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Pest Management Science Pest Manag Sci 63:000–000 (2007)

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Compounds from Ageratum conyzoides:isolation, structural elucidation andinsecticidal activityMarcio D Moreira,1 Marcelo C Picanco,1∗ Luiz Claudio A Barbosa,2

Raul Narciso C Guedes,1 Emerson C Barros1 and Mateus R Campos1

1Departamento de Biologia Animal, Universidade Federal de Vicosa, Vicosa, MG 36571-000, Brazil2Departamento de Quımica, Universidade Federal de Vicosa, Vicosa, MG 36571-000, Brazil

Abstract: This work aimed at identifying plant compounds with insecticidal activity against Diaphania hyalinata(L.) (Lepidoptera: Pyralidae), Musca domestica (L.) (Diptera: Muscidae), Periplaneta americana (L.) (Blattodea:Blattidae) and Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). The plant species used were: basil (Ocimumselloi Benth.), rue (Ruta graveolens L.), lion’s ear (Leonotis nepetaefolia L.), Jimson weed (Datura stramoniumL.), ‘baleeira’ herb (Cordia verbenaceae L.), mint (Mentha piperita L.), wild balsam apple (Mormodica charantiaL.) and billy goat weed (Ageratum conyzoides L.). Firstly, the insecticidal activities of hexane and ethanol plantextracts were evaluated against adults of R. dominica. Among them, only the hexane extract of A. conyzoidesshowed insecticidal activity. The hexane extract of this plant species was therefore fractionated by silica gel columnchromatography to isolate and purify its bioactive chemical constituents. Three compounds were identified usingIR spectra, 1H NMR, 13C NMR, HMBC and NOE after gel chromatography: 5,6,7,8,3′, 4′, 5′-heptamethoxyflavone,5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone and coumarin. The complete assignment of 13C NMRto 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone was successfully made for the first time. 5,6,7,8,3′-Pentamethoxy-4′, 5′-methylenedioxyflavone did not show any insecticidal activity against the four insect speciestested. 5,6,7,8,3′, 4′, 5′-Heptamethoxyflavone showed low activity against D. hyalinata and R. dominica and wasnot toxic to M. domestica or P. americana. In contrast, coumarin showed insecticidal activity against all fourinsect pest species tested, with the following order of susceptibility: R. dominica < P. americana < D. hyalinata <

M. domestica after 24 h exposure. 2007 Society of Chemical Industry

Keywords: botanical pesticide; insect control; secondary metabolites; bioactive compounds; Ageratum conyzoides

1 INTRODUCTIONAlthough recent innovations such as combinatorialchemistry enable 100 000 new chemicals per year tobe screened compared with 15 000 previously, newcompounds brought to the market are still decreasingat a rate of one per year.1 In addition, the highcost of development, $100–200 million per product,2

emphasizes the need for new tools of pest control.Tropical plants are recognized sources of bioac-

tive compounds, and less than 1% have beenchemically investigated.3 They can be used forpest control as plant extracts, horticultural oils(as either commercial or semi-commercial prod-ucts) or as a source of molecules for pes-ticide synthesis (e.g. pyrethroids and neonicoti-noids).

Ageratum conyzoides L. is an erect, herbaceousannual plant from the family Asteraceae (Compositae),native to tropical America, but with a distributionrange in tropical and subtropical areas around theworld.4 In spite of several studies on the bioactivityof A. conyzoides, only a few of these have isolated

and assessed the insecticidal activity of compoundsfrom this plant, and these efforts have focusedon human health pests and not crop and urbanpests. Since such studies were carried out witheither the crude extract or the essential oil of A.conyzoides, the potential use of this plant species forpest management as an insecticide requires furtherinvestigation.

The melonworm [Diaphania hyalinata (L.) (Lepido-ptera: Pyralidae)], the American cockroach [Peri-planeta americana (L.) (Blattodea: Blattidae)], thehouse fly [Musca domestica (L.) (Diptera: Muscidae)]and the lesser grain borer [Rhyzopertha dominica(F.) (Coleoptera: Bostrichidae)] represent four majororders of insect species of economic importance.Diaphania hyalinata is a key pest of Cucurbitaceae thatis found in most countries of the Americas.5,6 Rhyzop-ertha dominica is a cosmopolitan key pest of storedgrains, and M. domestica and P. americana are impor-tant household pests throughout the world.7,8 Theeconomic importance of these four insect pest species,

∗ Correspondence to: Marcelo C Picanco, Departamento de Biologia Animal, Universidade Federal de Vicosa, Vicosa, MG 3671-000, BrazilE-mail: picanco@ufv.br(Received 20 January 2006; revised version received 8 November 2006; accepted 30 November 2006)DOI: 10.1002/ps.1376

2007 Society of Chemical Industry. Pest Manag Sci 1526–498X/2007/$30.00

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aided by their easy maintenance and handling, makethem useful models for insecticide bioactivity assays.

Considering the potential of plant species as a sourceof potential insecticides and the importance of theseinsect pests, the present work aimed at recognizing theinsecticidal activity in extracts of eight plant speciesand at isolating and identifying the main bioactivecompounds present in these extracts.

2 MATERIALS AND METHODS2.1 Insect species and plant materialToxicity bioassays were carried out with first-instarlarvae of D. hyalinata, first-instar nymphs of P.americana, last-instar larvae of M. domestica and adultsof R. dominica. Rhyzopertha dominica was reared onwheat. Diaphania hyalinata, P. americana and M.domestica were reared as described elsewhere.6,8,9

The following plants were subject to extraction andtoxicity bioassays: basil (Ocimum selloi Benth.), rue(Ruta graveolens L.), lion’s ear (Leonotis nepetaefoliaL.), Jimson weed (Datura stramonium L.), ‘baleeira’herb (Cordia verbenaceae L.), mint (Mentha piperitaL.), wild balsam apple (Mormodica charantia L.) andbilly goat weed (Ageratum conyzoides L.).

Samples (500 g) of the canopy of each plant specieswere collected within the campus of the FederalUniversity of Vicosa, state of Minas Gerais, Brazil,where these plants are permanently cultivated. Eachsample was placed in a 1 L Erlenmeyer flask withenough hexane to submerge the plant material. Thesolvent was removed under filtration after 48 h.Ethanol extraction was carried out by grinding thesamples with the solvent and waiting for 48 h. Thehexane and ethanol extracts were concentrated underlow pressure and reduced temperature (<50 ◦C) andstored at low temperature for subsequent bioassays.

Ageratum conyzoides was selected for further extrac-tion with hexane and fractionation and structuralelucidation of its bioactive compounds. A total of5.31 kg of leaves of A. conyzoides was used for this pur-pose. The solvent (hexane) was changed at intervalsof 2 days for 45 days. The extraction continued untilthe solvent was colourless.

2.2 Gel chromatography and structuralelucidationThe hexane extract of A. conyzoides was concentratedunder low pressure and reduced temperature (<50 ◦C)and subjected to open column chromatography withsilica gel 60 (70–230 mesh). Two columns wereused in open column chromatography. The firstcolumn was eluted with pure hexane, hexane +diethyl ether (100 + 0.5 by volume), hexane + diethylether (50 + 50 by volume), pure diethyl ether andpure methanol. The second column was eluted withhexane + diethyl ether (100 + 10 by volume) andpure methanol. Seven fractions were obtained in thefirst column and four fractions in the second column.Thin-layer chromatography (TLC, silica gel 60 F254,

0.25 mm) spots were detected under UV (254 and365 nm) and heating the plates to 100 ◦C afterspraying with phosphomolybdic acid/ethyl alcohol.The IR spectra were recorded on potassium bromidein an infrared spectrometer (Paragon 1000 FTIR;Perkin Elmer, Wellesley, MA, USA) from 600 to4000 cm−1. The melting points (mp) were determinedin MQAPF-301 apparatus (MicroQuımica Equip.Ltda, Palhoca, SC, Brazil). 1H or 13C NMR spectra,recorded in either a Bruker WM 400 (Bruker OpticsInc., Billerica, MA, USA) or a Varian Mercury 300(Varian Inc., Palo Alto, CA, USA) spectrometer usingdeuterochloroform as solvent and tetramethyl silaneas internal standard, were used to identify the isolatedcompounds. The NMR assignments were made by 1HNMR, 13C NMR, HMBC and NOE.10

2.3 Toxicity bioassaysThree sets of bioassays were carried out. The first wasa set of screening bioassays to recognize the bioactiveplant extracts using adults of R. dominica. The secondset of bioassays aimed to select the most bioactivecompounds of the most active plant extract forsubsequent evaluation, using four insect pest speciesfor assessing insecticide activity – the melonwormDiaphania hyalinata, the house fly Musca domestica,the American cockroach Periplaneta americana andagain the lesser grain borer R. dominica. In thethird set of bioassays, the relative toxicity of theselected compounds was assessed by establishingdose–mortality curves for the same four insect pestspecies used in the second set of bioassays.

2.3.1 Bioassays with crude plant extractsThe stored extracts were diluted with either ethanol orhexane as solvent to a concentration of 20 mg mL−1.Three replicates were used in this bioassay. Eachreplicate encompassed a petri dish (9 cm diameter)with a filter paper impregnated with 1 mL of the testextract. The controls were treated with solvent only(either ethanol or hexane). Ten adults of R. dominicawere placed in each petri dish, which was maintainedat 25 ± 0.5 ◦C, 75 ± 5% RH and 12 h photophase, andinsect mortality was assessed after 4 and 24 h exposureto the impregnated filter paper. Mortality data weresubjected to analysis of variance, and the averages werecompared by the Scott–Knott groupment analysis test(P < 0.05).

2.3.2 Bioassays for selection of bioactive compoundsIn this second bioassay, the compounds 5,6,7,8,3′, 4′, 5′-heptamethoxyflavone, coumarin and 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone,which were identified in fractionation of the mostactive plant extract, were topically applied at a dose of10 mg g−1 fresh body mass to the four insect species.Three replicates were used in this bioassay. Each repli-cate encompassed a petri dish (9 cm diameter) with teninsects maintained at 25 ± 0.5 ◦C, 75 ± 5% RH and12 h photophase. The control was treated with the

2 Pest Manag Sci 63:000–000 (2007)DOI: 10.1002/ps

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same amount of solvent. Insect mortality was assessedat 6, 12, 24 and 48 h after treatment. Mortality datawere subjected to analysis of variance, and the aver-ages were compared by the Scott–Knott groupmentanalysis test (P < 0.05).

2.3.3 Dose–mortality curvesThe third set of bioassays was carried out with thefour insect pest species used in the previous setof bioassays to determine the potential insecticidalactivity of the compounds obtained from the mostbioactive plant extract. Four replicates were usedin this bioassay. Each replicate encompassed a petridish (9 cm diameter) with ten insects maintained at25 ± 0.5 ◦C, 75 ± 5% RH and 12 h photophase. Theinsects were submitted to 5–8 increasing doses of thecompound (plus a control with the application of thesolvent only). Insect mortality was evaluated 6, 12 and24 h after topical exposure. The data were correctedfor control mortality11 and probit analysis was carriedout.12

2.3.4 Bioassay of positive controlThis bioassay was carried out in order to compareresults from commercial (synthetic) coumarin and the(natural) coumarin obtained from A. conyzoides. Theobjectives were to confirm the activity of coumarinand to exclude interference by contaminants fromchromatography. The methodology was as statedin Section 2.3.2, but using commercial coumarin(synthetic; technical grade; 98% pure; Sigma-AldrichQuımica Brasil, Sao Paulo, Brazil). The dose causing90% mortality (LD90) against R. dominica and P.americana, estimated from the dose–mortality curve,was used in this bioassay.

3 RESULTS3.1 Bioactivity of plant extractsOnly the hexane extract from leaves of A. conyzoidesshowed insecticidal activity, causing 76.0 ± 2.35%(n = 30) and 88.7 ± 1.67% (n = 30) mean mortality± SEM in adult R. dominica after 4 and 24 h exposurerespectively. The mortality caused by the solvents wasalways negligible (<5%) in all sets of bioassays. Theethanol extract of A. conyzoides and the hexane andethanol extracts from R. graveolens, L. nepetaefolia,C. verbenacea, D. estramonium, M. charantia, O. selloiand M. piperita did not show any insecticidal activityagainst R. dominica.

3.2 Isolation and structural elucidationExtraction of 5.31 kg of A. conyzoides leaves withhexane yielded 86.13 g of crude extract. The crudeextract, when stored at low temperature, generatedan oily portion (77.37 g) and a crystallized portion(8.76 g). Compound 1 was isolated from the oilyportion using the first column in the open columnchromatography. This compound was collected in thesecond fraction. Compounds 2 and 3 were isolated

from the crystallized portion in the second and fourthfraction respectively using the second column in theopen column chromatography.

Compound 1, 5,6,7,8,3′, 4′, 5′-heptamethoxy-flavone (Fig. 1), was isolated as a yellow solid(0.8 g) with mp 115.3–116.9 ◦C. Its IR spectrumwas typical of non-phenolic flavones:13 2992, 2941and 2838 cm−1 (ν C–H3), 2890 cm−1 (νs C–H3),1643 cm−1 (ν C=O), 1589, 1570 and 1551 cm−1 (νs

C=C arom.) and 1247 and 1040 cm−1 (νas C–O–C).The 13C NMR spectrum gave rise to 22 carbon signals.Nine carbon signals were typical of the A and B ringof flavones: δ 161.00 (C-2), 107.86 (C-3), 177.56(C-4), 148.49 (C-5), 144.25 (C-6), 151.61 (C-7),138.12 (C-8), 147.75 (C-9) and 114.93 (C-10). Sixcarbon signals were typical of the C ring: δ 125.98(C-1′), 100.51 (C-2′), 149.66 (C-3′), 138.42 (C-4′),143.99 (C-5′) and 106.61 (C-6′). The signals of sevenmethoxy groups were present from δ 56 to 63. The1H NMR spectrum showed singlets at δ 6.63 (H-3),7.15 (H-2′ and H-6′) and 3.92, 3.94, 4.01 and 4.10respectively to 3H, 12H, 3H and 3H of the methoxygroups. The assignments of 13C NMR and 1H NMRsignals are in agreement with the literature.14,15

Compound 2, coumarin (Fig. 1), has a white needle-like appearance (5.28 g) with mp 66.7–68.9 ◦C. Its IRspectrum showed characteristic absorption bands at3045 cm−1 (νas C–H), 1706 cm−1 (ν C=O), 1619,1605 and 1562 cm−1 (ν C=C arom.) and 1259and 1229 cm−1 (νs C–O). The 13C NMR spectrumshowed characteristic carbon signals from δ 160.52(C=O), 116.5 (C=3), 143.3 (C=4), 153.71 (C-9)and 118.56 (C-10). The 1H NMR spectrum showedtwo doublets from δ 5.59 and 6.90 (J 9.6 Hz) oftwo proton cis-olefinics of the coumarinic ring. The13C NMR spectrum was consistent with the publisheddata.16

Compound 3, 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone (Fig. 1), was isolated as arose-blue solid (0.46 g), with mp 185–188.9 ◦C. ItsIR spectrum was 3078 cm−1 (ν C–H arom.), 2970and 2942 cm−1 (νas C–H3), 2890 cm−1 (νs C–H3),1631 cm−1 (ν C=O), 1584, 1560 and 1551 cm1

(ν C=C arom.), 1448 cm−1 (δas C–H3), 1370 cm−1

(δs C–H3), 1247 cm−1 (νas C–O–C) and 1040 cm−1

(νs C–O–C). The 13C NMR spectrum showed 21carbon signals, 15 of which were typical of flavones.The methylenedioxy group was observed at δ 102.45,and the signals from five methoxy groups were presentfrom δ 56.82 to 62.38. The other non-hydrogenated Cgave rise to δ 160.76 to 138.12. The complete assign-ment of 13C NMR was made for this compound forthe first time (Table 1). HMBC and NOE were usedin the assignments (Table 1). The 1H NMR spectrumshowed a singlet at δ 6.09 from methylenedioxy (H-16), and the singlet at δ 6.57 was from H-3. Thedoublets at δ 7.11 and 7.06 (J 1.7 Hz) were from 6′-Hand 2′-H. The methoxy groups (15 H) gave rise to δ

3.95 to 4.11. The assignments of 13C NMR and 1HNMR were confirmed by HMBC and NOE (Table 1).

Pest Manag Sci 63:000–000 (2007) 3DOI: 10.1002/ps

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Figure 1. Structures of compounds isolated from A. conyzoides:5,6,7,8,3′, 4′, 5′-heptamethoxyflavone (1), coumarin (2) and5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone (3).

The 1H NMR spectrum agrees with data previouslyreported by Quijano et al.17

3.3 Bioactivity of compounds isolated5,6,7,8,3′, 4′, 5′-Heptamethoxyflavone showed lowinsecticidal activity against D. hyalinata and R.dominica, but no activity was detected against M.domestica and P. americana at 48 h after topicalapplication (Table 2). Coumarin was toxic to M.domestica, P. americana, R. dominica and D. hyali-nata at 6, 12, 24 and 48 h after topical appli-cation. In contrast, 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone was not toxic to these insectpests.

Table 1. 13C NMR, 1H NMR, HMBC and NOE from

5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone

C atom 13C NMR 1H NMR HMBC C to H NOEa

2 160.76 3, 2′, 6′3 107.41 6.53 2′,∗ 6′∗4 177.37 35b 148.49 11, 36b 144.25 127 151.61 138 138.12 149 147.7510 114.93 311 62.38 3.9112 62.11 3.9213 61.78 4.07 1414 61.95 3.98 13, 2′+ 6′+15 56.82 3.9516 102.45 6.051′ 125.98 3, 2′, 6′2′ 100.51 7.06 (J 1.7 Hz) 6′ 3, 143′ 149.66 16, 2′, 3′4′ 138.42 16, 2′, 3′5′ 143.99 2′, 3′6′ 106.61 7.11 (J 1.7 Hz) 2′ 3, 14, 15

a∗ and + denote equal intensity.b This assignment can be inverted.

Dose–mortality curves for 5,6,7,8,3′, 4′, 5′-hepta-methoxyflavone and 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone against M. domestica, P. amer-icana, R. dominica and D. hyalinata were not obtainedowing to their low or lack of activity against thesespecies.

The toxicity of coumarin was higher in evaluationsat 24 h than at 6 and 12 h of exposure. The coumarindose–mortality curve for P. americana showed thesteepest slopes, suggesting a higher homogeneity ofresponse of this species to this compound comparedwith the other species (Table 3). The slopes obtainedfor R. dominica, D. hyalinata and M. domestica weregenerally smaller and similar to each other. Theincreasing order of susceptibility to coumarin was R.dominica < D. hyalinata < P. americana < M. domesticafor 12 h exposure and R. dominica < P. americana <

D. hyalinata < M. domestica for 24 h exposure.The mortality results obtained with the bioassays

of positive control using the same dose of thecommercial (synthetic) coumarin and the (natural)coumarin obtained from the hexane extract of A.conyzoides were very similar. The mortality of R.dominica was 92.00 ± 0.27% and 96.00 ± 0.18% tosynthetic and natural coumarin respectively after 24 hexposure. The mortality of nymphs of P. americanawas 95.00 ± 2.13% and 94.50 ± 1.97% to syntheticand natural coumarin respectively after 24 h exposure.

4 DISCUSSIONThe only plant species showing insecticidal activity inthe present study was the billy goat weed A. conyzoides,

4 Pest Manag Sci 63:000–000 (2007)DOI: 10.1002/ps

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Table 2. Mortality of Rhyzopertha dominica, Diaphania hyalinata, Musca domestica and Periplaneta americana 6, 12, 24 and 48 h after topical

application of 10 mg g−1 of 5,6,7,8,3′, 4′, 5′-heptamethoxyflavone (1); coumarin (2) or 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone (3)

extracted from leaves of Ageratum conyzoides (only solvent was used in the control)

Mortality (%) (± SEM)a

Insect species Control 1 2 3

6 h after topical applicationM. domestica 0.00 bA 0.00 bA 23.33 (±3.33) aB 0.00 ± 0.00 bAP. americana 0.00 bA 0.00 bA 66.16 (±2.36) aA 0.00 ± 0.00 bAR. dominica 0.00 bA 3.33 (±3.33) bA 16.67 (±4.67) aC 0.00 ± 0.00 bAD. hyalinata 0.00 bA 6.67 (±6.67) bA 63.33 (±8.82) aA 0.00 ± 0.00 bA

12 h after topical applicationM. domestica 0.00 bA 0.00 bB 23.33 (±3.33) aC 0.00 bAP. americana 0.00 bA 0.00 bB 80.44 (±3.53) aA 0.00 bAR. dominica 0.00 bA 3.33 (±3.33) bB 23.33 (±8.82) aC 0.00 bAD. hyalinata 0.00 cA 13.33 (±6.67) bA 70.00 (±5.77) aB 0.00 cA

24 h after topical applicationM. domestica 0.00 bA 0.00 bB 26.67 (±6.67) aD 3.33 (±3.33) bAP. americana 0.00 bA 0.00 bB 81.37 (±0.37) aA 0.00 bAR. dominica 0.00 bA 6.67 (±3.33) bB 43.33 (±14.53) aC 0.00 bAD. hyalinata 0.00 cA 16.67 (±3.33) bA 70.00 (±7.40) aB 0.00 cA

(48 hours after topical application)M. domestica 0.00 bA 0.00 bB 33.33 (±3.33) aC 3.33 (±3.33) bAP. americana 0.00 bA 0.00 bB 83.01 (±2.56) aA 0.00 bAR. dominica 0.00 cA 13.33 (±6.67) bA 68.89 (±11.6) aB 0.00 cAD. hyalinata 0.00 cA 20.00 (±5.77) bA 68.18 (±7.40) aB 0.00 cA

a Means followed by the same lower-case letter in a row or by the same upper-case letter in a column are not significantly different by the Scott–Knottgroupment analysis test at P < 0.05.

Table 3. Toxicity of coumarin extracted from leaves of Ageratum conyzoides against Rhyzopertha dominica, Diaphania hyalinata, Musca domestica

and Periplaneta americana

Insect species Slope (± SE) LD50 (mg g−1) (95% FL) LD90 (mg g−1) (95% FL) χ2 Probability

6 h after topical applicationR. dominica 2.76 (±0.25) 39.72 (35.90–45.00) 115.19 (88.89–173.90) 6.27 0.09

12 h after topical applicationR. dominica 2.87 (±0.18) 20.82 (18.70–23.08) 58.04 (49.40–71.65) 3.17 0.37D. hyalinata 2.65 (±0.07) 3.80 (3.27–4.34) 11.54 (9.95–13.78) 0.75 0.86M. domestica 1.62 (±0.20) 2.28 (1.73–3.44) 14.07 (7.81–36.00) 2.97 0.60P. americana 3.19 (±0.27) 3.05 (3.16–3.96) 8.82 (7.07–12.33) 0.85 0.66

24 h after topical applicationR. dominica 2.37 (±0.26) 11.82 (10.07–13.59) 42.94 (34.67–50.30) 6.93 0.07D. hyalinata 2.14 (±0.09) 2.21 (1.75–2.64) 8.72 (7.31–10.92) 0.52 0.92M. domestica 2.60 (±0.10) 1.18 (0.98–1.52) 3.67 (2.56–6.29) 1.18 0.76P. americana 3.38 (±0.07) 2.49 (2.29–2.70) 5.15 (4.54–6.08) 1.89 0.61

and such activity was observed only in the hexaneextract, not in the ethanol extract. The insecticidalactivity of A. conyzoides extract has previously beenreported against Culex quinquefasciatus Say (Diptera:Culicidae).18 Fagoonee and Umrit19 observed thepresence of precocenes I and II in the acetone-diethylether extract of A. conyzoides and reported ovarianinhibition in Dysdercus flavidus Signoret (Heteroptera:Pyrrhocoridae). However, the insecticidal activity ofthe hexane extract against R. dominica is not due to theprecocenes, since these compounds have antijuvenilehormone activity interfering with insect moulting andreproduction, while the insecticide mortality observedin the present study was acute and quickly apparent

(within 24 h), unlike what would be expected withprecocenes.

Three compounds were identified in the bioac-tive fractions of the hexane extract of A. conyzoides:two flavonoids (5,6,7,8,3′, 4′, 5′-heptamethoxyflavoneand 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxy-flavone) and coumarin. Flavonoids are a major classof phytochemicals found in plants, with beneficialaction to humans, including antimicrobial, pharma-cological and antioxidant activity, besides adverselyaffecting insect pests.20 These compounds have beenmainly reported as antifeedant and growth inhibitorsin insects, probably because of their interference withendocrine regulation.20 Maysin, a C-glycosil flavone,

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MD Moreira et al.

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has been implicated in maize resistance to the cornearworm, Helicoverpa zea (Boddie) (Lepidoptera: Noc-tuidae), and in this case is thought to interferewith amino acid metabolism in the insect gut.21

The flavone meliternantin (3,5-dimethoxy-3′, 4′,6,7-bismethylenedioxyflavone) is a feeding deterrent thatreduces the growth rate and food consumption rate,and inhibits 50% of feeding by Sitophilus zeamaisMotsch. (Coleoptera: Curculionidae) at 125 ppm.22

Flavonoids possess a catecholic B-ring responsiblefor their toxic activity to insects.23 The activityvaries according to the chemical structure of thesecompounds.24 Thus, 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone did not show toxicity, while5,6,7,8,3′, 4′, 5′-heptamethoxyflavone was toxic to R.dominica and D. hyalinata. The structural diversity ofthis group emphasizes the need for structure–activitystudies to guide the search for higher toxicity to insects.The diversity in mode of action and the range of effectsof these compounds against insects are likely to slowdown the evolution of resistance to them.

The results with the commercial (synthetic)coumarin closely resembled the effect of the (natural)coumarin extracted from A. conyzoides, confirming itsactivity against the tested insect pest species, which washigher than the activity of the two flavonoids obtainedin the bioactive fractions of the hexane extract of thisplant species. The difference in coumarin toxicity to D.hyalinata, M. domestica, P. americana and R. dominicais due to the differential susceptibility of these species.Such variation in insecticidal activity against differentinsect species is frequent and it was expected.

The active compound ryanodine, extracted fromplants of the genus Ryania, has an LD50 of 0.39 µg g−1

on adults of M. domestica and several of its chemicalanalogues have LD50 from 0.11 to 100 µg g−1.25

Coumarin was less toxic to R. dominica (11.82 µg g−1)than deltamethrin (0.3 ng g−1),26 but the authorswere not able to find any information about thetoxicity of coumarin and related compounds toD. hyalinata. Although coumarin and its relatedcompounds are not used as insecticides, their lowertoxicity to humans27 and significant insecticidalactivity, capable of enhancement through quantitativestructure–activity relationship studies, make thempotential insect control agents for pest management.

There is a lack of studies on the mode of action ofcoumarins, but surangin• B has been subjected to such

AQ1

investigation. The coumarin surangin B is an inhibitorof mitochondrial electron transport, probably target-ing cytochrome c oxidoreductase (complex III) andcytochrome b, leading to a reduction in ATP synthe-sis and bioenergetic muscle disruption.28 Respirationin the cricket Acheta domestica L. (Orthoptera: Gryl-lidae) and blowfly Phaenicia sericata Meig (Diptera:Calliphoridae) flight muscle mitochondria are blockedby surangin B.28

Coumarins and mainly furanocoumarins inhibitthe cytochrome P450 detoxication enzymes, disrupt-ing the insect detoxication capability.29,30 Coumarins

have a reversible inhibition of cytochrome P450,and furanocoumarins show reversible or irreversibleinhibition.30 Letteron et al.29 suggested that thecoumarin xanthotoxin is metabolically activated onthe outer double bond on the furan ring to form anextremely unstable radicaloid species. Cytochrome-P450-mediated radicaloid formation requires oxida-tion of xanthotoxin. The unstable radicaloid may bindcovalently to the active site of the P450 or be releasedfrom the active site.

Other compounds with potential pesticidal activityare also likely to be present in A. conyzoides and theycertainly deserve attention, as does coumarin as apotential pest management tool.

5 CONCLUSIONThe present work identified three compounds in thebioactive fractions of the hexane extract from leaves ofAgeratum conyzoides; hexane and ethanol extracts fromseven other plant species did not show any insecticidalactivity. Among these three compounds from A.conyzoides, identified as 5,6,7,8,3′-pentamethoxy-4′, 5′-methylenedioxyflavone, 5,6,7,8,3′, 4′, 5′-hepta-methoxyflavone and coumarin, only the last twoshowed insecticidal activity. 5,6,7,8,3′, 4′, 5′-Hepta-methoxyflavone was less active and showed insecticidalactivity only against D. hyalinata and R. dominica.In contrast, coumarin was active against all fourinsect pest species tested. The increasing order ofsusceptibility to coumarin was R. dominica < D.hyalinata < P. americana < M. domestica at 12 h andR. dominica < P. americana < D. hyalinata < M.domestica at 24 h. Therefore, coumarin is a potentialpest management tool likely to have its insecticidalactivity improved through organic synthesis guided byquantitative structure–activity relationship studies.

ACKNOWLEDGEMENTSThe authors are grateful to the National Council ofScientific and Technological Development (CNPq)and the Minas Gerais State Foundation for ResearchAid (FAPEMIG) for scholarships and grants received.

REFERENCES1 Tait J, AgBioForum 4:36–67 (2001).2 Grapov A, Novel insecticides and acaricides.• Rus Chem Rev

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68:697–707 (1999).3 Balick MJ, Elisabetsky E and Laird SA, Medicinal Resources of the

Tropical Forest: Biodiversity and its Importance to Human Health.Columbia University Press, New York, 440 pp.• (1995).

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4 Okunade AL, Ageratum conyzoides L. (Asteraceae). Fitoterapia73:1–16 (2002).

5 Boyhan GE and Brown JE, Reflective mulch reduces infestationof melonworm in squash. J Veg Crop Prod 4:17–22 (1998).

6 Mendes ACB and Berti-Filho E, Biologia da broca das cucur-bitaceas Diaphania nitidalis (Cramer, 1781) (Lepidoptera:Pyralidae). An Soc Entomol Bras 10:141–146 (1981).

7 Guedes RN, Kambhampati S and Dover SK, Allozyme varia-tion among Brazilian and U.S. populations of Rhyzopertha

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dominica resistant to insecticides. Entomol Exp Appl 84:49–57(1997).

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11 Abbott WS, A method of computing the effectiveness of aninsecticide. J Econ Entomol 18:265–267 (1925).

12 Finney DJ, Probit Analysis, 3rd edition. Cambridge UniversityPress, London, 256 pp.• (1971).

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13 Mabry TJ, Markham KR and Thomas MB, The SystematicIdentification of Flavonoids. Springer, New York, 354 pp.•AQ7

(1970).14 Horie T, Ohtsuru Y, Shibata K, Yamashita K, Tsukayama M

and Kawamura Y. 13C NMR spectral assignment of the A-ring of polyoxygenated flavones. Phytochemistry 47:865–874(1998).

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16 Breitmaier E and Voelter W, Carbon 13 NMRSpectroscopy – High Resolution Methods and Applicaions inOrganic Chemistry and Biochemistry. VCH Publishers, NewYork, 515 pp.• (1990).

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17 Quijano L, Calderon JS, Gomez F, Soria IE and Rios T,Highly oxygenated flavonoids from Ageratum corymbosum.Phytochemistry 19:2439–2442 (1980).

18 Saxena RC, Dixit OP and Sukumaran P, Laboratory assessmentof indigenous plant extracts for antijuvenile hormone activityin Culex quinquefasciatum. Indian J Med Res 95:204–206(1992).

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22 Ho SH, Wang J, Sim KY, Ee GCL, Imiyabir Z, Yap KF, et al,Meliternatin: a feeding deterrent and larvicidal polyoxy-genated flavone from Melicope subunifoliolata. Phytochemistry62:1121–1124 (2003).

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24 Larsson S, Lundgren L, Ohmart CP and Gref R, Weakresponses of pine sawfly larvae to high needle flavonoid con-centrations in scots pine. J Chem Ecol 18:271–282 (1992).

25 Jefferies PR, Yu P and Casida JE. Structural modificationsincrease the insecticidal activity of ryanodine. Pestic Sci51:33–38 (1997).

26 Lorini I and Galley DJ. Relative effectiveness of topical, filterpaper and grain application of deltamethrin, and associatedbehaviour of Rhyzoperha dominica (F.) strains. J Stored ProdRes 34:377–383 (1998).

27 Lake BG, Coumarin metabolism, toxicity and carcinogenicity:relevance for human risk assessment. Food Chem Toxicol37:423–453 (1999).

28 Zheng J, Leong D, Lees G and Nicholson A, Studies on theinteraction of surangin B with insect mitochondria, insectsynaptosomes, and rat cortical neurons in primary culture.Pestic Biochem Physiol 61:1–13 (1998).

29 Letteron P, Descatoir P, Larrey D, Tinel M, Geneve J andPessayre D, Inactivation and induction of cytochrome P450by various psoralens derivatives in rats. J Pharmacol Exp Ther238:685–692 (1986).

30 Neal JJ and Wu D, Inhibition of insect cytochromes P450 byfuranocoumarins. Pestic Biochem Physiol 50:43–50 (1994).

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