216

VECTOR-BORNE AND ZOONOTIC DISEASESVolume 6, Number 2, 2006© Mary Ann Liebert, Inc.

Research Paper

Larvicidal Diterpenes from Pterodon polygalaeflorus

M.C. DE OMENA,1 E.S. BENTO,1 J.E. DE PAULA,2 and A.E.G. SANT’ANA1

ABSTRACT

Ethanolic extract from seeds of Pterodon polygalaeflorus (Benth) has been shown to possess significant larvicidalactivity against the mosquito Aedes aegypti. Bioassay-guided fractionation of the extract led to the isolation andcharacterization of the know diterpenoid furans 6�-hydroxyvouacapan-7�,17�-lactone (1), 6�,7�-dihydroxyvoua-capan-17�-oic acid (2) and methyl 6�,7�-dihydroxyvouacapan-17�-oate (3). The structures were established frominfrared (IR), ultraviolet (UV), 1H–nuclear magnetic resonance (NMR), 13C-NMR, and mass spectral data: full NMRassignments are presented for compounds 1–3 and the diacetyl derivative of 3. Compounds 1–3 exhibited LC50values of 50.08, 14.69, and 21.76 �g/mL against fourth-instar Aedes aegypti larvae. Key Words: Diterpenes—Larvi-cidal—activities—Aedes aegypti—Pterodon polygalaeflorus. Vector-Borne Zoonotic Dis. 6, 216–222.

INTRODUCTION

THE WIDESPREAD USE of plants for the pur-poses of primary health care and disease

prevention in underdeveloped regions of LatinAmerica is mainly based on tradition, and isdriven by poverty, necessity, and the generallylow availability of conventional medications. Inmany of these areas, there are insufficient med-icines with which to treat the major endemicdiseases, and a lack of appropriate insecticidesin order to control the main disease carriers. Inthis context, mosquitoes are responsible for thetransmission of more diseases than any othergroup of arthropods. Of particular concern isAedes aegypti, which is a vector for the arbovirusresponsible for yellow fever in Central andSouth American and in West Africa, and forhemorrhagic dengue fever that is endemic to these areas and also to South East Asia andthe Pacific Islands (Monath 1994). The currentrecrudescence of diseases transmitted by thismosquito is due mainly to the greater avail-

ability of breeding places and to the increasingresistance of the insect to commercial insecti-cides.

The ideal method of control of A. aegyptiwould be the systematic treatment of its breed-ing places using larvicides derived from nat-ural sources (Cipleanu 1993). Despite the suc-cessful and selective application of the nat-ural insecticide Bacillus thuringiensis (BT) toxinagainst insects of the order Lepidoptera, its useagainst A. aegypti is not applicable. However,surveys of ethnobotanic and chemosystematicdata indicate that numerous higher plants re-putedly exhibit general or specific biocidal ef-fects as expressed in the forms of anti-parasitic,anti-microbial, vermifugal, insecticidal, or cyto-toxic activities. Based on the discovery of phy-totoxins in Chrysanthemum cinnerariaefolium, itappears that genera of the family Asteraceaeare the most promising candidates for a searchfor new natural insecticides, and a number ofresearch groups have focused on this area(Green et al. 1991, Slimestad et al. 1995). How-

1Departamento de Química, Universidade Federal de Alagoas, Maceió-AL, Brazil.2Departamento de Botánica, Universidade de Brasília, Brasília-DF, Brazil.

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LARVICIDES FROM PTERODON POLYGALAEFLORUS 217

ever, phytotoxins have been discovered in var-ious other plant families (Roth et al. 1998, Iosetet al. 2000).

We have recently surveyed 51 plants from 23families for activity against fourth-instar larvaeof A. aegypti (de Omena et al. 2006), and haveselected the highly active ethanolic extract ofseeds of Pterodon polygalaeflorus (Leguminosae)for a study of the active principles involved.The use in Brazilian folk medicine of alcoholicinfusions of the fruits of this plant (knownlocally as “sucupira branca”) for the treatmentof rheumatism and throat infections is wellknown (Nunan 1982). A number of diterpenoidfurans belonging to the vouacapane grouphave been isolated from Pterodon species (Ma-hajan and Monteiro 1972, 1973, Fascio et al.1970, 1976, Campos et al. 1994), and some havebeen found to possess anti-inflammatory andanalgesic activities, amongst others. We havecarried out an activity-guided fractionation ofthe ethanolic extract of seeds of P. polygalae-florus and report here the isolation of threevouacapane diterpenoids that possess signifi-cant larvicidal activities

METHODS

Reagents and general techniques

Analytical and spectroscopic grade solventswere obtained from Grupo Química (Rio deJaneiro, Brazil). All other Analytical Grade rea-gents were purchased from Aldrich (Milwaukee,WI). Silica gel 60 (particle size 0.063–0.200 mm;70–230 mesh ASTM) for column chromatogra-phy (CC) and silica gel 60 F254 layers (20 � 20cm; 0.2 mm thickness) pre-coated onto alumin-ium backing sheets for thin-layer chromatogra-phy (TLC) were purchased from Merck (Darm-stadt, Germany). Fractions obtained during theactivity-guided separation of extracts were ana-lysed by TLC using n-hexane/ethyl acetate (7:3)as the mobile phase: components were vizual-ized by spraying with ceric sulphate in sulfuricacid or with anisaldehyde reagent, followed byheating in an oven at 100°C for 5–10 min.

Melting points were determined using aKofler block (Microquímica, Florianópolis, SC,Brazil). Fourier-Transform infrared (FTIR) spec-

tra were measured in KBr discs using a Perkin-Elmer (Palo Alto, CA) model FTIR 1600 spec-trometer. Ultraviolet (UV)–visible spectra wererecorded in ethanol solution on a Perkin-Elmermodel Lambda 3 spectrometer. Nuclear mag-netic resonance (NMR) spectra were measuredwith samples dissolved in deuterochloroform(15 mg/mL) using a Bruker Analytic (Ettlingen,Germany) model DRX operating at 300 MHz(1H) and 75 MHz (13C) or model WH-400 op-erating at 400 MHz (1H) and 100 MHz (13C),with tetramethylsilane (TMS) as internal stan-dard. EIMS spectra were obtained using a MSShimadzu QP 5050 (Shimadzu, Kyoto, Japan).

Assay of larvicidal activity

Eggs of A. aegypti were hatched by submer-sion in dechlorinated tap water at a tempera-ture of 26 � 1°C, and fourth stage larvae werecollected 72 h after hatching. Test compoundswere dissolved in an appropriate mixture of di-methylsulfoxide (DMSO) and dechlorinatedwater, and the resulting solution diluted withdechlorinated water to give a final concentra-tion of DMSO of 1%. An appropriate volumeof this test solution was added to a test tubecontaining 25 A. aegypti larvae in sufficientdechlorinated water (containing 1% DMSO) inorder to give a final volume of 10 mL. The testtubes were incubated in darkness at 25–27°Cfor 24 h, and larval mortality was observedunder laboratory lighting conditions. A 3 �gmL�1 solution of Themephos was used as apositive control, and the negative control waswater containing 1% DMSO following the pro-tocol of de Omena et al. (2005).

Plant material

Plants of Pterodon polygalaeflorus Benth werecollected in August 1999 in Brasília, DF, Braziland identified by Professor José Elias de Paula(Departamento de Botânica, Universidade deBrasília, Brasília-DF, Brazil). Voucher speci-mens are deposited in the herbarium of theUniversidade de Brasília with the referencenumbers JEP 3410, 3421, and 3684 UB.

Immediately after harvesting, seeds (720 g)were ground in a Nogueira (Itapira, SP, Brazil)laboratory mill to yield a coarse powder (meshsize 2.5 mm) that was extracted three times,

6271_13_p216-222 6/13/06 9:20 AM Page 217

each with 7 L of 90% ethanol, in a percolatorfor 3 days. The extracts were concentrated un-der reduced pressure in a rotary evaporator toyield 138 g (19.2%) of dry crude extract that wassubmitted to biological assay. Crude ethanolicextracts of leaves, wood, wood bark, root, rootbark, and pericarp—in yields of 95.6 g (19.1%),313 g (4.8%), 116 g (5.22%), 150 g (3.0%), 113 g(13.3%), and 29.7 g (9.8%), respectively—wereobtained using the same protocol.

Bioassay-guided isolation of active components

It was established in a preliminary assay(Table 1) that only the crude ethanolic extractobtained from seeds of P. polygalaeflorus showed

activity against larvae of A. aegypti. One portion(110 g) of the crude seed extract was suspendedin methanol/water (4:6) and extracted succes-sively with n-hexane, chloroform, and ethyl ac-etate (1.5 L each) to yield, after solvent removal,PTP-1 (80.47 g; 73.0%), PTP-2 (23.55 g; 21.4%),and PTP-3 (2.02 g; 1.8%), respectively, togetherwith the remaining water-soluble material PTP-4 (1.54 g; 1.4%). Preliminary assays (Table 1) in-dicated that only those residues obtained by ex-traction of the crude seed extract with n-hexane(PTP-1) and chloroform (PTP-2) exhibited activ-ity against larvae of A. aegypti.

A portion of extract PTP-1 (72.0 g) was sub-jected to CC over silica gel (350 g) with step-wise elution with 1.5 L each of n-hexane (yield-

DE OMENA ET AL.218

TABLE 1. LARVICIDAL ACTIVITIES OF EXTRACTS, FRACTIONS, AND COMPOUNDS DERIVED FROM PTERODON

POLYGALAEFLORUS AGAINST LARVAE OF AEDES AEGYPTI FOLLOWING ACTIVITY-GUIDED FRACTIONATION

Concentration assayed Percentage deathSamplea (�g/mL) after 24 h

Ethanolic extractsLeaves 100 0Wood 100 0Wood bark 100 0Pericarp 100 0Root wood 100 0Seeds 100 100

Fractions from the ethanolic seed extractn-Hexane (PTP-1) 100 100Chloroform (PTP-2) 100 100Ethyl acetate (PTP-3) 100 0Hydromethanolic (PTP-4) 100 0

Fractions from hexane extract PTP-1n-Hexane (PTP-1a) 100 100n-Hexane/chloroform (1�1) (PTP-1b) 100 75Chloroform (PTP-1c) 100 90Methanol (PTP-1d) 100 72

Fractions from chloroform extract PTP-2n-Hexane (PTP-2a) 50 70n-Hexane/chloroform (1�1) (PTP-2b) 50 —Chloroform (PTP-2c) 50 —Methanol (PTP-2d) 50 —

Fractions from extract PTP-2an-Hexane (PTP-2a1) 50 60n-Hexane/chloroform (1�1) (PTP-2a2) 50 80Chloroform (PTP-2a3) 50 100Chloroform/methanol (99�1) (PTP-2a4) 50 —Methanol (PTP-2a5) 50 —

Fractions from extract PTP-2a2n-Hexane 50 80n-Hexane/chloroform (1�1) 50 100Chloroform/methanol (99�1) 50 60Methanol 50 0

Positive control (Themephos) 3 100Negative control (water 1% DMSO) 0

aSamples dissolved in water containing 1% DMSO: assays conducted at 26 � 1°C and relative humidity 77–85%.DMSO, dimethylsulfoxide.

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LARVICIDES FROM PTERODON POLYGALAEFLORUS 219

ing 25.92 g of dry extract PTP-1a), n-hexane/chloroform (1:1; yielding 13.26 g of PTP-1b),chloroform (yielding 12.95 g of PTP-1c), andmethanol (yielding 18.02 g of PTP-1d). A sam-ple (5.0 g) of the chloroform fraction (PTP-1c)was crystalized and re-crystalized with diethylether to give colorless crystaline needles of 1(0.740 g). A portion (8.0 g) of the methanol frac-tion (PTP-1d) was crystalized and re-crystal-ized with diethyl ether to yield white crystalineneedles of 2 (0.585 g).

A portion of the chloroform fraction (PTP-2;20.0 g) was subjected to CC over silica gel (150 g) eluted with 1.2 L of n-hexane (yielding15.01 g of dry extract PTP-2a), 1 L of n-hexane/chloroform (1:1; yielding 1.1 g of PTP-2b), 1 L ofchloroform (yielding 1.3 g of PTP-2c) and 1 L ofmethanol (yielding 2.1 g of PTP-2d). Preliminaryassays (Table 1) indicated that only fraction PTP-2a showed activity against larvae of A. aegypti.The bulk of this fraction (14.0 g) was re-chro-matographed over silica gel (300 g) eluted with700 mL each of n-hexane (yielding 1.22 g of dry extract PTP-2a1), n-hexane/chloroform (1:1;yielding 5.40 g of PTP-2a2), chloroform (yield-ing 4.44 g of PTP-2a3), chloroform/methanol(99:1; yielding 0.13 g of PTP-2a4), and methanol(yielding 2.1 g of PTP-2a5). Only fractions PTP-2a1, PTP-2a2, and PTP-2a3 showed larvicidal ac-tivity (Table 1). A portion (1.2 g) of fraction PTP-2a2 was chromatographed over silica gel (30 g)eluted with 500 mL each of n-hexane (yielding288 mg of dry extract), n-hexane/chloroform(1:2; yielding 372 mg of extract), chloroform/methanol (99:1; yielding 149 mg of extract) andmethanol (yielding 356 mg of extract). Crystal-

ization of the n-hexane/chloroform (1:2) fractionwith diethyl ether produced colorless needles of3 (255 mg), whilst crystalization of the chloro-form/methanol (99:1) fraction produced whitecrystaline needles of 2 (58.3 mg).

Preparation of the acetyl derivative of 3

Freshly distilled acetic anhydride (5 mL) wasadded to a solution of 3 (200 mg; 0.55 mmol)in anhydrous pyridine (1 mL): the mixture waskept at room temperature for 96 h, and the re-action was followed by TLC analyis. For thework-up, the reaction mixture was poured overice, and following hydrolysis of excess aceticanhydride, the product was extracted withchloroform (4 � 100 mL). The organic solutionwas washed with 0.1 M hydrochloric acid (3 �100 mL), water (2 � 200 mL), 0.1 M sodium bi-carbonate (3 � 100 mL), and finally with water(2 � 100 mL). The organic layer was dried overanhydrous sodium sulfate and the chloroformremoved by distillation. After purification byCC, the diacetyl derivative 4 was obtained as awhite crystaline solid from ethanol (225 mg,yield 92%).

RESULTS AND DISCUSSION

Interest in the genus Pterodon was promotedby the knowledge that the fruit oil of P. pubes-cens inhibited the penetration of schistosomecercaria through the skin, a property that wasattributed to the diterpenoid 14,15-epoxy ger-anylgeraniol and later to the accompanying lin-

FIG. 1. The main compounds isolated from ethanolic extract of seeds of P. polygalaeflorus.

6271_13_p216-222 6/13/06 9:20 AM Page 219

TA

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6271_13_p216-222 6/13/06 9:20 AM Page 220

LARVICIDES FROM PTERODON POLYGALAEFLORUS 221

ear diterpenoid 14,15-dihydroxy-14,15-dihy-drogeranylgeraniol (Dos Santos et al. 1972, Fas-cio et al. 1976, Mors et al. 1966, Gilbert et al.1970). Subsequently, a number of diterpenoidfurans belonging to the vouacapane groupwere isolated from members of the genus (Fas-cio et al. 1970, 1976, Mahajan and Monteiro1972, 1973, Campos et al. 1994), and one ofthem, 6�,7�-dihydroxyvouacapan-17�-oic acid,presented significant anti-inflammatory andanalgesic activities (Maltha et al. 1995).

When ethanolic extracts obtained from vari-ous parts of P. polygalaeflorus were assayed forlarvicidal activity against A. aegypti (Table 1),that derived from seed material was signifi-cantly active (Table 1). Initial chromatographicseparation of the seed extract by CC producedtwo active fractions, namely, that eluting withn-hexane (PTP-1) and that eluting with chlo-roform (PTP-2). Both of these fractions werefurther separated by CC eluting with n-hexane, n-hexane/chloroform (1:1), chloroform, andmethanol. The sub-fractions derived from PTP-1 all exhibited larvicidal activity (Table 1), andTLC analysis indicated that each contained twomajor compounds in varying amounts. Com-pounds 1 and 2 could be crystalized from PTP-1c and PTP-1d, respectively, in good yield.Only the n-hexane sub-fraction (PTP-2a) de-rived from PTP-2 showed larvicidal activity,and compound 3 was obtained in pure crys-taline form following further fractionation ofthis extract. The diacetyl derivative of 3 (com-pound 4) was prepared in order to assist withthe identification of this compound (Fig. 1). Theidentities of 1–4 were determined from theirFTIR, UV, 1H- and 13C-NMR, and MS data incomparison with those reported by Nunan(1982), Mahajan and Monteiro (1972, 1973) andFascio et al (1976).

6�-Hydroxyvouacapan-7�,7�-lactone (1)

Melting point [from diethyl ether], 229–232°C(Rubinger 1991; 226.1–227.9°C); molecular for-mula C20H26O4; IR (KBr, �� cm�1), 3497, 2929,2862, 1767, 1463, 1365, 1120, 1051; MS (EIMS)m/z, M� 330; 1H and 13C-NMR (Table 2).

6�,7�-Dihydroxyvouacapan-17�-oic acid (2)

Melting point [from diethyl ether], 272–278°C(Mahajan and Monteiro 1973; 272–274°C); mo-lecular formula, C20H28O5; IR (Kbr, �� cm�1)3506, 3435, 2990, 2950, 1720, 1520, 1395, 1204,1187, 1153, 1050; MS (EIMS) m/z, M� 348; 1H�

and 13C-NMR (Table 2).

Methyl 6�,�-dihidroxyvouacapan-17�-oate (3)

Melting point [from diethyl ether], 202–204°C(Fascio et al. 1976; 204–205°C); molecular for-mula, C21H30O5; IR (KBr, �� cm�1), 3518, 3001,2921, 2300, 1726, 1436, 1390, 1281, 1194, 1019; MS(EIMS), M� 362(45); 1H� and 13C-NMR (Table 2).

Diacetyl derivative of methyl 6�,7�-dihidroxyvouacapan-17�-oate (4)

Melting point [from ethanol], 202–204°C (Mahajan and Monteiro 1973; 206–207°C); mo-lecular formula, C25H34O7; IR (KBr, �� cm�1)3000, 2947, 2860, 1743, 1577, 1436, 1248, 1034; MS(EIMS) m/z, M� 446; 1H� and 13C-NMR (Table 2).

Full assignments of the NMR spectra of com-pounds 1–4 are provided in Table 2 and weredetermined from one- and two-dimensionalspectra data.

The LC10, LC50, and LC90 values for com-pounds 1–3 are presented in Table 3. 6�,7�-Dihydroxyvouacapan-17�-oic acid (2) showedthe highest larvicidal activity with an LC50 valueof 14.69 �g/mL, which is comparable with other

TABLE 3. LARVICIDAL ACTIVITIESa OF COMPOUNDS 1–3 AGAINST AEDES AEGYPTI LARVAE

Compound LC90 (�g/mL) LC50 (�g/mL) LC10 (�g/mL)

6�-Hydroxyvouacapan-7�, 7�-lactone (1) 87.31 50.08 28.736�,7�-Dihydroxyvouacapan-17�-oic acid (2) 27.50 14.69 7.85Methyl 6�,7�-dihidroxy-vouacapan-17�-oate (3) 25.13 21.76 18.85

aValues determined after 24 h of treatment with compounds dissolved in water containing 1% DMSO: assays con-ducted at 26 � 1°C and relative humidity 77–85%. Positive control: Themephos 3 �g mL�1; negative control: water1% DMSO.

DMSO, dimethylsulfoxide.

6271_13_p216-222 6/13/06 9:20 AM Page 221

active natural substances, including three limo-noids isolated from Turraea wakefieldii (Meliaceae)with LC50 values of 7.83, 7.07, and 7.05 �g/mL(Ndung’n et al. 2003) and a very active flavonefrom Melicope subunifoliolata (Rutaceae) with anLC50 of 0.47 �g/mL (Hung Ho et al. 2003).

The present results show that vouacapanediterpenoids possess larvicidal activity. SinceP. polygalaeflorus is a common plant in Brazil,seed material is readily available on a largescale, and the seed oil itself, or crude extractsderived therefrom, could have immediate ap-plication as an efficient biocontrol agent againstlarvae of the mosquito A. aegypti.

ACKNOWLEDGMENTS

We wish to thank the Conselho Nacional deDesenvolvimento Tecnológico (CNPq, Brazil),Fundação Coordenação de Aperfeiçoamentode Pessoal de Nível Superior CAPES and Fun-dação de Amparo a Pesquisa do Estado deAlagoas FAPEAL for support of this work.

REFERENCES

Campos, AM, Silveira, ER, Braz-Filho, R, et al. Diter-penoids from Pterodon polygalaeflorus. Phytochem-istry 1994; 36:403–406.

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Address reprint requests to:Dr. A.E.G. Sant’Ana

Department of ChemistryFederal University of Alagoas

Tabuleiro do Martins57072-970 Maceió, AL, Brasil

E-mail: aegs@qui.ufal.br

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