Pest Management Science
Pest Manag Sci 62:1072–1081 (2006)
Bioactivity of extracts and isolated compounds 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 ˜ B Fernandes,1 M Fatima ´ Joao das GF da Silva1 and Luciane G Batista-Pereira1 1 Departamento
˜ Carlos (UFSCar), CP 676, 13565-905, Sao ˜ Carlos-SP, Brazil de Qu´ımica, Universidade Federal de Sao ˜ de Entomologia, Fitopatologia e Zoologia Agr´ıcola, Escola Superior de Agronomia Luiz de Queiroz, Universidade de Sao Paulo (ESALQ/USP), CP 09, 13418-900, Piracicaba-SP, Brazil 2 Departamento
Abstract: The effects of crude extracts, fractions and isolated compounds from Vitex polygama Cham. and Siphoneugena densiflora Berg were evaluated on the development of Spodoptera frugiperda JE Smith, a destructive insect pest of corn and several other crops. The extracts and fractions were incorporated into an artificial diet at 1 mg g−1 and offered to the insect during its larval stage. Length and viability of larval and pupal stages as well as pupal weight were assessed. Isolated compounds were tested through superficial contamination of the diet at 0.1 mg g−1 . Weight and viability of ten-day-old larvae were determined. Methanolic and hydroalcoholic S. densiflora extracts caused 100% larval mortality, while leaf and fruit hydroalcoholic extracts from V. polygama were the most active. Among the isolated compounds, flavonoids presented the best insecticidal results, and tannins the best larval growth inhibition. 2006 Society of Chemical Industry
Keywords: Tarum˜a; Uvatinga; Hoja Menuda; fall armyworm; Vitex polygama; Siphoneugena densiflora; Spodoptera frugiperda; botanical insecticide; pest control
1 INTRODUCTION With a projected increase in world population to 10 billion over the next four decades, an immediate priority for agriculture is to achieve maximum production of food and other products in a manner that is environmentally sustainable and cost effective.1 One major limiting factor to global food production is damage by pests, during both growth and storage stages. The use of synthetic pesticides has been an effective way to control pests, but their efficacy is constantly weakened by development of resistance in economically important pests.2 Increasing interest in the application of plant secondary metabolites in insect pest management, as an alternative to the use of synthetic insecticides, has led to the search for active plant compounds, less poisonous to the environment and with low mammalian toxicity.3 Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae), the model insect pest selected, has a record of injuring over 80 plants, among them important field crops such as corn, cotton, soybean and wheat.
Larvae destroy the plant growth potential by consuming foliage and burrowing into its growing points. The control of the damage requires high volumes of insecticide, resulting in resistance to several kinds of insecticidal compound.4 Of the two Brazilian plant species chosen to be bioassayed and phytochemically investigated, Vitex polygama Cham. (Verbenaceae) had already been studied, and many of its compounds characterized, but none was considered as a potential insecticide.5 – 9 This plant is commonly called ‘Maria-Preta’, ‘Tarum˜a’, ‘Congonha-cinco-folhas’ and the like.10,11 Its leaf tea has been used in folk medicine to treat kidney diseases.11 The other plant, Siphoneugena densiflora Berg (Myrtaceae), belongs to a genus comprising only eight species, with two of them vulnerable to extinction in S˜ao Paulo State.12,13 Popularly named ‘Uvatinga’,13 it has been pointed out as a source of energy generation in a recent investigation,14 and herbarium observations by the present authors indicate that this species shows strong resistance to insect attack.
∗ ˜ Carlos (UFSCar), CP 676, 13565-905, Sao ˜ Carlos-SP, Brazil Correspondence to: Margareth BC Gallo, Departamento de Qu´ımica, Universidade Federal de Sao E-mail:
[email protected] † 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 not been ordinarily employed in pest control, their families comprise several species with injurious properties to insects reported in the literature.15 – 21 The present paper specifically deals with the effects of crude extracts, fractions and isolated compounds from V. polygama and S. densiflora against fall armyworm (S. frugiperda). Aspects examined include insecticidal and growth regulatory activities.
2 MATERIALS AND METHODS 2.1 General Heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bonding correlation (HMBC, J 8.0 Hz) experiments as well as 1D and 2D nuclear magnetic resonance (NMR) spectra were recorded in deuterated solvents (deuteropyridine, acetone-d6 or deuteromethanol) from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA), or Merck¨ Schuchardt (8011 Hohenbrunn bei Munchen), using TMS (tetramethylsilane) as internal reference. The equipment employed was either a Bruker Avance DRX-400 spectrometer (Karlsruhe, Germany; 1 H, 400 MHz; 13 C, 100 MHz) or a Bruker ARX-200 spectrometer (1 H, 200 MHz; 13 C, 50 MHz). Lowresolution electrospray mass spectroscopy (ESI/MS) was carried out on a Micromass Quattro LC triplequadrupole instrument (Manchester, UK). IR spectra were measured on a Bomem MB-102 spectrophotometer (Quebec, Canada) in potassium bromide pellets. UV spectra were obtained on a Varian Cary 500 Scan/UV-Vis-Nir spectrophotometer (Mulgrave, Australia). The high-performance liquid chromatography (HPLC) system consisted of a Shimadzu LC-10AD pump (Kyoto, Japan), an SPD-10A UV-Vis detector and a CBM-10A interface, using a Shodex Asahipack GS-310 preparative column (50.0 × 2.5 cm ID). Data acquisition was performed on CLASS LC10 software. Column chromatography (CC) was performed on silica 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 Chemical Company, Inc., Milwaukee, WI, USA). Spots were visualized under 254 nm UV light and by spraying with either vanillin sulfuric acid solution followed by heating or FeCl3 acid solution. Methanol (JT Baker, Philipsburg, PA, USA) used for the mobile phase in HPLC analysis was HPLC grade. All other solvents used in extraction and isolation of compounds were of analytical grade (Mallinkrodt Baker, SA, Xalostoc, Mexico) or home distilled. 2.2 Plants Vitex polygama and Siphoneugena densiflora were collected in July 2000, in the city of Po¸cos de Caldas, Minas Gerais, Brazil. Voucher specimens were deposited at the Universidade Federal de Juiz de Fora Herbarium (Minas Gerais) and at the Botany Pest Manag Sci 62:1072–1081 (2006) DOI: 10.1002/ps
Institute Herbarium, Universidade de S˜ao Paulo, Brazil, respectively. 2.3 Extraction and isolation The powder of the air-dried plant organs was extracted successively with hexane and methanol by percolation at room temperature. Crude extracts were obtained after filtration and removal of the solvents under vacuum at 40 ◦ C. Hydroalcoholic extracts were obtained by stirring 100 g of the residue with methanol + water (50 + 50 by volume; 3 × 300 mL) with ultrasonic mixing for 10 min. The resulting filtrates were combined, evaporated under vacuum (40 ◦ C) and lyophilized (Table 1). Some crude extracts were dissolved in water + methanol (1 + 1 by volume) and fractionated through liquid–liquid partition using solvents of increasing polarity (dichloromethane, ethyl acetate, butanol). The corresponding labeled layers are characterized in Table 1. Compounds 1 to 43 were obtained by submitting the most active extracts (see Table 2) to several chromatographic techniques. Procedures to isolate the substances bioassayed (1, 9, 14 to 18, 25, 26 and 43; see Fig. 1) are described below. Some of them, like compounds 15 and 16, were isolated from more than one different extract of the same plant. 2.3.1 Isolation of compounds 13 to 19 from Siphoneugena densiflora (SD-EMR layer) A quantity of 9 g of SD-EMR (Table 1) was subjected to CC using silica gel (38.0 × 5.0 cm) with dichloromethane + ethyl acetate (7 + 3 by volume) as eluent and gradient elution. A total of 65 fractions of 150 mL were collected, and the similar ones were pooled into 22 fractions (R1 to R22) according to their composition determined by thin-layer chromatography (TLC) and visualized under UV light and spraying with color reagent. Fraction 11 (R11, 317.0 mg) was chromatographed over Sephadex LH-20 (52.0 × 3.0 cm) using methanol as eluent. A total of 40 fractions of 30 mL were collected 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 chromatographed over Sephadex LH-20 (66.0 × 2.0 cm) and eluted with methanol + acetone (8 + 2 by volume). A total of 22 fractions of 30 mL were obtained and combined into 11 fractions (R12b to R12m). The constituent of fraction 3 (R12d) was identified as compound 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 to R12f9). Fraction 4 (R12f4, 50.0 mg) afforded a substance identified as 13, and fraction 7 (R12f7, 60.0 mg) was identified as compound 17. Fraction 18 (R18, 698.0 mg) was chromatographed over XAD7 (67.0 × 4.0 cm) using methanol as eluent. Ten 1073
MBC Gallo et al. Table 1. Extracts and fractions obtained from Siphoneugena densiflora (SD) and Vitex polygama (VP)
Plant material (g) SD leaves (923)
SD stem (2247)
SD twigs (1288)
SD root bark (400)
VP leaves (423)
VP twigs (415)
VP fruit (171)
Extraction solventa
Extract code
Amount obtained (g)
H M
SD-HL SD-ML
8.2 164.8
HA H M
SD-HAL SD-HS SD-MS
22.1 0.9 177.6
HA H M HA H M
SD-HAS SD-HT SD-MT SD-HAT SD-HR SD-MR
5.5 1.0 131.0 5.7 0.3 73.2
HA H M HA
SD-HAR VP-HL VP-ML VP-HAL
2.4 5.1 40.4 31.0
H M
VP-HT VP-MT
0.85 19.3
HA H M HA
VP-HAT VP-HF VP-MF VP-HAF
2.3 1.1 2.0 0.9
Liquid/liquid partition solventa
Layer code
Amount obtained (g)
D E B A
SD-DML SD-EML SD-BML SD-AML
9.5 14.6 12.0 12.5
D E B A
SD-DMS SD-EMS SD-BMS SD-AMS
1.62 7.67 12.7 35.4
D E A
SD-DMR SD-EMR SD-AMR
0.9 10.5 52.0
E B A
VP-EHAL VP-BHAL VP-AHAL
2.2 12.8 22.6
D E A
VP-DMT VP-EMT VP-AMT
2.6 3.9 8.4
Solvents: H, hexane; M, methanol; HA, methanol + water (1 + 1 by volume) (hydroalcoholic); D, dichloromethane; E, ethyl acetate; B, butanol; A, aqueous residue.
a
fractions of 200 mL were collected and pooled into four fractions (R18a to R18d). Fraction 1 (R18a) was further purified by R-HPLC (column ASAHIPAK GS-310 SHODEX, mobile phase methanol, flowrate 2 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 31 fractions of 30 mL were obtained and pooled into eight fractions (R19a to R19h). Fraction 8 (R19h, 127.6 mg) was rechromatographed over Sephadex LH-20 (52.0 × 3.0 cm) using methanol as eluent. A total of 36 fractions of 30 mL were collected and pooled into ten fractions (R19h1 to R19h10). Fractions 8 (R19h8) and 10 (R19h10) produced brown 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 from Siphoneugena 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 1074
(30.0 × 5.0 cm) and eluted in a step gradient from acetone + ethyl acetate (95 + 5 by volume) to 100% methanol. A total of 47 fractions of 150 mL were collected and pooled into 15 fractions (L1 to L15). Fraction 2 (L2, 96.4 mg) was submitted to CC using Sephadex LH-20 (100.0 × 2.5 cm) and methanol as eluent, resulting in 24 fractions of 30 mL each, which were combined into ten fractions (L2a to L2j). The constituent of fraction 5 (L2e) was identified as compound 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 LH20 (38.0 × 3.0 cm) and methanol as eluent, yielding 17 fractions of 30 mL each, which were pooled into eight fractions (L5a to L5h). Fraction 5 (L5e) afforded the flavonoid 26 (8.0 mg). Fraction 6 (L5f) was identified as compounds 27 and 28 in mixture (13.3 mg). Fraction 6 (L6, 784.0 mg) was chromatographed over Sephadex LH-20 (100.0 × 2.5 cm) using methanol as eluent. A total of 25 fractions of 30 mL were collected and pooled into eight fractions (L6a to L6h). The constituent of fraction 2 (L6b) was identified as compound 29 (61.0 mg). Pest Manag Sci 62:1072–1081 (2006) DOI: 10.1002/ps
Natural products active against S. frugiperda OH OH
O HO
O OH
O
HO
HO
HO OH
HO
OH
OH
HO
1
9
O
OH OH
OHOH
OH
OH
HO
OH
HO
OR
COOC O O
O CO
HO
OH
OH
O OC CO
25 R = H 26 R = rha
OH OH
OHOH
OH
OH HO
O
OH
O
HO OH
OH OH
HO
OH
OH
O
14
HO
COOC O O
HO
CO
HO
CO
O O
OH
O CO
OH
HO
O
HO
O OC
O OC CO
OH
HO
OC
OH
OH
HO
OH
OH OH OH
OH
O
HO
HO
OH
O
OH
15
OH
COOH
OH
43
OH O 18 R = H 17 R = rha
RO
O O
OR OH
O
HO
13 R = H 16 R = CH3
OR Figure 1. Chemical structures of compounds tested on Spodoptera frugiperda.
2.3.3 Isolation of compounds 15 and 43 from Siphoneugena 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) and methanol + acetone (1 + 1 by volume) (XMA). The dried fraction XWA (672.8 mg) was chromatographed over Sephadex LH-20 (46.0 × 3.0 cm) using methanol as eluent. A total of 29 fractions of 30 mL were obtained and combined into ten fractions (XWA1 to XWA10). Both fractions 6 (XWA6) and 8 (XWA8) Pest Manag Sci 62:1072–1081 (2006) DOI: 10.1002/ps
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 Vitex polygama (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 of the dried fraction MV (6.75 g) was chromatographed over Sephadex LH-20 (53.0.0 × 3.0 cm) using MeOH 1075
MBC Gallo et al. 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 weight
Treatment (plant extract)
Larval mortality (%)b
Pupation time (days)
(mg)b,c (±SEM)
SD-HL SD-ML SD-HAL SD-HS SD-MS SD-HAS SD-HT SD-MT SD-HAT SD-HR SD-MR SD-EMR SD-AMR SD-HAR VP-HL VP-ML VP-HAL VP-EHAL VP-BHAL VP-AHAL VP-HT VP-MT VP-HAT VP-HF VP-MF VP-HAF
10.0 100.0 100.0 20.0 100.0 100.0 10.0 100.0 100.0 30.0 100.0 100.0 100.0 100.0 40.0 0 60.0 0 100.0 42.9 20.0 0 10.0 90.0 40.0 100.0
21 – – 21 – – 20 – – 20 – – – – 33∗ 21 29 18 – 24 27 21 25 42∗ 30 –
249.2 (±6.84)∗ – – 279.3 (±6.07) – – 266.4 (±6.43) – – 274.3 (±9.00) – – – – 207.6 (±7.47)∗ 302.9 (±5.91) 251.0 (±8.31) 243.8 (±2.52)∗ – 236.3 (±13.68) 258.3 (±6.23)∗ 295.3 (±13.43) 230.4 (±7.46) 160.0 (±0)∗ 216.8 (±8.41) –
(%)d
Survival pupation (%)e
Emergence time (days)c
Emergence (%)f
89 – – 100 – – 96 – – 99 – – – – 88 107 107 91 – 97 110 104 98 68 92 –
60 – – 80 – – 90 – – 70 – – – – 70 100 40 60 – 60 80 100 90 10 60 –
32 – – 31 – – 30 – – 29 – – – – 44∗ 30 39 32∗ – 35 40 32∗ 35 50∗ 39 –
50
100
100
100
85.7 70 75 100 – 100 87.5 80 77.8 100 66.7 –
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 were set 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. a
as eluent, resulting in 41 fractions of 30 mL, which were pooled into seven fractions (MV1 to MV7). Fraction 4 (MV4, 168.1 mg) was rechromatographed over the same column containing Sephadex LH-20 and eluted with methanol. A total of 23 fractions of 30 mL were collected and pooled into seven fractions (MV4s to MV4z). Fraction 4 (MV4v, 57.9 mg) was further purified by R-HPLC (column ASAHIPAK GS-310 SHODEX, mobile phase methanol, flowrate 3 mL min−1 ; λ 330 nm) to yield compound 1 (first peak, 5.5 mg) and substances 7 and 8, identified as a mixture, (second peak, 3.1 mg), after three cycles of 54 min. 2.3.5 Isolation of compounds 9 to 11 from Vitex polygama (VP-EMT layer) A quantity of 1.9 g and 900 mg of VP-EMT layer (Table 1) were submitted to column chromatography (3.0 × 46.0 cm) using Sephadex LH-20 and methanol as eluent. A total of 23 fractions of 50 mL were collected. Similar fractions were combined, yielding seven fractions (EMT1 to EMT7). Fraction 3 1076
(EMT3, 551.0 mg) was rechromatographed over silica gel (230–400 mesh) using gradient elution from ethyl acetate + dichloromethane (9 + 1 by volume) to methanol; 60 fractions of 50 mL were obtained and combined into 17 fractions (EMT3a to EMT3r). Fraction 5 (EMT3e, 140.5 mg) was purified by preparative TLC on silica gel 60 F254 with double development, using dichloromethane + acetone (1 + 1 by volume) as mobile phase and UV detection, yielding compounds 9 (77.7 mg), 10 (11.0 mg) and 11 (14.3 mg) respectively. 2.4 Insect rearing Larvae of S. frugipereda used in the experiments were obtained from cultures at the Departamento de Entomologia, Fitopatologia e Zoologia Agr´ıcola of the Escola Superior de Agronomia Luiz de Queiroz, Universidade de S˜ao Paulo (ESALQ/USP), and maintained in environmental chambers at 25 ± 1 ◦ C, 70 ± 5% RH and 12:12 h light:dark photoperiod. The artificial diet used for the bioassay and previously described22 contained cooked ‘carioca’ bean 37.5 g, Pest Manag Sci 62:1072–1081 (2006) DOI: 10.1002/ps
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 phydroxybenzoate 1.5 g, formaldehyde (40% v/v) 1.8 mL and distilled water 600 mL, in a total volume of 720 mL. 2.5 Toxicity bioassays by ingestion against Spodoptera frugiperda 2.5.1 Crude extracts and layers A quantity of 100 mg of crude extracts or fractions was diluted in a small volume of acetone (e.g. 2 mL) along with ascorbic acid (ingredient of the artificial diet). The solvent was allowed to evaporate for 1 h, and the resultant mixture was ground to powder and incorporated into the liquid diet (100 g), at a final concentration of 1 mg g−1 . The liquid diet was distributed 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 temperature to eliminate excess humidity. One first-instar S. frugiperda larva (from 0 to 3 h old) was then placed in each tube and held at 25 ± 1 ◦ C, 70 ± 5% RH and 12:12 h light:dark photoperiod for about 20 days until pupation (each experiment comprised ten glass tubes, each containing a single first-instar S. frugiperda larva, totaling 10 larvae). Mortality was determined for each larva every 24 h; cessation of movement followed by color change to black was the criterion used for judging mortality. The percentage insect mortality was corrected using Abbot’s formula.23 On the day after pupation, live pupae were weighed and transferred to transparent plastic vials (6.0 × 6.0 cm) containing filter paper at the bottom (4.0 × 4.0 cm), wetted by two drops of distilled water and maintained at the same conditions as the larvae until adults emerged (10–20 additional days). Other developmental factors were recorded, such as time to pupation and adult emergence. The control diet was prepared by adding the mixture of ascorbic acid and acetone previously evaporated and ground. Methanolic and hydroalcoholic extracts insoluble in acetone were prepared in methanol and water respectively, as well as their respective controls. 2.5.2 Isolated compounds Four well plates (24 × 1.5 mL wells) were filled with the liquid diet and left for 1 h at room temperature in a decontaminated (UV light) laminar flow hood. Each compound (12.0 mg) was dissolved in 3 mL of distilled water, and 30 µL aliquots were layered on the top of each well containing the artificial congealed diet. The final concentration was about 0.1 mg g−1 . The surface excess solvent was allowed to evaporate for about 1 h under sterile conditions. Subsequently, a single second-instar S. frugiperda larva was placed on the diet mixture in each well and maintained for Pest Manag Sci 62:1072–1081 (2006) DOI: 10.1002/ps
10 days, under the same conditions as in Section 2.5.1. The percentage insect mortality was corrected using Abbott’s formula.23 Each single experiment contained a total of 96 larvae (each plate of 24 wells with four replicates). Controls were carried out in a similar way without addition of the test compound. 2.6 Statistical analysis Data consisting of the average value for each experimental unit were subjected to analysis of variance (ANOVA). Differences between treatment means were established by Tukey’s test.24 Results are given in the text as probability values, with P < 0.05 adopted as the criterion of significance. Complete statistical analysis was performed by means of the SAS program.25
3 RESULTS AND DISCUSSION 3.1 Structure elucidation Substances 1 to 43 were identified by comparison of their NMR, MS, UV and IR spectroscopic data with data previously reported in the literature. Data are given only for those compounds that were tested independently (see Table 3). 3.1.1 Compounds identified in the VP-BHAL layer Caffeoyl 6-O-β-D-glucopyranoside (1) and caffeoyl 6-O-α-D-glucopyranoside (2; C15 H18 O9 ): amorphous yellow solid; ESI/MS m/z 341 [M − H]− ; 1 H NMR (400 MHz, acetone-d6 ): aglycone: δ 7.18 (H-2; d; J 2.5 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 11.8 and 6.2 Hz); 4.43 (H-6a; dd; J 11.8 and 2.0 Hz); βglucose: δ 4.53 (H-1; d; J 8.0 Hz); 3.17 (H-2; t; J 8.0 Hz); 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 13 C NMR data were identical to data previously reported.26 Isoorientin (3) and orientin (4; C21 H20 O11 ); carlinoside (5) and isocarlinoside (6; C26 H28 O15 ); schaftoside (7) and isoschaftoside (8; C26 H28 O14 ). 3.1.2 Compounds identified in the VP-EMT layer 20-hydroxyecdysone (9; C27 H44 O7 ): amorphous colorless solid; ESI/MS m/z 479 [M − H]− ; 13 C 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 (C11); 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 1 H NMR data were identical to data previously reported.27 Polypodine B (10; C27 H44 O8 ); stachysterone (11; C27 H42 O6 ) and shidasterone (12; C27 H42 O6 ). 1077
MBC Gallo et al. 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
1 (VP) 9 (VP) 14 (SD) 15 (SD) 16 (SD) 17 (SD) 18 (SD) 25 (VP) 26 (VP) 43 (SD)
2.8 (±2.77) a 0a 3.1 (±1.04) a 1.6 (±1.56) a 26.1 (±6.13) a 21.0 (±1.85) a 8.9 (±1.55) a 78.0 (±0) b 85.0 (±0) b 0a
(mg) (±SEM)c,d
(%)e
293.1 (±15.09) ab 262.6 (±3.91) b 100.8 (±12.14) b 300.2 (±20.07) ab 199.7 (±30.10) ab 308.6 (±2.52) ab 192.3 (±4.44) ab ne ne 121.4 (±25.24)b
85.6 76.6 39.9 87.6 91.3 90.1 76.2
48.1
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 controls were 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’s test). d ne = not evaluated because live larvae were too small to be weighed. e Larval weight as percentage of control. a
3.1.3 Compounds identified from the SD-EMR layer Gallic acid (13; C7 H6 O5 ). Casuarinin (14; C41 H28 O26 ): amorphous brown powder; ESI/MS m/z 935 [M − H]− ; 1 H NMR (400 MHz, acetoned6 /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 (H5; dd; J 8.7 and 2.6 Hz); 4.80 (H-6a; dd; J 13.7 and 3.8 Hz); 4.02 (H-6b; d; J 13.7 Hz); IR, UV and 13 C NMR data were similar to those reported in the literature.28 Castalagin (15; C41 H26 O26 ): amorphous brown powder; ESI/MS m/z 933 [M − H]− ; 1 H NMR (400 MHz, acetone-d6 /deuterium oxide): δ 6.70, 6.69 and 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); 13 C NMR data were similar to those reported in the literature.28 Syringic acid (16; C9 H10 O5 ): amorphous white powder; ESI/MS m/z 197 [M − H]− ; 1 H NMR (200 MHz, acetoned6 ): δ 7.25 (H-2 and 6; s); 3.61 (OCH3-3 and 5; s); 13 C NMR and UV data were similar to those reported in the literature.28 Ellagic acid 4-Oα-L-rhamnopyranoside (17; C20 H16 O12 ): pale yellow powder; ESI/MS m/z 447 [M − H]− ; 1 H NMR (400 MHz, CD3 OD): 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; J 1.6 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 9.5 and 6.2 Hz); 1.27 (H-6 ; d; J 6.2 Hz); IR, UV and 13 C NMR data were similar to those reported in the literature.28 Ellagic acid (18; C14 H6 O8 ): amorphous pale yellow solid; ESI/MS m/z 301 [M − H]− ; 1 H NMR (200 MHz, CD3 OD): δ 7.47 (H-5 and 5 ; s); 13 C NMR and IR data were similar to those reported in the literature.28 Daucosterol (19; C35 H60 O6 ); ellagic 1078
acid 4-O-α-L-2 -O-acetylrhamnopyranoside (20) and ellagic acid 4-O-α-L-3 -O-acetylrhamnopyranoside (21; C22 H18 O13 ); siphoneugenin (22; C21 H22 O14 ); 3, 4 -di-O-methylellagic acid 4-O-β-D-6 -O-acetylglucopyranoside (23; C24 H22 O14 ) and 3,4 -di-O-methylellagic acid 4-O-β-D-3, 6 -di-O-acetylglucopyranoside (24; C26 H24 O15 ). 3.1.4 Compounds identified in the SD-EML layer Gallic acid (13); casuarinin (14); syringic acid (16). Quercetin (25; C15 H10 O7 ): yellow powder; ESI/MS m/z 301 [M − H]− ; 1 H 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 and 13 C NMR data were similar to those reported in the literature.28 Quercetin-3-O-α-L-rhamnopyranoside or quercitrin (26; C21 H20 O11 ): yellow solid; ESI/MS m/z 447 [M − H]− ; 1 H NMR (400 MHz, CD3 OD): 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; J 9.3 and 3.3 Hz); 3.41 (H-5 ; m); 3.35 (H-4 ; d; J 9.3 Hz); 0.93 (H-6 ; d; J 6.1 Hz); 13 C NMR data were similar to those reported in the literature.28 Quercetin3-O-α-L-arabinopyranoside or guiajaverin (27) and quercetin-3-O-β-D-xylopyranoside or reynoutrin (28; C20 H18 O11 ); chebuloside II (29; C36 H58 O11 ) and 28-β-D-glucopyranosyl-6β-hydroxymaslinate (30; C36 H58 O10 ). 3.1.5 Compounds identified in the SD-DML layer α-Amyrin (31), β-amyrin (32) and lupeol (33; C30 H50 O); terminolic (34) and madecassic acids (35; C30 H48 O6 ); asiatic acid (36; C36 H58 O11 ). Pest Manag Sci 62:1072–1081 (2006) DOI: 10.1002/ps
Natural products active against S. frugiperda
3.1.6 Compounds identified in the SD-DMS layer Gallic (13) and arjunolic acids (37; C30 H48 O5 ); vanillic acid (38; C8 H8 O4 ); 2,4,6-trimethoxybenzoic acid (39; C10 H12 O5 ); 2,4,6-trimethoxybenzaldehyde (40; C10 H12 O4 ) and trans-2,4,6-trimethoxyphenylpropenaldehyde (41; C12 H14 O4 ); 5-hydroxymethyl-2-furfuraldehyde (42; C6 H6 O3 ).
compounds tested (Fig. 1) showed poor results, with a maximum larval mortality of 26%, a value statistically not significantly different (P < 0.05) from the control (Table 3). Compound 17, when layered on the top of the diet, became dark-brownish after a few hours, probably indicating oxidation and loss of activity.
3.1.7 Compounds identified in the SD-BMS layer Casuarinin (14). β-Pedunculagin (43; C34 H24 O22 ): amorphous off-white solid; ESI/MS m/z 783 [M − H]− ; 1 H NMR (200 MHz, acetone-d6 ): δ 6.71 and 6.56 (HHDP group; brs); 5.53 (anomeric proton; d; J 6.9 Hz); IR, UV and 13 C NMR data were similar to those reported in the literature.29
3.3 Developmental effects In general, no statistically significant differences in pupation time and emergence were observed in the tests with extracts and fractions, except for two, VPHL and VP-HF, which showed significantly extended larval periods of 6 and 15 days respectively above those of the controls (Table 2). Moreover, VP-HL caused 10% of malformed pupae, 20% of uncompleted second-instar larvae and 30% of pupae that did not reach adult stage. Compound 9, originating from an inactive extract (VP-MT, Table 2), caused a moderate but statistically significant larval weight loss in relation to the control after 10 days of treatment (P < 0.05; Table 3). The effects of ecdysteroids are known to be surprisingly complex and dependent on the concentration of compound and the developmental stage of the insect,31 which could explain that result. Although the bioassay used does not address behavioral responses (inhibition of feeding), the results suggest that larval weight loss caused by compounds 14 and 43 (Table 3) may be correlated with feeding deterrence. In fact, it is known that tannins reduce plant palatability and act as inhibitors of digestion by precipitating food protein and digestive enzymes.36 In addition, there is evidence that some species of Lepidoptera have taste receptors that respond to some phenolics.32 However, tannins 15 and 17 were practically inactive in this respect, probably because some tannins may be oxidized37 or hydrolyzed during passage through the insect gut,36 reducing their effectiveness. According to UrreaBulla et al.,38 gallic acid (13), the compound isolated from several extracts of S. densiflora and also a probable product of hydrolysis of compound 14, significantly reduces larval growth of S. frugiperda at 1 mg g−1 .
3.1.8 Compounds identified in the SD-EMS layer Gallic acid (13); casuarinin (14); castalagin (15); ellagic acid 4-O-α-L-rhamnopyranoside (17). 3.2 Larval mortality Very large differences in larval stage mortality, ranging from zero to 100% of the exposed larvae, resulted from treatments with different extracts (Table 2). S. densiflora polar extracts (SD-ML, SD-HAL, SDMS, SD-HAS, SD-MT, SD-HAT, SD-MR, SDHAR) 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 active secondary metabolites and were further submitted to liquid–liquid partition. Partition of VP-HAL produced a VP-BHAL fraction with increased activity (100%), whereas the resultant fractions in the SD-MR partition (SD-EMR and SDAMR respectively) retained the same potent activity as 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 literature reports that a similar extract of Vitex madiensis caused death by disruption of the moulting cycle in S. frugiperda.30 Ecdysteroids 9 to 12 (Fig. 1), known for their various effects on insects,31 were obtained in the fractionation of this extract. Flavonoid 7, isolated from the VP-BHAL layer, is reported in the literature32 to have an insecticidal effect, but it was not isolated in sufficient amounts to be bioassayed. The flavonoids quercetin (25) and quercitrin (26) were highly active substances causing larval mortality of 78 and 85% (Table 3) respectively after 10 days of treatment. This result could be explained by earlier reports indicating that quercetin (25) may be a potent inhibitor of mitochondrial ATPase,33 of cytochrome P450dependent mixed function oxidases (MFO)34 and of midgut 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 Pest Manag Sci 62:1072–1081 (2006) DOI: 10.1002/ps
4 CONCLUSIONS Polar extracts from S. densiflora and the VP-BHAL layer displayed considerable insecticidal activity, and may be efficient alternatives to conventional synthetic insecticides in the integrated pest management of S. frugiperda. Moreover, the feeding deterrence caused by tannin 14 along with the insecticidal effects of flavonoids 25 and 26 might explain the 100% outcome when the SD-ML extract was tested. In addition, compound 14 might be acting synergistically with 43, and in concert with daucosterol (19), known for its insecticidal action,15 phenolic acid 13 and other 1079
MBC Gallo et al.
compound(s) not tested or characterized, emphasizing the excellent results obtained with extracts SD-MS and SD-MR respectively.
ACKNOWLEDGEMENTS The authors are grateful to Dr F´atima R SalimenaPires (Universidade Federal de Juiz de Fora – UFJF) and Marcos Sobral (Universidade Federal de Minas Gerais – UFMG) for botanical identifications, and to ALCOA Alum´ınio S/A for facilitating plant collections. This study has been financially supported by Coordena¸ca˜ o de Aperfei¸coamento de Pessoal de N´ıvel Superior (CAPES), Conselho Nacional de ´ Desenvolvimento Cient´ıfico e Tecnologico (CNPq) and Funda¸ca˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP).
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19
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21
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