occurring widely throughout the world (KO; ELSTON, 2004). Humans are infested by this parasite for several reasons. In modern society, the main cause of the infestation is not the influence of socio-economic conditions; the predominant factor contributing to its increase is the resistance to insecticides (HODGDON et al., 2010). The problem encountered with the control of this parasitic disease is strictly related to all the stages of the parasite’s life along with the mode of transmission; therefore, several products and methodologies have been tried to remove, kill or prevent the infestation of lice (GRATZ et al., 1997; BURKHART et al., 2003). Insecticidal products, undoubtedly, have been the simplest and most effective method to control the disease. However, side effects and resistance to insecticides, along with the toxicity of currently available treatments, are serious problems to treatment compliance. Thus, plants may provide potential alternatives to the currently used insect- control agents because they constitute a rich source of bioactive chemicals. Therefore, searching for pediculicidal compounds from several sources is necessary; nevertheless, the in-vitro pediculicidal protocol used to assess this activity is, in general, complicated (OLADIMEJI et al., 2000; YANG et al., 2004; WILLIAMSON et al., 2007). The bioassays for pediculosis described in literature promote the evolution of more complexity in the parasite itself if used. Several difficulties in the protocols, such as collecting and feeding the lice during the experiment, have been described. In addition to the process of assay development, application of the product to be tested, and the interpretation of the results have been also considered (OLADIMEJI et al., 2000; YANG et al., 2003, 2004). In folk medicine, pulverised seeds of Duguetia furfuracea, a shrub typical of the cerrado of Central Brazil and which is popularly known as “Araticum-seco” are diluted in water and are commonly used to eliminate parasites, particularly louses (CORREA, 1978; SILBERBAUER- GOTTSBERGER, 1981/1982). The purpose of this Received: 10/01/12 Accepted: 06/09/12 256 Brine shrimp... VIDOTTO, C. et al. Biosci. J., Uberlândia, v. 29, n. 1, p. 255-263, Jan./Feb. 2013 study was to achieve a good correlation between brine shrimp lethality test (BSL test) and the pediculicidal activity of some compounds and extracts from plants, in order to use the BSL test as a convenient preliminary protocol for the screening of the pediculicidal effect. Some compounds and essential oils described as being active on louse were tested; moreover, the essential oil and the extracts of D. furfuracea were also tested. MATERIAL AND METHODS Preparation of essential oil and extracts from D. furfuracea Benzyl benzoate (BzB), deltametrine (∆) and the essential oil of Eucalyptus globulus Labill. (Eucal) and buds of Eugenia caryophyllata Thunb. were purchased commercially. The essential oil of E. caryophyllata (clove) was obtained by hydrodistillation. The leaves, fruit and underground parts (stem bark and wood) of Duguetia furfuracea (A. St.-Hil.) Benth. e Hook f. were collected in March 2007, on the UFMS campus in Campo Grande, MS, Brazil, and identified by Prof. R. Mello-Silva. A voucher specimen (No. 023) was deposited in the CGMS Herbarium. The essential oils and extracts of D. furfuracea (Annonaceae), which are described in folk medicine as having pediculicidal properties, were obtained according to the following procedure. The dried natural fruit pulp (211.0 g) was pulverised and subjected to an exhaustive extraction in a Soxhlet apparatus with chloroform for six hours, yielding 277.4 mg (0.13%, Dfp) of extract. The seeds (141.8 g) were subjected to the same methodology and 55.9 mg (0.04%, Dfs) of the chloroform extract was obtained. Fresh leaves (400 g) and the underground stem bark (300 g) of D. furfuracea were pulverised and subjected to an exhaustive extraction by hydrodistillation, and they yielded 2.9 mL (0.73%, w/v, Dfl) and 3.6 mL (1.20%, w/v, Dfusb) of the essential oil, respectively. All extracts and volatile oils were subjected to a BSL bioassay. BSL test as a screening test for pediculicidal activity With the aim of developing a simple methodology for use as a preliminary screening test for pediculicidal activity, various substances and herb extracts previously described as active were subjected to the BSL test, using a protocol similar to that used in the P. capitis assay, but with some adaptations. Each flask was examined every five minutes in the first half hour and then every ten minutes until all the naupli were dead or no movements were observed (knockdown). The tested sample was considered ineffective if any modification of Artemia salina. behaviour was observed in a span of two hours (120’;OLADIMEJI et al., 2000; YANG et al., 2003, 2004). Assays for the essential oils were also developed, with at least three dilutions and in triplicate, with 1% of dimethyl sulphoxide (DMSO; v/v). Thus, assays with BzB, ∆, and the essential oils clove and Eucal were carried out. In addition, the essential oils and chloroform extracts of D. furfuracea were also tested. Samples with four dilutions, in triplicate, of BzB were prepared in the following concentrations: 5.6, 2.8, 1.4, 0.7 and 0.35 mg/cm3, as described in literature (OLADIMEJI et al. 2000; YANG et al. 2004). Thus, these samples were solubilised in 20 mL of marine solution with 1% DMSO (v/v), and ten naupli (second instar) of brine shrimp were added in each flask and maintained in direct contact with the solutions. Most of the solutions tested had a milky appearance, but were translucent enough for the development of the bioassay. To avoid the volatilisation of the substances, the flasks were covered with a polyvinyl chloride film. A magnifying glass, a light focus and a dark background were used to conduct the readings. The same procedure was followed for ∆, with three dilutions and in triplicate, that is, 0.20, 0.10 and 0.05 mg/cm3 in 20 mL of marine solution with 1% DMSO (v/v). These concentrations are almost the same as those described by Yang (2004) with pyrethroid derivatives in the P. capitis assay. The essential oils from E. globulus and E. caryophyllata were diluted at concentrations of 0.20, 0.10 and 0.05 mg/cm3 in marine solutions with 1% DMSO (v/v). Samples from D. furfuracea were diluted at concentrations of 1.00, 0.50 and 0.25 mg/cm3. Gas chromatography–mass spectrometry Analysis The samples of volatile oil were subjected to gas chromatography–mass spectrometry (GC–MS) analysis on a Shimadzu QP2010. The apolar extract was purified using a cartridge of silica gel (AccuBondII SPE) with hexane, dichloromethane and methanol. The chloroform fractions were subjected to GC–MS analysis. The GC column was a DB-5MS (30 m in length, 0.25 µm in thickness, 0.25 mm i.d.). The carrier gas was helium, with pressure of 81.9 kPa and flow rate of 1.33 mL/min. 257 Brine shrimp... VIDOTTO, C. et al. Biosci. J., Uberlândia, v. 29, n. 1, p. 255-263, Jan./Feb. 2013 Injector temperature was 250 °C and the split ratio was 1: 50. The temperature was programmed in the range of 60–240 °C increasing at 3 °C/min. The mass spectra were recorded in the electron ionisation mode, with ionisation energy of 70 eV. The temperature of the ion source was 250 °C. Identification of the constituents Identification of each individual constituent of the samples (Table 1) was achieved based on the retention indices (calculated using C9 to C22 alkanes), by comparison of the mass spectra data with a computer databank (WILEY 7 and NIST 62) and with reference to published data (ADAMS, 1995). Statistical methodology The statistical analysis of the estimated survival time was performed with the application of the Kaplan-Meier nonparametric method. In the present analysis has been not used any assumption about the probability of survival time, and the concept of independence among the events was applied. The survival curves were compared with the log-rank test, and level significance of 5% was considered statistically significant. The values to determine the lethal concentrations were analyzed by using the regression model probit program. RESULTS AND DISCUSSION The results of the BSL test using the essential oils and other active substances are described in the Tables 1 and 2, and they are essentially in agreement with those described in literature for pediculicidal activity (YANG et al., 2003, 2004). During the A. salina test, it was also possible to observe the effect of a particular lethal dose or only a knockdown in the arthropod, as occurred in the test with lice (OLADIMEJI et al., 2000; YANG et al., 2003, 2004). Substances and extracts were active for a period of up to two hours in the A. salina assay. Only the E. globulus sample, among those described as active, was relatively less active; however, it still falls within the expected time interval (<2 hours). Table 1. Relative toxicity of compounds, extracts and essential oils against brine shrimp larvae Compound, extract or essential oil Concentration in marine solution (mg/cm3) Estimate Medians for Survival Time (min) 95% Confidence Interval Lower Bound Upper Bound 5.6 10 - - 2.8 10 - - Bzb 1.40 16 9.62 22.38 0.70 20 3.93 36.07 0.35 40 29.36 50.64 ∆ 0.20 5 - 0.10 15 - 0.05 5 - clove 0.20 25 22.36 27.64 0.10 50 39.26 60.74 0.05 NE - - Eucal 0.20 30 22.49 37.51 0.10 210 - 0.05 210 - Dfusb 1.00 30 16.61 43.39 0.50 90 67.20 112.80 0.25 210 146.16 273.84 Dfp (*) NE - - Dfs (*) NE - - Dfl (*) NE - - (*) Concentrations of 1.00, 0.50 and 0.25 mg/cm3. NE: no effectiveness. Dfusb: essential oil from underground of stem bark of D. furfuracea, Dfl: essential oil from leaves of D. furfuracea, Dfp: chloroform extract from pulp fruit of D. furfuracea, Dfs: chloroform extract from seed of D. furfuracea, Eucal: essential oil from leaves of E. globulus, clove: essential oil from bud of E. caryophyllata. 258 Brine shrimp... VIDOTTO, C. et al. Biosci. J., Uberlândia, v. 29, n. 1, p. 255-263, Jan./Feb. 2013 Table 2. Lethal dose (DL50) by compound, extract or essential oil in mg/cm 3, against brine shrimp larvae until 50 minutes exposure Compound, extract or essential oil DL50 IC 95% R 2 Bzb 226.1 102.5 – 498.7 0.09 ∆ # - - Clove 102.0 89.9 – 115.8 0.9 Eucal 141.4 127.1 – 157.3 1.0 Dfusb 715.2 608.3 – 840.9 0.85 Dfp* NE - - Dfs* NE - - Dfl* NE - - # Total mortality at all concentrations up to 50 minutes exposure;* Concentration of 1.00, 0.50, 0.25 mg/cm3 NE: no effectiveness. Dfusb: essential oil from underground of stem bark of D. furfuracea, Dfp: chloroform extract from pulp fruit of D. furfuracea, Dfl: essential oil from leaves of D. furfuracea, Dfs: chloroform extract from seed of D. furfuracea, Eucal: essential oil from leaves of E. globulus, clove: essential oil from bud of E. caryophyllata. Theoretically, the A. salina toxicity assay can be used as a pre-evaluation model for pediculicidal activity as long as the bioassay is developed as described in the present work, which follows the protocol proposed for the parasite assay, and the requirements of knockdown and/or death of the naupli stage must be in the range from 50% to 100% during the interval of 120 minutes, as described for lice (YANG et al., 2003, 2004). Extracts and essential oils of plants are mainly characterised by their complex chemical mixture. In a preliminary assay, the results can be diffuse and not well bio-directed; however, continuing the phytochemical fractionation and testing the sub-fractions using the protocol described previously can identify potential pediculicidal substances. The chemical profiles of the active substances within the active essential oils, extracts and fractions have been investigated to define the possible potential pediculicidal pattern in terms of monoterpenes, sesquiterpenes and other classes of compounds. Mono-oxygenated nonocyclical monoterpenes have shown more efficiency against lice (OLADIMEJI et al., 2000, PRIESTLEY et al., 2006); moreover, some phenolic compounds, especially benzaldehyde and salicylaldehyde, have shown 30- and 17-fold potency compared to pyrethrin, and eugenol and methyl salicylate, the constituents present in E. Caryophyllata have been reported to be active (YANG et al., 2003, 2004). Data relating to pediculicidal or insecticidal activity of sesquiterpene pediculicides are scarce; nevertheless, β-caryophyllene, α-humulene and caryophyllene oxide, for instance, present in the essential oil obtained from the leaves of D. furfuracea (Table 3) have been described as inactive against pediculosis (YANG et al., 2003, 2004). For D. furfuracea, only the essential oil from the underground stem bark (Dfusb) showed relative activity, whereas the other extracts from the plant were not active at the concentrations tested. α-Asarone was isolated from Dfusb, and this substance has been described as a potential pesticide (LEE et al., 2002, PARK et al., 2003). Artemia naupli have been suggested for use as a model for several preliminary evaluations of pharmacological and ecotoxicological activities of compounds of greater complexity (MCLAUGHLIN 1993; DVORAK et al., 2010). The BSL test has been used for insecticidal, acaricidal, anaesthetic, and anti-tumour activity evaluations, using different methodologies. (AREEKUL et al., 1960; ROBINSON et al., 1965; HARWING et al., 1971; MCLAUGHLIN 1993) For the correlations cited above, the tests are conducted and the readings taken twenty-four hours after application of the substance. In the present work, readings have been taken every five minutes in the first 30 minutes, followed by further readings every ten minutes, which proved to be a useful search for the substances and extracts with pediculicidal potential. The extracts and essential oil obtained from the aerial parts of D. furfuracea did not show any activity (Table 4); however, the essential oil from the underground stem bark was active; α- asarone has already been isolated from the underground stem and has been previously described to possess insecticidal activity (LEE et al., 2002; PARK et al., 2003) 259 Brine shrimp... VIDOTTO, C. et al. Biosci. J., Uberlândia, v. 29, n. 1, p. , Jan./Feb. 2013 Table 3. Results of analysis of the survival of the Artemia naupli for concentration in marine solution,* Log-rank test compared with p value of < 0.05 was considered statistically significant Dfusb: essential oil from underground of stem bark of D. furfuracea, Dfl: essential oil from leaves of D. furfuracea, Dfp: chloroform extract from pulp fruit of D. furfuracea, Eucal: essential oil from leaves of E. globulus, clove: essential oil from bud of E. caryophyllata. Compound Concentration in marine solution (mg/cm3) 2.80 mg/cm3 1.40 mg/cm3 0.70 mg/cm3 0.50 mg/cm3 0.35 mg/cm3 0.25 mg/cm3 0.10 mg/cm3 0.05 mg/cm3 X2 Sig. X2 Sig. X2 Sig. X2 Sig. X2 Sig. X2 Sig. X2 Sig. X2 Sig. BzB 5.60 17.9 <0.01* 17.7 <0.01* 52.1 <0.01* 65.3 <0.01 * 2.80 3.0 0.08 26.1 <0.01 * 60.3 <0.01 * 1.40 15.2 <0.01 * 32.2 <0.01 * 0.70 3.5 0.06 ∆ 0.20 44.1 <0,01 * 2.98 0.08 0.10 11.39 <0.01 0.05 clove 0.20 39.3 <0,01 * 68.39 <0.01 0.10 67.70 <0.01 Eucal 0.20 60.61 <0,01 * 66.26 <0.01 0.10 2.03 0.15 Dfusb 1.00 23.12 <0.01 * 56.7 <0,01 * 0.50 20.0 <0,01 * Dfp 1.00 2.03 0.15 15.3 <0,01 * 0.50 9.6 <0,01 * Dfl 1.00 1.00 0.32 2.0 0.15 0.50 0.3 0.56 260 Brine shrimp... VIDOTTO, C. et al. Biosci. J., Uberlândia, v. 29, n. 1, p. 255-263, Jan./Feb. 2013 Table 4. Chemical constituents of extracts, essential oils identified by gas chromatograph-mass spectrometer. Relative content (%) RI Dfusb Dfl Dfp Dfs Eucal clove α-pinene - - - - 2.3 - 932 β-pinene - - - - 0.3 - 978 β-myrcene 0.6 - - - 0.2 - 988 δ-2-carene 1.6 - - - - - 992 trans-m-mentha-4,8-diene 6.5 - - - - - 995 o-cymene - - - - 6.7 - 1023 Limonene 0.1 - - - 9.2 - 1028 β-phellandrene 0.9 - - - - - 1030 Eucaliptol - - - - 79.0 - 1032 Linalool - - - - 0.1 - 1099 cis-limonene oxide - - - - 0.1 - 1132 trans-limonene oxide - - - - - 1137 trans-pinocarveol - - - - 0.1 - 1139 Terpin-4-ol 0.3 - - - 0.3 - 1180 p-cymen-8-ol - - - - 0.1 - 1185 α-terpineol - - - - 1.3 - 1189 trans-carveol - - - - 0.1 - 1218 Carvone - - - - 0.2 - 1242 (E,E)-2,4-decadienal - - - 1.5 - - 1317 Bicycloelemene - 0.2 - - - - 1329 δ-elemene - 3.4 - - - - 1332 Eugenol - - - - - 89.4 1354 Cyclosativene 0.1 - - - - - 1364 α-copaene 0.1 0.5 - - - - 1372 β-elemene 0.9 3.6 - - - - 1385 Cyperene 16.0 - - - - 1397 α-gurjunene 22.2 - - - - - 1403 NI 1.2 - - - - - 1408 β-caryophyllene 2.6 11.5 - - - 4.1 1415 β-gurjunene 0.2 0.5 - - - - 1428 Aromadendrene 0.1 0.6 - - - - 1434 α-humulene 0.5 1.3 - - - 0.5 1451 NI 1.5 - - - - - 1457 γ-gurjunene 1.9 - - - - - 1470 α-amorphene - 1.1 - - - - 1471 γ-muurolene 1.0 - - - - - 1473 Germacrene D 0.8 13.0 - - - - 1476 β-selinene 0.4 - - - - - 1484 Viridiflorene - 1.0 - - - - 1487 Bicyclogermacrene 1.4 16.2 - - - - 1491 α-muurolene 0.6 1.0 - - - - 1495 δ-guaiene 0.2 - - - - - 1498 γ-cadinene 0.6 0.5 - - - - 1514 Eugenol Acetate - - - - - 5.8 1518 δ-Cadinene - 0.6 - - - - 1519 trans-calamenen 1.3 - - - - - 1540 Germacrene B - 2.2 - - - - 1553 2,4,5-trimethoxy-styrene 19.7 - - - - - 1554 Dodecanoic acid - - - 2.1 - - 1564 Ledol 1.0 0.1 - - - - 1565 Spathulenol 0.2 17.8 15.0 25.2 - - 1572 Caryophyllene oxide 0.1 3.6 - - - 0.2 1577 Globulol - 3.5 - - - - 1580 Viridiflorol - 4.0 - 1.7 - - 1589 261 Brine shrimp... VIDOTTO, C. et al. Biosci. J., Uberlândia, v. 29, n. 1, p. 255-263, Jan./Feb. 2013 Cedrol 1.1 - - - - - 1600 Rosifoliol - 0.4 - - - - 1601 NI 1.5 - 7.5 - - - 1605 NI - 1.6 2.6 - - - 1615 NI - 1.9 - - - - 1624 δ-cadinol 0.4 - - - - - 1625 Isospathulenol - 1.1 15.2 3.2 - - 1628 Hinesol 0.4 - - - - - 1635 epi-α-cadinol - 1.3 - - - - 1638 epi-α-muurolol 0.2 1.3 - - - - 1640 α-muurolol 0.9 0.6 3.9 1.7 - - 1642 trans-isoelemicin 0.2 - - - - - 1645 NI - - 1.0 - - - 1649 α-cadinol 0.4 2.6 1.8 - - - 1651 Caryophylla-4(12),8(13)-diene- 5β-ol - 0.6 - - - - 1658 NI - 2.4 - - - - 1664 α-asarone 10.1 - - - - - 1672 ΝΙ - - 4.5 - - - 1688 ΝΙ - - 4.6 - - - 1692 Asaraldehyde - - 14.1 15.5 - - 1708 Aromadendrene Oxide II - - - 4.1 - - 1714 ΝΙ - - 3.4 - - - 1727 NI - - - 2.8 - - 1734 NI - - - 2.7 - - 1736 (E,Z)-farnesol - - 1.2 - - - 1743 NI - - 2.4 - - - 1748 Tetradecanoic acid - - - 1.12 - - 1762 NI - - 4.6 - - - 1802 NI - - 6.5 - - - 1855 Hexadecanoic acid - - 7.9 20.0 - - 1966 Ethyl Hexadecanoate - - - 2.5 - - 1994 n-Octadecanol - - 3.5 1.7 - - 2084 (Z)-9-Octadecenoic acid methyl ester - - - 1.4 - - 2097 (Z,Z)-9,12-Octadecadienoic acid methyl ester - - - 1.7 - - 2125 (Z)-9-Octadecenoic acid - - 0.4 4.0 - - 2145 (Z,Z)-9,12-Octadecadienoic acid - - - 2.5 - - 2158 Dfusb: essential oil from underground of stem bark of D. furfuracea, Dfl: essential oil from leaves of D. furfuracea, Dfp: chloroform extract from pulp fruit of D. furfuracea, Dfs: chloroform extract from seed of D. furfuracea, Eucal: essential oil from leaves of E. globulus, clove: essential oil from bud of E. caryophyllata, NI: no identification. ACKNOWLEDGEMENTS This work was financially supported by FUNDECT-MS. The author JMS acknowledges CNPq for a research grant. They are also grateful to FCFRP-USP-SP and Professor Norberto Peporine Lopes for making their facilities available for additional analysis. RESUMO: O teste de toxicidade frente a Artemia salina (TAS) foi utilizado como modelo biológico preliminar na busca de substâncias potencialmente pediculicidas a partir de fontes naturais. Foi encontrada uma boa correlação entre a atividade pediculicida e o TAS, várias substâncias e óleos essenciais descritos como pediculicidas foram testados sobre o microcrustáceo, com o objetivo de se obter um protocolo preliminar e apropriado para detectar aquela atividade. Benzoato de benzila, deltametrina, e óleos essenciais de eucalipto e cravo foram obtidos comercialmente e/ou por extração. Além desses, extratos e óleos essenciais de Duguetia furfuracea também foram testados frente a Artemia. Todas as amostras foram conduzidas utilizando o mesmo protocolo proposto para atividade pediculicida, ou seja, os frascos testes foram lidos a cada cinco minutos na primeira meia hora e depois a cada dez minutos até os nauplios estarem mortos ou sem 262 Brine shrimp... VIDOTTO, C. et al. Biosci. J., Uberlândia, v. 29, n. 1, p. 255-263, Jan./Feb. 2013 movimento (knockdown). Durante o TAS foi possível observar a dose letal ou somente o knockdown na larva, como ocorrido no teste com piolho. Os resultados do TAS para os óleos essencial e os demais compostos ativos estão essencialmente de acordo com o descrito na literatura para a atividade pediculicida. Extratos das sementes e óleos essenciais das folhas e sementes D. furfuracea não apresentaram atividade, mas o óleo das cascas do caule subterrâneo foi ativo α-asarona foi isolado das cascas do caule e esta substância apresenta atividade inseticida. PALAVRAS-CHAVES: Atividade pediculicida. Bioensaio. Óleo essentical. Duguetia furfuracea. REFERENCES ADAMS, R. P. Identification of essential oil components by Gas Chromatography/Mass Spectroscopy. Illinois: Allured Publishing Corporation, 1995. 469 p. AREEKUL, S.; HARWOOD, R. F. Two organisms suitable for bioassaying specific acaricides. J. Agric. Food Chem., Columbus, v. 8, n. 1, p. 32-36, jan. 1960. DVORAK, P.; BENOVÁ, K.; ŽD´ARSKY, M.; SKLENÁR, Z.; HAVELKOVÁ, A. Use of the Crustacean Artemia franciscana for Alternative Biotests. Acta Vet. Brno, Brno, v. 79, Suppl. 9, 47–53, aug. 2010. BURKHART, C. N. Fomite transmission with head lice: A continuing controversy. The Lancet, London, v. 361, n. 9352, p. 99-100, jan. 2003. CORREA, M. P. Dicionário das Plantas Úteis do Brasil e das Exóticas Cultivadas, 2 ed. Rio de Janeiro: Imprensa Nacional (6 vols), 1978. 4329p. GRATZ, N. G. Human lice: Their prevalence, control and resistance to insecticides. A review 1985-1997. Geneva: World Health Organization, Division of Control of Tropical Diseases, WHO Pesticide Evaluation Scheme, 1997. HARWING, J.; SCOTT, P. Brine shrimp (Artemia salina) larvae as screening system for fungal toxins. Appl. Microbiol., Washington, v. 21, n. 6, p. 1011-1016, june. 1971. HODGDON H. E.; YOON, K-S.; PREVITE, D. J.; KIM, H. J.; ABOELGHAR, G. E.; LEED, S. H.; CLARKA, M. Determination of knockdown resistance allele frequencies in global human head louse populations using the serial invasive signal amplification reaction. Pest Manag. Sci., London, v. 66, n. 9, p. 1031–1040, sept. 2010. KO, C. J.; ELSTON, D. M. Pediculosis. J. Am. Acad. Dermatol., Schaumburg, v. 50, n. 1, p.1-12, jan. 2004. LEE, H-K.; PARK, C.; AHN, Y-J. Insecticidal activities of asarones identified in Acorus gramineus rhizome against Nilaparvata lugens (Homoptera: Delphacidae) and Plutella xylostella (Lepidoptera: Yponomeutoidae). Appl. Entomol. Zool., Tsukuba, v. 37, n. 3, p. 459–464, aug. 2002. MCLAUGHLIN, J. L.; CHANG, C-J.; SMITH, D. L. Simple bench-top biossays (brine shrimp and potato disk) for the discovery of plant antitumor compounds - Review of Recent Progress. In: KINGHORN, A. D.; BALANDRIN, M. F. (Eds). Human Medicinal Agents from Plants, Symposium Series No. 534, New York: American Chemical Society, 1993. p. 112-137. ROBINSON A. B.; NANLY, K. F.; ANTHONY, M. P.; CATCHPOOL, J. F.; PAULING, L. Anesthesia of Artemia larvae: Method for quantitative study. Science, Washington, v. 149, n. 3689, p. 1255-1258, sept. 1965. OLADIMEJI, F. A.; ORAFIDIYA, O. O.; OGUNNIYI, T. A. B.; ADEWUNMI, T. A. Pediculocidal and scabicidal of Lippia multiflora essential oil. J. Ethnopharmacol., Leiden, v. 72, n. 1-2, p. 305-311, sept. 2000. 263 Brine shrimp... VIDOTTO, C. et al. Biosci. J., Uberlândia, v. 29, n. 1, p. 255-263, Jan./Feb. 2013 PARK, C.; KIM, S.; AHN, Y. J. Insecticidal activity of asarones identified in Acorus gramineus rhizome against three coleoptera stored-product insects. J. Stored Prod. Res., London, v. 39, n. 3, p. 333-342, june. 2003. PRIESTLEY, C. M.; BURGESS, I. F.; WILLIAMSON, E. M. Lethality of essential oil constituents towards the human louse. Fitoterapia, Novara, v. 77, n. 4, p. 303-309, june. 2006. YANG Y.C.; LEE, H. S.; LEE, S. H.; CLARK, M.; AHN, Y. J. Ovicidal and adulticidal activities of Cinnamomum zeylanicum bark essential oil compounds and related compounds against Pediculus humanus capitis (Anoplura: Pediculicidae). Int. J. Parasitol., Cairns, v. 35, n. 14, p. 1595-1600, dec. 2005. YANG Y. C.; LEE, H. S.; CLARK, M.; AHN, Y. J. Insecticidal Activity of Plant Essential Against Pediculus humanus capitis (Anoplura: Pediculidae). J. Med. Entomol., Columbia, v. 41, n. 4, p. 699-704, jul. 2004. YANG Y. C.; LEE, S. H.; CHOI, D. H.; AHN, Y. J. Ovicidal and Adulticidal of Eugenia caryophillata Bud and Leaf Oil Compounds on Pediculus capitis. J. Agric. Food Chem., Columbus, v. 51, n. 17, p. 4884-4888, aug. 2003. SILBERBAUER-GOTTSBERGER, I. O cerrado como potencial de plantas medicinais e tóxicas, Oréades, Belo Horizonte, v. 8, n. 14-15, p. 15-30, sept.1981/82. WILLIAMSON, E. M.; PRIESTLEY, C. M.; BURGESS, I. F. An investigation and comparison of the bioactivity of selected essential oils on human lice and house dust mites. Fitoterapia, Novara, v. 78, n. 7-8, p. 521-525, dec. 2007.