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Industrial Crops and Products 44 (2013) 600– 602

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

jo ur nal homep age: www.elsev ier .com/ locate / indcrop

hort communication

imaroubaceae and Picramniaceae as potential sources of botanical pesticides

aría Laura Martíneza,∗, Gilsane von Poserb, Amelia Henriquesb, Martha Gattusoa, Carmen Rossini c

Cátedra de Botánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 531, S2002LRK Rosario, ArgentinaLaboratório de Farmacognosia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av Ipiranga 2752, CP 90.610-000 Porto Alegre, BrazilLaboratorio de Ecología Química, Facultad de Química, Universidad de la República, Gral. Flores 2124, CP 11800 Montevideo, Uruguay

r t i c l e i n f o

rticle history:eceived 5 June 2012eceived in revised form8 September 2012ccepted 23 September 2012

eywords:icramniaceaeimaroubaceaeotanical antifeedant agentpilachna paenulatapodoptera frugiperda

a b s t r a c t

Prospection for new sources of botanical pesticides has shown a revival in the last decades due notonly to the fast development of resistance among different pests around the world but also by the needto use less eco-toxic products to control etiological agents of different pest-related problems found inagro-production. In this work, extracts from members of the families Simaroubaceae (Ailanthus altissima,Castela coccinea and Picrasma crenata) and Picramniaceae (Alvaradoa subovata and Picramnia sellowii)were evaluated for their toxicity against the cattle tick and for their antifeedant activity against insects.At the tested doses, none of the extracts exhibited a good toxicity against larvae of the common cattletick. On the other hand, antifeedant activity was detected in various extracts. Foliage consumption wascompletely deterred in adults of the specialist Epilachna paenulata in the cases of the C. coccinea leaf andwood extracts and P. crenata wood extract. For larvae of the generalist Spodoptera frugiperda antifeedanteffects were also detected, although in a lesser extent. Phytochemical analyses of the extracts showed the

presence of alkaloids in P. crenata (including canthin-6-one) and in C. coccinea. Besides, GC/MS analysesof the wood extract from C. coccinea showed the presence of several steroids (ergot 5-en-3-ol-acetate,stigmastan-3,5 diene and stigmasta-3,5dien-7-one). Anthraquinones (emodin and chrysophanol) as wellas chlorogenic acid were detected in the cases of A. subovata and P. sellowii extracts. The differences indeterrent activity could not be atributed to differences in the HPLC chemical profiles of the differentextracts.

. Introduction

The indiscriminate worldwide use of synthetic chemicals forrop protection has led to environmental contamination, pest resis-ance and negative impact on non-target organisms (Philogènet al., 2005). In contrast, plant-derived pesticides are eco-friendlynd have low persistence. Strategies for studying new insecticidalatural products include screening on the most traditionally usedioactive families. Among them, the Simaroubaceae family hasemonstrated to possess insecticidal properties (e.g. Lü and Shi,012). Ailanthus, Picrasma and Castela genera belong to this family.icramnia and Alvaradoa although formerly belonged to this fam-ly, both were segregated and included in the recently establishedicramniaceae family (Fernando and Quinn, 1995).

In the present work the deterrent and toxic activities of leavesnd wood extracts of three species of the Simaroubaceae fam-

ly (Ailanthus altissima, Castela coccinea and Picrasma crenata)nd two species of the Picramniaceae family (Alvaradoa subo-ata and Picramnia sellowii) were assessed. Deterrent activity was

∗ Corresponding author. Tel.: +54 341 4804592; fax: +54 341 4804592.E-mail address: mlmartin1967@gmail.com (M.L. Martínez).

926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2012.09.015

© 2012 Elsevier B.V. All rights reserved.

evaluated against the specialist Epilachna paenulata (Coleoptera:Coccinellidae) and the generalist Spodoptera frugiperda (Lepi-doptera: Noctuidae), pest species that were selected due to theirdifferent diet breath. Both species are themselves important agri-cultural pests, either in conventional or in organic production(Scatoni and Bentancourt, 1999). Toxicity was evaluated againstlarvae of the cattle tick, Rhipicephalus (Boophilus) microplus (Acari:Ixodidae) which represents a major sanitary problem for the cat-tle industry in tropical and subtropical regions. They have beentraditionally controlled with pyrethroids and formamidines (SardáRibeiro et al., 2010).

2. Materials and methods

2.1. Plant material and extracts

Leaf and wood were collected with flowers and/or fruits toenable the identification (carried out by the authors). Voucherspecimens (numbers indicated below) were deposited at the UNR

herbarium. The locations and date of collection were the following:A. subovata Cronquist ARGENTINA Prov. Tucumán: Dpto. Burruyacú,7-VII-2007, Gattuso M. 248 (UNR). P. sellowii Planch. ARGENTINA.Prov. Chaco: Dpto. Bermejo, 23-XI-2007, Oakley et al., 53 (UNR) C.

M.L. Martínez et al. / Industrial Crops a

Table 1Yields of the plant extracts assayed and their bioactivities against different arthro-pods (feeding activity in the cases of E. paenulata adults and S. frugiperda larvae andcontact toxicity against R. microplus larvae). Results are shown as mean ± standarderror.

Species Organ Yield (%) E. paenulataFR#

S. frugiperdaFR#

R. microplus% Mortality�

A. altisima Leaf 3.2 0.0 ± 0.3 −0.1 ± 0.3 7 ± 13 a

Wood 0.1 0.7 ± 0.1* −0.2 ± 0.3 30 ± 1 b

C. coccinea Leaf 10.0 0.97 ± 0.02* −0.5 ± 0.4 9 ± 14 a

Wood 1.0 1 ± 0* 0.5 ± 0.2** 0 ± 2 a

P. crenata Leaf 0.8 0.2 ± 0.3 −0.05 ± 0.11 5 ± 5 a

Wood 0.5 1 ± 0* 0.5 ± 0.2** 2 ± 2 a

A. subovata Leaf 8.0 0.71 ± 0.02* 0.5 ± 0.3** 14 ± 3 a

Wood 0.5 0.7 ± 0.2 0.53 ± 0.08* 16 ± 4 a

P. sellowii Leaf 3.6 0.95 ± 0.05* 0.2 ± 0.2 0 ± 5 a

Wood 0.37 0.95 ± 0.04* 0.5 ± 0.3 7 ± 3 a

Controlpositive

– – 0.82 ± 0.01¥ – 100 ± 0 c

# FR: feeding reduction index (see text). Data were analyzed by the Wilcoxon ranktest (p < 0.05 for * 2-tailed tests, and ** 1-tailed tests).

� % Mortality: mortality corrected following the Abbott’s correction (Abbott,1925). Different letters indicate significant differences among treatments in post-A

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NOVA pair wise comparisons (p < 0.05, Tukey’s error rate).¥ Data for nicotine from Castillo et al. (2009).

occinea Griseb. ARGENTINA. Prov. Santiago del Estero: Dpto. Robles,entura, 11-I-2008, Oakley 66 (UNR). Prov. Santa Fe: Dpto. Gral. Obli-ado, 17-XII-2004, Pensiero 6943 (SF). A. altissima (Mill) SwingleRGENTINA. Prov. Santa Fe: Dpto. Rosario, 18-IV-2009, Gattuso S,014 (UNR). P. crenata (Vell.) Engl. ARGENTINA. Prov. Misiones: Dpto.ral. M. Belgrano, 15/X/2009, Gattuso S, 2020 (UNR).

For each species, leaves and wood were separately extractedhree times with dichloromethane (DCM) at room temperature,nder stirring during 24 h. The three extracts were joined, filterednd concentrated under reduced pressure to give a dried residue.ields are shown in Table 1.

.2. Insects

E. paenulata Germar (Coleoptera: Coccinellidae): a laboratoryolony was maintained on squash (Cucurbita maxima L. var. zapal-ito (Carr.) Millán) under controlled conditions of temperature20 ± 2 ◦C) and photophase (14L:10D) (Camarano et al., 2006).

S. frugiperda Boisduval (Lepidoptera: Noctuidae) colonies wereeared on an artificial diet (Poitout and Bues, 1974) maintained at5 ± 1 ◦C, >70% relative humidity, and a 16L:8D photoperiod.

.3. Deterrent activity

Extracts were evaluated in Petri dishes (9 cm × 1 cm) lined athe bottom with a layer of agar (2%). Insects were offered four leafiscs (1 cm2) of the appropriate host plant (C. maxima var. zapallitor L. sativa for E. paenulata adults and S. frugiperda larvae, respec-ively). Two of the discs (T) were coated with 100 mg of the extract10 �L of a 10% MeOH solution), and the other two (C) with 10 �Lf MeOH. One individual of each species was placed into individuallates: adults (15 and 10 replicates per extract for E. paenulata and. frugiperda, respectively) (Castillo et al., 2009). A visual score ofhe leaf area consumed (0, 25, 50, 75 or 100%) was assigned for alliscs and a feeding reduction index (FR) was determined for each

eplicate using the formula FR = (control − treatment consump-ion)/(control + treatment consumption) (Bellomo et al., 2009) Aegative value indicates a phagostimulant and a positive value anntifeedant effect.

nd Products 44 (2013) 600– 602 601

2.4. Ixodicide activity against Rhipicephalus (Boophilus)microplus larvae

Tests were run with 24-day old larvae as described by Gonzalez-Coloma et al. (in press). Briefly, 50 �L of extracts (10 �g/�Lmethanol solutions) were applied on 25 mg of microcellulose (E.Merck, Darmstadt, F.R.G.). Positive (amitraz, 10 �g/mL) and nega-tive (MeOH) controls were also included. After complete solventremoval, 20 larvae were placed in contact with the microcellulose(N = 3 per sample). Larval mortality was checked after 24 h.

2.5. Phytochemical analysis

HPLC separations were carried out on a Waters 2690 separationmodule equipped with a binary pump, vacuum degasser, autosam-pler and a PDA detector (Waters 996). The solvent system usedwas water with 0.025% trifluoroacetic acid (solvent A) and MeCN(solvent B) at a flow rate of 0.8 mL/min. The solvent gradient wasdesigned to decrease solvent A from 95% at 0 min to 0% at 60 min.The injection volume was 10 �L.

GC–MS analyses were carried out on an Agilent 7890Agas chromatograph attached to a 5975C MSD. A DB-5(30 m × 0.25 mm × 0.25 �m) column was used with helium(1 mL/min) as the carrier gas. Oven temperature was programmedfrom 60 ◦C to 310 ◦C at a rate of 3 ◦C/min. The mass-scanning wasdone by electron impact (EI) at 70 eV (m/z range 35–450). Theidentification of compounds was based by comparison of retentionindices and mass spectra with those of authentic samples, anddata from the NIST GS–MS library.

Extracts were subjected to TLC examination on aluminumsheets pre-coated with silica gel 60 F254 (Merck). Differ-ent mobile phase were used: I ethylacetate:methanol:water(100:13.5:10), II ethylacetate:acetic acid:formic acid:water(100:11:11:26), III chloroform:methanol:NH4OH (4:0.2) or IVdichloromethane:methanol (9:1). Chromatograms were devel-oped using different spray reagents as described in Wagner andBladt (2001).

3. Results and discussion

Table 1 shows the results for all plant extracts evaluated againstthe three arthropod species. Only the wood extract from A. altissimashowed some degree of ixodicidal activity, although lower thanthe positive control used at the same doses. Results were morepromissory when extracts were tested against insects followinga dual-choice bioassay method. As a general pattern, the extractsexhibited higher activity against the specialist E. paenulata thanagainst the generalist S. frugiperda. The feeding reduction observedfor these extracts against E. paenulata is similar to or even greaterthan the reduction produced by two commonly used botanicals(nicotine, FR = 0.82 ± 0.01; rotenone % FR = 0.87 ± 0.07) (Castilloet al., 2009).

C. coccinea wood and A. subovata leaf and wood extracts showedactivity against S. frugiperda. Interestingly, if larvae consumed theleaves treated with C. coccinea wood extracts, they immediatelyregurgitated indicating a separate toxic systemic effect. Previouswork with this bioassay indicates that good antifeeding activityis achieved only with FR greater than 0.70 (García et al., 2007),therefore these extracts can be considered as moderately active asdeterrent. A potential systemic effect would strengthen the anti-insect properties of this extract.

Reversed phase HPLC separation was developed to analyzethe wood extracts (chromatograms showed as Supplementarymaterial). The chromatograms were quite different, suggest-ing a different chemical composition. Therefore, a correlation

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etween activity and chemical composition is not straightfor-ard. In contrast, the five species are intensely bitter especially in

imaroubaceae woods and Picramniaceae leafs and woods (Jacobs,003). This may have been a contributing factor to their deter-ent activity. Chlorogenic and 3,4-dihydroxybenzoic acids and thenthraquinones emodin and chrysophanol were detected as theain components in P. sellowii and A. subovata extracts. Chryso-

hanol, physcion and emodin were reported previously from P.ellowii (Jacobs, 2003).

A coincident elution time and UV spectrum with theanthin 6-one were detected in A. altissima and P. cre-ata extracts and the coumarins scopoletin and ostol andhe alkaloid harmine in C. coccinea extract. Canthin 6-oneas previously described in the first two species (Kundu

nd Laskar, 2010; Sanchez and Comin, 1971). By GC–MS 8ydrophobic compounds were detected, among wich ergot-en-3-ol-acetate, stigmastan-3,5 diene and stigmasta-3,5dien--one steroids were the most abundant ones. in C. coccineaxtract.

C. coccinea and P. crenata extracts could play an impor-ant role for managing E. paenulata. Results obtained here openn avenue for future research because added to the inter-sting activities of the extracts, they do not pose risks touman beings and other mammals, which is evidence by the

acts that the wood is commonly used as a natural medicineor digestive problems in several South American countriesnd its fruits are commonly eaten by goats (Xifreda andeo, 2006; Novello et al., 2008). The rest of active extractsave previously showed some degree of toxicity (Furlan et al.,994).

cknowledgments

Work was supported by ANPCyT (BID-PICT 1494), CYTED307AC0512 and Programa Escala Docente Convocatoria010–2011 de la Asociación de Universidades Grupo Montevideo.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.ndcrop.2012.09.015.

nd Products 44 (2013) 600– 602

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