Pest Management Science Pest Manag Sci 62:1072–1081 (2006)

Bioactivity of extracts and isolatedcompounds from Vitex polygama(Verbenaceae) and Siphoneugena densiflora(Myrtaceae) against Spodoptera frugiperda(Lepidoptera: Noctuidae)†Margareth BC Gallo,1∗ Waldireny C Rocha,1 Uemerson S da Cunha,2

Fernanda A Diogo,2 Fernando C da Silva,1 Paulo C Vieira,1 Jose D Vendramim,2

Joao B Fernandes,1 M Fatima das GF da Silva1 and Luciane G Batista-Pereira1

1Departamento de Quımica, Universidade Federal de Sao Carlos (UFSCar), CP 676, 13565-905, Sao Carlos-SP, Brazil2Departamento de Entomologia, Fitopatologia e Zoologia Agrıcola, Escola Superior de Agronomia Luiz de Queiroz, Universidade de SaoPaulo (ESALQ/USP), CP 09, 13418-900, Piracicaba-SP, Brazil

Abstract: The effects of crude extracts, fractions and isolated compounds from Vitex polygama Cham. andSiphoneugena densiflora Berg were evaluated on the development of Spodoptera frugiperda JE Smith, a destructiveinsect pest of corn and several other crops. The extracts and fractions were incorporated into an artificial dietat 1 mg g−1 and offered to the insect during its larval stage. Length and viability of larval and pupal stages aswell as pupal weight were assessed. Isolated compounds were tested through superficial contamination of thediet at 0.1 mg g−1. Weight and viability of ten-day-old larvae were determined. Methanolic and hydroalcoholicS. densiflora extracts caused 100% larval mortality, while leaf and fruit hydroalcoholic extracts from V. polygamawere the most active. Among the isolated compounds, flavonoids presented the best insecticidal results, andtannins the best larval growth inhibition. 2006 Society of Chemical Industry

Keywords: Taruma; Uvatinga; Hoja Menuda; fall armyworm; Vitex polygama; Siphoneugena densiflora; Spodopterafrugiperda; botanical insecticide; pest control

1 INTRODUCTIONWith a projected increase in world population to10 billion over the next four decades, an immediatepriority for agriculture is to achieve maximumproduction of food and other products in a mannerthat is environmentally sustainable and cost effective.1

One major limiting factor to global food productionis damage by pests, during both growth and storagestages. The use of synthetic pesticides has been aneffective way to control pests, but their efficacy isconstantly weakened by development of resistancein economically important pests.2 Increasing interestin the application of plant secondary metabolitesin insect pest management, as an alternative tothe use of synthetic insecticides, has led to thesearch for active plant compounds, less poisonousto the environment and with low mammaliantoxicity.3

Spodoptera frugiperda JE Smith (Lepidoptera: Noc-tuidae), the model insect pest selected, has a recordof injuring over 80 plants, among them importantfield crops such as corn, cotton, soybean and wheat.

Larvae destroy the plant growth potential by con-suming foliage and burrowing into its growing points.The control of the damage requires high volumes ofinsecticide, resulting in resistance to several kinds ofinsecticidal compound.4

Of the two Brazilian plant species chosen to bebioassayed and phytochemically investigated, Vitexpolygama Cham. (Verbenaceae) had already beenstudied, and many of its compounds characterized, butnone was considered as a potential insecticide.5–9 Thisplant is commonly called ‘Maria-Preta’, ‘Taruma’,‘Congonha-cinco-folhas’ and the like.10,11 Its leaftea has been used in folk medicine to treat kidneydiseases.11 The other plant, Siphoneugena densifloraBerg (Myrtaceae), belongs to a genus comprisingonly eight species, with two of them vulnerable toextinction in Sao Paulo State.12,13 Popularly named‘Uvatinga’,13 it has been pointed out as a sourceof energy generation in a recent investigation,14 andherbarium observations by the present authors indicatethat this species shows strong resistance to insectattack.

∗ Correspondence to: Margareth BC Gallo, Departamento de Quımica, Universidade Federal de Sao Carlos (UFSCar), CP 676, 13565-905, Sao Carlos-SP, BrazilE-mail: marejor@uol.com.br†This paper was given in part at the XX Brazilian Congress of Entomology, Gramado-RS-Brazil, 5-10 September 2004, Abstract EN-335, p. 341.(Received 15 November 2005; revised version received 12 April 2006; accepted 9 May 2006)Published online 4 September 2006; DOI: 10.1002/ps.1278

2006 Society of Chemical Industry. Pest Manag Sci 1526–498X/2006/$30.00

Natural products active against S. frugiperda

Even though the selected plant species have notbeen ordinarily employed in pest control, their familiescomprise several species with injurious properties toinsects reported in the literature.15–21 The presentpaper specifically deals with the effects of crudeextracts, fractions and isolated compounds fromV. polygama and S. densiflora against fall armyworm(S. frugiperda). Aspects examined include insecticidaland growth regulatory activities.

2 MATERIALS AND METHODS2.1 GeneralHeteronuclear single-quantum correlation (HSQC)and heteronuclear multiple-bonding correlation(HMBC, J 8.0 Hz) experiments as well as 1Dand 2D nuclear magnetic resonance (NMR) spectrawere recorded in deuterated solvents (deuteropyridine,acetone-d6 or deuteromethanol) from Aldrich Chemi-cal Company, Inc. (Milwaukee, WI, USA), or Merck-Schuchardt (8011 Hohenbrunn bei Munchen), usingTMS (tetramethylsilane) as internal reference. Theequipment employed was either a Bruker AvanceDRX-400 spectrometer (Karlsruhe, Germany; 1H,400 MHz; 13C, 100 MHz) or a Bruker ARX-200spectrometer (1H, 200 MHz; 13C, 50 MHz). Low-resolution electrospray mass spectroscopy (ESI/MS)was carried out on a Micromass Quattro LC triple-quadrupole instrument (Manchester, UK). IR spectrawere measured on a Bomem MB-102 spectropho-tometer (Quebec, Canada) in potassium bromide pel-lets. UV spectra were obtained on a Varian Cary 500Scan/UV-Vis-Nir spectrophotometer (Mulgrave, Aus-tralia). The high-performance liquid chromatography(HPLC) system consisted of a Shimadzu LC-10ADpump (Kyoto, Japan), an SPD-10A UV-Vis detectorand a CBM-10A interface, using a Shodex AsahipackGS-310 preparative column (50.0 × 2.5 cm ID). Dataacquisition was performed on CLASS LC10 software.

Column chromatography (CC) was performed onsilica gel (70–230 or 230–400 mesh; Merck KGaA,64 271, Darmstadt, Germany), Sephadex LH-20(25–100 µm; Pharmacia Fine Chemical Co. Ltd,Uppsala, Sweden) or XAD7 (Aldrich ChemicalCompany, Inc., Milwaukee, WI, USA). Spots werevisualized under 254 nm UV light and by sprayingwith either vanillin sulfuric acid solution followed byheating or FeCl3 acid solution. Methanol (JT Baker,Philipsburg, PA, USA) used for the mobile phase inHPLC analysis was HPLC grade. All other solventsused in extraction and isolation of compounds wereof analytical grade (Mallinkrodt Baker, SA, Xalostoc,Mexico) or home distilled.

2.2 PlantsVitex polygama and Siphoneugena densiflora werecollected in July 2000, in the city of Pocos deCaldas, Minas Gerais, Brazil. Voucher specimens weredeposited at the Universidade Federal de Juiz deFora Herbarium (Minas Gerais) and at the Botany

Institute Herbarium, Universidade de Sao Paulo,Brazil, respectively.

2.3 Extraction and isolationThe powder of the air-dried plant organs wasextracted successively with hexane and methanol bypercolation at room temperature. Crude extracts wereobtained after filtration and removal of the solventsunder vacuum at 40 ◦C. Hydroalcoholic extracts wereobtained by stirring 100 g of the residue with methanol+ water (50 + 50 by volume; 3 × 300 mL) withultrasonic mixing for 10 min. The resulting filtrateswere combined, evaporated under vacuum (40 ◦C)and lyophilized (Table 1). Some crude extracts weredissolved in water + methanol (1 + 1 by volume)and fractionated through liquid–liquid partition usingsolvents of increasing polarity (dichloromethane, ethylacetate, butanol). The corresponding labeled layersare characterized in Table 1.

Compounds 1 to 43 were obtained by submittingthe most active extracts (see Table 2) to severalchromatographic techniques. Procedures to isolate thesubstances bioassayed (1, 9, 14 to 18, 25, 26 and 43;see Fig. 1) are described below. Some of them, likecompounds 15 and 16, were isolated from more thanone different extract of the same plant.

2.3.1 Isolation of compounds 13 to 19 fromSiphoneugena densiflora (SD-EMR layer)A quantity of 9 g of SD-EMR (Table 1) wassubjected to CC using silica gel (38.0 × 5.0 cm)with dichloromethane + ethyl acetate (7 + 3 byvolume) as eluent and gradient elution. A totalof 65 fractions of 150 mL were collected, and thesimilar ones were pooled into 22 fractions (R1 toR22) according to their composition determinedby thin-layer chromatography (TLC) and visualizedunder UV light and spraying with color reagent.Fraction 11 (R11, 317.0 mg) was chromatographedover Sephadex LH-20 (52.0 × 3.0 cm) using methanolas eluent. A total of 40 fractions of 30 mL werecollected and combined according to their similarities,affording 15 fractions (R11a to R11p). Fraction 6(R11f) was identified as compound 16 (14.7 mg).Fraction 12 (R12, 900.0 mg) was chromatographedover Sephadex LH-20 (66.0 × 2.0 cm) and elutedwith methanol + acetone (8 + 2 by volume). Atotal of 22 fractions of 30 mL were obtained andcombined into 11 fractions (R12b to R12m). Theconstituent of fraction 3 (R12d) was identified ascompound 19 (23.0 mg). Fraction 6 (R12f, 176.0 mg)was chromatographed over Sephadex LH-20 (60.0 ×2.0 cm) using methanol + acetone (7 + 3 by volume)as eluent and yielded 14 fractions of 30 mL each,which were pooled into nine fractions (R12f1 toR12f9). Fraction 4 (R12f4, 50.0 mg) afforded asubstance identified as 13, and fraction 7 (R12f7,60.0 mg) was identified as compound 17. Fraction 18(R18, 698.0 mg) was chromatographed over XAD-7 (67.0 × 4.0 cm) using methanol as eluent. Ten

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Table 1. Extracts and fractions obtained from Siphoneugena densiflora (SD) and Vitex polygama (VP)

Plantmaterial (g)

Extractionsolventa

Extractcode

Amountobtained (g)

Liquid/liquidpartition solventa

Layercode

Amountobtained (g)

SD leaves (923) H SD-HL 8.2M SD-ML 164.8 D SD-DML 9.5

E SD-EML 14.6B SD-BML 12.0A SD-AML 12.5

HA SD-HAL 22.1SD stem (2247) H SD-HS 0.9

M SD-MS 177.6 D SD-DMS 1.62E SD-EMS 7.67B SD-BMS 12.7A SD-AMS 35.4

HA SD-HAS 5.5SD twigs (1288) H SD-HT 1.0

M SD-MT 131.0HA SD-HAT 5.7

SD root bark (400) H SD-HR 0.3M SD-MR 73.2 D SD-DMR 0.9

E SD-EMR 10.5A SD-AMR 52.0

HA SD-HAR 2.4VP leaves (423) H VP-HL 5.1

M VP-ML 40.4HA VP-HAL 31.0 E VP-EHAL 2.2

B VP-BHAL 12.8A VP-AHAL 22.6

VP twigs (415) H VP-HT 0.85M VP-MT 19.3 D VP-DMT 2.6

E VP-EMT 3.9A VP-AMT 8.4

HA VP-HAT 2.3VP fruit (171) H VP-HF 1.1

M VP-MF 2.0HA VP-HAF 0.9

a Solvents: H, hexane; M, methanol; HA, methanol + water (1 + 1 by volume) (hydroalcoholic); D, dichloromethane; E, ethyl acetate; B, butanol; A,aqueous residue.

fractions of 200 mL were collected and pooled intofour fractions (R18a to R18d). Fraction 1 (R18a) wasfurther purified by R-HPLC (column ASAHIPAKGS-310 SHODEX, mobile phase methanol, flowrate2 mL min−1; λ 330 nm) to yield compound 18(second peak, 109.4 mg) after one cycle of 74 min.A quantity of 240 mg of fraction 19 (R19, 434.5 mg)was chromatographed over Sephadex LH-20 (52.0 ×3.0 cm) using methanol as eluent. A total of 31fractions of 30 mL were obtained and pooled intoeight fractions (R19a to R19h). Fraction 8 (R19h,127.6 mg) was rechromatographed over SephadexLH-20 (52.0 × 3.0 cm) using methanol as eluent.A total of 36 fractions of 30 mL were collectedand pooled into ten fractions (R19h1 to R19h10).Fractions 8 (R19h8) and 10 (R19h10) producedbrown solids identified as compounds 15 (75.0 mg)and 14 (19.6 mg) respectively.

2.3.2 Isolation of compounds 16 and 25 to 29 fromSiphoneugena densiflora (SD-EML layer)A quantity of 4.7 g and 700 mg of SD-EML (Table 1)were chromatographed on a silica gel 60 column

(30.0 × 5.0 cm) and eluted in a step gradient fromacetone + ethyl acetate (95 + 5 by volume) to 100%methanol. A total of 47 fractions of 150 mL werecollected and pooled into 15 fractions (L1 to L15).Fraction 2 (L2, 96.4 mg) was submitted to CC usingSephadex LH-20 (100.0 × 2.5 cm) and methanol aseluent, resulting in 24 fractions of 30 mL each, whichwere combined into ten fractions (L2a to L2j). Theconstituent of fraction 5 (L2e) was identified ascompound 16 (57.3 mg), and that of fraction 10(L2j) as compound 25 (4.0 mg). Fraction 5 (L5,236.0 mg) was submitted to CC using Sephadex LH-20 (38.0 × 3.0 cm) and methanol as eluent, yielding 17fractions of 30 mL each, which were pooled into eightfractions (L5a to L5h). Fraction 5 (L5e) afforded theflavonoid 26 (8.0 mg). Fraction 6 (L5f) was identifiedas compounds 27 and 28 in mixture (13.3 mg).Fraction 6 (L6, 784.0 mg) was chromatographed overSephadex LH-20 (100.0 × 2.5 cm) using methanolas eluent. A total of 25 fractions of 30 mL werecollected and pooled into eight fractions (L6a to L6h).The constituent of fraction 2 (L6b) was identified ascompound 29 (61.0 mg).

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Natural products active against S. frugiperda

O

HO

HO

O

O

OHHO

HOOH

HO

HO

O

OH

OHOH

OH

O

OO

O

OH

OH

OR

HO

COOH

RO

OH

OR

O

O

HO

OH

OR

OH

OH

OC OC

O

CO

CO O

O

O O

OH

HO

HO OH OH

OH

OH

HO

HO

HO

HO

OH

OH

HO

HO

OH

OH

HO

OH

OH

OC

OHOH

COO

O

O

O

CO OCO

CO

OH

OH

OH

OHOH

HO

OH

HO

HO

OH

OH

HO

OH

OH

OC

OHOH

CO

O

O

O

O

CO OC

O

CO

OH

OH

OH

OHOH

HO

OH

91

14

15

43

25 R = H26 R = rha

18 R = H17 R = rha 13 R = H

16 R = CH3

Figure 1. Chemical structures of compounds tested on Spodoptera frugiperda.

2.3.3 Isolation of compounds 15 and 43 fromSiphoneugena densiflora (SD-HAS extract)A quantity of 1.3 g and 300 mg of SD-HAS extract(Table 1) were subjected to CC using XAD7 (63.0 ×4.5 cm) and subsequent elution with 500 mL of water(XA), water + acetone (1 + 1 by volume) (XWA) andmethanol + acetone (1 + 1 by volume) (XMA). Thedried fraction XWA (672.8 mg) was chromatographedover Sephadex LH-20 (46.0 × 3.0 cm) using methanolas eluent. A total of 29 fractions of 30 mL wereobtained and combined into ten fractions (XWA1to XWA10). Both fractions 6 (XWA6) and 8 (XWA8)

afforded brown powders identified as compounds 43(78.6 mg) and 15 (97.3 mg) respectively.

2.3.4 Isolation of compounds 1, 7 and 8 from Vitexpolygama (VP-BHAL layer)A quantity of 10.7 g and 700 mg of VP-BHAL layer(Table 1) were subjected to CC using XAD7 (46.0.0 ×4.0 cm) and subsequent elution with 500 mL of water(WV) and water + methanol (1 + 1 by volume)(WMV) and 2 L of methanol (MV). A quantity of 1 g ofthe dried fraction MV (6.75 g) was chromatographedover Sephadex LH-20 (53.0.0 × 3.0 cm) using MeOH

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Table 2. Activities of extracts and fractions from Siphoneugena densiflora (SD) and Vitex polygama (VP) on growth parameters of first-instar

Spodoptera frugiperda larvaea

Pupal weightTreatment(plantextract)

Larvalmortality

(%)bPupation

time (days) (mg)b,c (±SEM) (%)d

Survivalpupation

(%)eEmergencetime (days)c

Emergence(%)f

SD-HL 10.0 21 249.2 (±6.84)∗ 89 60 32 50SD-ML 100.0 – – – – –SD-HAL 100.0 – – – – –SD-HS 20.0 21 279.3 (±6.07) 100 80 31 100SD-MS 100.0 – – – – –SD-HAS 100.0 – – – – –SD-HT 10.0 20 266.4 (±6.43) 96 90 30 100SD-MT 100.0 – – – – –SD-HAT 100.0 – – – – –SD-HR 30.0 20 274.3 (±9.00) 99 70 29 100SD-MR 100.0 – – – – –SD-EMR 100.0 – – – – –SD-AMR 100.0 – – – – –SD-HAR 100.0 – – – – –VP-HL 40.0 33∗ 207.6 (±7.47)∗ 88 70 44∗ 85.7VP-ML 0 21 302.9 (±5.91) 107 100 30 70VP-HAL 60.0 29 251.0 (±8.31) 107 40 39 75VP-EHAL 0 18 243.8 (±2.52)∗ 91 60 32∗ 100VP-BHAL 100.0 – – – – – –VP-AHAL 42.9 24 236.3 (±13.68) 97 60 35 100VP-HT 20.0 27 258.3 (±6.23)∗ 110 80 40 87.5VP-MT 0 21 295.3 (±13.43) 104 100 32∗ 80VP-HAT 10.0 25 230.4 (±7.46) 98 90 35 77.8VP-HF 90.0 42∗ 160.0 (±0)∗ 68 10 50∗ 100VP-MF 40.0 30 216.8 (±8.41) 92 60 39 66.7VP-HAF 100.0 – – – – – –

a Each datum represents the mean of ten replicates each set up with one larvae (n = 10). Extracts were tested at 1.0 mg g−1. Different controls wereset up depending on the solvents used for extract dilutions and the date of the experiment.b After 20 days of treatment; – , not evaluated; mortality corrected according to Abbott’s formula.23

c Means within a column followed by ∗ are significantly different from the control at P < 0.05 (ANOVA followed by Tukey’s test).d Pupal weight as percentage of control.e Survival pupation (%) = number of surviving pupae ×100/total larvae for pupation.f Emergence (%) = number of adults emerged ×100/total number of pupae.

as eluent, resulting in 41 fractions of 30 mL, whichwere pooled into seven fractions (MV1 to MV7).Fraction 4 (MV4, 168.1 mg) was rechromatographedover the same column containing Sephadex LH-20and eluted with methanol. A total of 23 fractions of30 mL were collected and pooled into seven fractions(MV4s to MV4z). Fraction 4 (MV4v, 57.9 mg) wasfurther purified by R-HPLC (column ASAHIPAKGS-310 SHODEX, mobile phase methanol, flowrate3 mL min−1; λ 330 nm) to yield compound 1 (firstpeak, 5.5 mg) and substances 7 and 8, identified as amixture, (second peak, 3.1 mg), after three cycles of54 min.

2.3.5 Isolation of compounds 9 to 11 from Vitexpolygama (VP-EMT layer)A quantity of 1.9 g and 900 mg of VP-EMT layer(Table 1) were submitted to column chromatogra-phy (3.0 × 46.0 cm) using Sephadex LH-20 andmethanol as eluent. A total of 23 fractions of 50 mLwere collected. Similar fractions were combined, yield-ing seven fractions (EMT1 to EMT7). Fraction 3

(EMT3, 551.0 mg) was rechromatographed over sil-ica gel (230–400 mesh) using gradient elution fromethyl acetate + dichloromethane (9 + 1 by volume) tomethanol; 60 fractions of 50 mL were obtained andcombined into 17 fractions (EMT3a to EMT3r). Frac-tion 5 (EMT3e, 140.5 mg) was purified by preparativeTLC on silica gel 60 F254 with double development,using dichloromethane + acetone (1 + 1 by volume)as mobile phase and UV detection, yielding com-pounds 9 (77.7 mg), 10 (11.0 mg) and 11 (14.3 mg)respectively.

2.4 Insect rearingLarvae of S. frugipereda used in the experimentswere obtained from cultures at the Departamentode Entomologia, Fitopatologia e Zoologia Agrıcola ofthe Escola Superior de Agronomia Luiz de Queiroz,Universidade de Sao Paulo (ESALQ/USP), andmaintained in environmental chambers at 25 ± 1 ◦C,70 ± 5% RH and 12:12 h light:dark photoperiod. Theartificial diet used for the bioassay and previouslydescribed22 contained cooked ‘carioca’ bean 37.5 g,

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Natural products active against S. frugiperda

wheat germ 30.0 g, soy meal 15.0 g, casein 15.0 g,yeast extract 18.75 g, tetracycline 56.5 mg, agar 11.5 g,Vanderzant vitamin mixture for insects 4.5 mL,ascorbic acid 1.8 g, sorbic acid 0.9 g, methyl p-hydroxybenzoate 1.5 g, formaldehyde (40% v/v)1.8 mL and distilled water 600 mL, in a total volumeof 720 mL.

2.5 Toxicity bioassays by ingestion againstSpodoptera frugiperda2.5.1 Crude extracts and layersA quantity of 100 mg of crude extracts or fractionswas diluted in a small volume of acetone (e.g. 2 mL)along with ascorbic acid (ingredient of the artificialdiet). The solvent was allowed to evaporate for 1 h,and the resultant mixture was ground to powderand incorporated into the liquid diet (100 g), at afinal concentration of 1 mg g−1. The liquid diet wasdistributed in portions (10 g) into ten glass tubes(8.5 × 2.5 cm, previously sterilized for 1 h at 170 ◦C),covered with sterile hydrofugous (not absorbent)cotton and then left for 24 h at room temperatureto eliminate excess humidity. One first-instar S.frugiperda larva (from 0 to 3 h old) was then placedin each tube and held at 25 ± 1 ◦C, 70 ± 5% RHand 12:12 h light:dark photoperiod for about 20 daysuntil pupation (each experiment comprised ten glasstubes, each containing a single first-instar S. frugiperdalarva, totaling 10 larvae). Mortality was determined foreach larva every 24 h; cessation of movement followedby color change to black was the criterion used forjudging mortality. The percentage insect mortality wascorrected using Abbot’s formula.23 On the day afterpupation, live pupae were weighed and transferredto transparent plastic vials (6.0 × 6.0 cm) containingfilter paper at the bottom (4.0 × 4.0 cm), wetted bytwo drops of distilled water and maintained at thesame conditions as the larvae until adults emerged(10–20 additional days). Other developmental factorswere recorded, such as time to pupation and adultemergence.

The control diet was prepared by adding the mixtureof ascorbic acid and acetone previously evaporated andground.

Methanolic and hydroalcoholic extracts insolublein acetone were prepared in methanol and waterrespectively, as well as their respective controls.

2.5.2 Isolated compoundsFour well plates (24 × 1.5 mL wells) were filled withthe liquid diet and left for 1 h at room temperaturein a decontaminated (UV light) laminar flow hood.Each compound (12.0 mg) was dissolved in 3 mL ofdistilled water, and 30 µL aliquots were layered onthe top of each well containing the artificial congealeddiet. The final concentration was about 0.1 mg g−1.The surface excess solvent was allowed to evaporatefor about 1 h under sterile conditions. Subsequently,a single second-instar S. frugiperda larva was placedon the diet mixture in each well and maintained for

10 days, under the same conditions as in Section 2.5.1.The percentage insect mortality was corrected usingAbbott’s formula.23 Each single experiment containeda total of 96 larvae (each plate of 24 wells with fourreplicates). Controls were carried out in a similar waywithout addition of the test compound.

2.6 Statistical analysisData consisting of the average value for eachexperimental unit were subjected to analysis ofvariance (ANOVA). Differences between treatmentmeans were established by Tukey’s test.24 Results aregiven in the text as probability values, with P < 0.05adopted as the criterion of significance. Completestatistical analysis was performed by means of the SASprogram.25

3 RESULTS AND DISCUSSION3.1 Structure elucidationSubstances 1 to 43 were identified by comparisonof their NMR, MS, UV and IR spectroscopic datawith data previously reported in the literature. Dataare given only for those compounds that were testedindependently (see Table 3).

3.1.1 Compounds identified in the VP-BHAL layerCaffeoyl 6-O-β-D-glucopyranoside (1) and caffeoyl6-O-α-D-glucopyranoside (2; C15H18O9): amorphousyellow solid; ESI/MS m/z 341 [M − H]−; 1H NMR(400 MHz, acetone-d6): aglycone: δ 7.18 (H-2; d; J2.5 Hz); 6.88 (H-5; d; J 8.2 Hz); 7.08 (H-6; m);7.56 (H-7; d; J 15.9 Hz); 6.31 (H-8; d; J 15.9 Hz);α-glucose: δ 5.12 (H-1; d; J 3.6 Hz); 3.38 (H-2; m);3.71 (H-3; t; J 9.1 Hz); 3.36 (H-4; m); 3.54 (H-5;ddd; J 9.1, 6.2 and 2.0 Hz); 4.26 (H-6b; dd; J 11.8and 6.2 Hz); 4.43 (H-6a; dd; J 11.8 and 2.0 Hz); β-glucose: δ 4.53 (H-1; d; J 8.0 Hz); 3.17 (H-2; t; J 8.0Hz); 3.42 (H-3; m); 3.39 (H-4; m); 4.03 (H-5; m);4.30 (H-6b; dd; J 11.8 and 5.8 Hz); 4.49 (H-6a; dd;J 11.8 and 2.0 Hz); UV and 13C NMR data wereidentical to data previously reported.26 Isoorientin(3) and orientin (4; C21H20O11); carlinoside (5) andisocarlinoside (6; C26H28O15); schaftoside (7) andisoschaftoside (8; C26H28O14).

3.1.2 Compounds identified in the VP-EMT layer20-hydroxyecdysone (9; C27H44O7): amorphous col-orless solid; ESI/MS m/z 479 [M − H]−; 13C NMR(50 MHz, deuteropyridine): δ 38.1 (C-1); 68.5 (C-2);68.3 (C-3); 32.7 (C-4); 51.6 (C-5); 204.1 (C-6); 121.9(C-7); 166.6 (C-8); 34.7 (C-9); 38.9 (C-10); 21.4 (C-11); 32.3 (C-12); 48.4 (C-13); 84.5 (C-14); 31.9(C-15); 21.7 (C-16); 50.4 (C-17); 18.2 (C-18); 24.7(C-19); 77.2 (C-20); 21.9 (C-21); 77.9 (C-22); 27.7(C-23); 42.8 (C-24); 70.1 (C-25); 30.4 (C-26); 30.1(C-27); UV and 1H NMR data were identical to datapreviously reported.27 Polypodine B (10; C27H44O8);stachysterone (11; C27H42O6) and shidasterone (12;C27H42O6).

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Table 3. Activity of compounds 1, 9, 14 to 18, 25 to 26 and 43 from Siphoneugena densiflora (SD) and Vitex polygama (VP) on viability and weight of

second-instar Spodoptera frugiperda larvaea after 10 days of incubation

Larval weight

Treatment (plant) Larval mortality (%) (±SEM)b,c (mg) (±SEM)c,d (%)e

1 (VP) 2.8 (±2.77) a 293.1 (±15.09) ab 85.69 (VP) 0 a 262.6 (±3.91) b 76.6

14 (SD) 3.1 (±1.04) a 100.8 (±12.14) b 39.915 (SD) 1.6 (±1.56) a 300.2 (±20.07) ab 87.616 (SD) 26.1 (±6.13) a 199.7 (±30.10) ab 91.317 (SD) 21.0 (±1.85) a 308.6 (±2.52) ab 90.118 (SD) 8.9 (±1.55) a 192.3 (±4.44) ab 76.225 (VP) 78.0 (±0) b ne26 (VP) 85.0 (±0) b ne43 (SD) 0 a 121.4 (±25.24)b 48.1

a Each datum represents the mean of four replicates, each one set up with 24 larvae (n = 96). Compounds were tested at 0.1 mg g−1. The controlswere different, depending on the date of the experiment and the solvents used for compound dilution.b Larval mortality corrected according to Abbott’s formula.23

c Means (±SE) within a column followed by the same letters are not significantly different from the control at P < 0.05 (ANOVA followed by Tukey’stest).d ne = not evaluated because live larvae were too small to be weighed.e Larval weight as percentage of control.

3.1.3 Compounds identified from the SD-EMR layerGallic acid (13; C7H6O5). Casuarinin (14; C41H28

O26): amorphous brown powder; ESI/MS m/z935 [M − H]−; 1H NMR (400 MHz, acetone-d6/deuterium oxide): δ 7.13 (galloyl group; s); 6.88,6.58 and 6.53 (HHDP groups; s); glucose moiety:5.53 (anomeric proton; d; J 5.0 Hz); 4.58 (H-2; dd;J 2.0 and 5.0 Hz); 5.39 (H-3 and 4; m); 5.26 (H-5; dd; J 8.7 and 2.6 Hz); 4.80 (H-6a; dd; J 13.7and 3.8 Hz); 4.02 (H-6b; d; J 13.7 Hz); IR, UV and13C NMR data were similar to those reported in theliterature.28 Castalagin (15; C41H26O26): amorphousbrown powder; ESI/MS m/z 933 [M − H]−; 1H NMR(400 MHz, acetone-d6/deuterium oxide): δ 6.70, 6.69and 6.55 (HHDP groups; s); glucose moiety: 5.60(anomeric proton; d; J 4.6 Hz); 4.94 (H-2, 3 and 6a;m); 5.12 (H-4; t; J 7.1 Hz); 5.49 (H-5; brd; J 6.5 Hz);3.95 (H-6b; d; J 12.6 Hz); 13C NMR data were similarto those reported in the literature.28 Syringic acid(16; C9H10O5): amorphous white powder; ESI/MSm/z 197 [M − H]−; 1H NMR (200 MHz, acetone-d6): δ 7.25 (H-2 and 6; s); 3.61 (OCH3-3 and5; s); 13C NMR and UV data were similar tothose reported in the literature.28 Ellagic acid 4-O-α-L-rhamnopyranoside (17; C20H16O12): pale yellowpowder; ESI/MS m/z 447 [M − H]−; 1H NMR(400 MHz, CD3OD): δ 7.84 (H-5; s); 7.49 (H-5′;s); 5.52 (H-1′′; d; J 1.6 Hz); 4.20 (H-2′′; dd; J1.6 and 3.4 Hz); 4.02 (H-3′′; dd; J 9.5 and 3.4 Hz);3.48 (H-4′′; t; J 9.5 Hz); 3.77 (H-5′′; dd; J 9.5and 6.2 Hz); 1.27 (H-6′′; d; J 6.2 Hz); IR, UV and13C NMR data were similar to those reported in theliterature.28 Ellagic acid (18; C14H6O8): amorphouspale yellow solid; ESI/MS m/z 301 [M − H]−; 1HNMR (200 MHz, CD3OD): δ 7.47 (H-5 and 5′; s);13C NMR and IR data were similar to those reported inthe literature.28 Daucosterol (19; C35H60O6); ellagic

acid 4-O-α-L-2′′-O-acetylrhamnopyranoside (20) andellagic acid 4-O-α-L-3′′-O-acetylrhamnopyranoside(21; C22H18O13); siphoneugenin (22; C21H22O14); 3,4′-di-O-methylellagic acid 4-O-β-D-6′′-O-acetylglu-copyranoside (23; C24H22O14) and 3,4′-di-O-methyl-ellagic acid 4-O-β-D-3′′, 6′′-di-O-acetylglucopyrano-side (24; C26H24O15).

3.1.4 Compounds identified in the SD-EML layerGallic acid (13); casuarinin (14); syringic acid (16).Quercetin (25; C15H10O7): yellow powder; ESI/MSm/z 301 [M − H]−; 1H NMR (200 MHz, acetone-d6):δ 12.17 (OH; s); 7.82 (H-2′; d; J 2.0 Hz); 7.70 (H-6′;dd; J 8.0 and 2.0 Hz); 7.00 (H-5′; d; J 8.0 Hz); 6.52(H-8; d; J 2.0 Hz); 6.26 (H-6; d; J 2.0 Hz); UV and13C NMR data were similar to those reported in theliterature.28 Quercetin-3-O-α-L-rhamnopyranoside orquercitrin (26; C21H20O11): yellow solid; ESI/MS m/z447 [M − H]−; 1H NMR (400 MHz, CD3OD): δ 7.33(H-2′; d; J 2.1 Hz); 7.31 (H-6′; dd; J 8.3 and 2.1 Hz);6.91 (H-5′; d; J 8.3 Hz); 6.36 (H-8; d; J 2.1 Hz);6.19 (H-6; d; J 2.1 Hz); 5.34 (H-1′′; d; J 1.5 Hz);4.21 (H-2′′; dd; J 3.3 and 1.5 Hz); 3.75 (H-3′′; dd; J9.3 and 3.3 Hz); 3.41 (H-5′′; m); 3.35 (H-4′′; d; J 9.3Hz); 0.93 (H-6′′; d; J 6.1 Hz); 13C NMR data weresimilar to those reported in the literature.28 Quercetin-3-O-α-L-arabinopyranoside or guiajaverin (27) andquercetin-3-O-β-D-xylopyranoside or reynoutrin (28;C20H18O11); chebuloside II (29; C36H58O11) and28-β-D-glucopyranosyl-6β-hydroxymaslinate (30; C36

H58O10).

3.1.5 Compounds identified in the SD-DML layerα-Amyrin (31), β-amyrin (32) and lupeol (33;C30H50O); terminolic (34) and madecassic acids (35;C30H48O6); asiatic acid (36; C36H58O11).

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3.1.6 Compounds identified in the SD-DMS layerGallic (13) and arjunolic acids (37; C30H48O5); vanillicacid (38; C8H8O4); 2,4,6-trimethoxybenzoic acid(39; C10H12O5); 2,4,6-trimethoxybenzaldehyde (40;C10H12O4) and trans-2,4,6-trimethoxyphenylpropen-aldehyde (41; C12H14O4); 5-hydroxymethyl-2-furfur-aldehyde (42; C6H6O3).

3.1.7 Compounds identified in the SD-BMS layerCasuarinin (14). β-Pedunculagin (43; C34H24O22):amorphous off-white solid; ESI/MS m/z 783 [M −H]−; 1H NMR (200 MHz, acetone-d6): δ 6.71 and6.56 (HHDP group; brs); 5.53 (anomeric proton; d;J 6.9 Hz); IR, UV and 13C NMR data were similar tothose reported in the literature.29

3.1.8 Compounds identified in the SD-EMS layerGallic acid (13); casuarinin (14); castalagin (15);ellagic acid 4-O-α-L-rhamnopyranoside (17).

3.2 Larval mortalityVery large differences in larval stage mortality, rangingfrom zero to 100% of the exposed larvae, resultedfrom treatments with different extracts (Table 2).S. densiflora polar extracts (SD-ML, SD-HAL, SD-MS, SD-HAS, SD-MT, SD-HAT, SD-MR, SD-HAR) were responsible for the highest mortalities(100%), followed by VP-HAL (60%), VP-HF (90%)and VP-HAF (100%) extracts from Vitex polygama,taking into account the treatment period (Table 2).They were then considered promising sources of activesecondary metabolites and were further submitted toliquid–liquid partition.

Partition of VP-HAL produced a VP-BHAL fractionwith increased activity (100%), whereas the resultantfractions in the SD-MR partition (SD-EMR and SD-AMR respectively) retained the same potent activityas the original extract (100%, Table 2).

The VP-MT extract (Table 2) was also fractionated,in spite of its lack of activity, on the basis of literaturereports that a similar extract of Vitex madiensiscaused death by disruption of the moulting cycle inS. frugiperda.30 Ecdysteroids 9 to 12 (Fig. 1), knownfor their various effects on insects,31 were obtained inthe fractionation of this extract.

Flavonoid 7, isolated from the VP-BHAL layer,is reported in the literature32 to have an insecticidaleffect, but it was not isolated in sufficient amountsto be bioassayed. The flavonoids quercetin (25)and quercitrin (26) were highly active substancescausing larval mortality of 78 and 85% (Table 3)respectively after 10 days of treatment. This resultcould be explained by earlier reports indicatingthat quercetin (25) may be a potent inhibitorof mitochondrial ATPase,33 of cytochrome P450-dependent mixed function oxidases (MFO)34 and ofmidgut glutathione S-transferases from S. frugiperda.35

In addition, quercitrin (26), a flavonol glycoside,may be hydrolysed when ingested by some insects,so releasing its aglycone, quercetin.32 The other

compounds tested (Fig. 1) showed poor results,with a maximum larval mortality of 26%, a valuestatistically not significantly different (P < 0.05) fromthe control (Table 3). Compound 17, when layeredon the top of the diet, became dark-brownish after afew hours, probably indicating oxidation and loss ofactivity.

3.3 Developmental effectsIn general, no statistically significant differences inpupation time and emergence were observed in thetests with extracts and fractions, except for two, VP-HL and VP-HF, which showed significantly extendedlarval periods of 6 and 15 days respectively abovethose of the controls (Table 2). Moreover, VP-HLcaused 10% of malformed pupae, 20% of uncompletedsecond-instar larvae and 30% of pupae that did notreach adult stage.

Compound 9, originating from an inactive extract(VP-MT, Table 2), caused a moderate but statis-tically significant larval weight loss in relation tothe control after 10 days of treatment (P < 0.05;Table 3). The effects of ecdysteroids are knownto be surprisingly complex and dependent on theconcentration of compound and the developmen-tal stage of the insect,31 which could explain thatresult.

Although the bioassay used does not addressbehavioral responses (inhibition of feeding), theresults suggest that larval weight loss caused bycompounds 14 and 43 (Table 3) may be correlatedwith feeding deterrence. In fact, it is known thattannins reduce plant palatability and act as inhibitorsof digestion by precipitating food protein and digestiveenzymes.36 In addition, there is evidence that somespecies of Lepidoptera have taste receptors thatrespond to some phenolics.32 However, tannins 15and 17 were practically inactive in this respect,probably because some tannins may be oxidized37 orhydrolyzed during passage through the insect gut,36

reducing their effectiveness. According to Urrea-Bulla et al.,38 gallic acid (13), the compound isolatedfrom several extracts of S. densiflora and also aprobable product of hydrolysis of compound 14,significantly reduces larval growth of S. frugiperda at1 mg g−1.

4 CONCLUSIONSPolar extracts from S. densiflora and the VP-BHALlayer displayed considerable insecticidal activity, andmay be efficient alternatives to conventional syntheticinsecticides in the integrated pest management ofS. frugiperda. Moreover, the feeding deterrence causedby tannin 14 along with the insecticidal effects offlavonoids 25 and 26 might explain the 100% outcomewhen the SD-ML extract was tested. In addition,compound 14 might be acting synergistically with43, and in concert with daucosterol (19), known forits insecticidal action,15 phenolic acid 13 and other

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MBC Gallo et al.

compound(s) not tested or characterized, emphasizingthe excellent results obtained with extracts SD-MSand SD-MR respectively.

ACKNOWLEDGEMENTSThe authors are grateful to Dr Fatima R Salimena-Pires (Universidade Federal de Juiz de Fora – UFJF)and Marcos Sobral (Universidade Federal de MinasGerais – UFMG) for botanical identifications, andto ALCOA Alumınio S/A for facilitating plantcollections. This study has been financially supportedby Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior (CAPES), Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq)and Fundacao de Amparo a Pesquisa do Estado deSao Paulo (FAPESP).

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