Research Article Received: 29 February 2008
Revised: 16 June 2008
Accepted: 20 June 2008
Published online in Wiley Interscience: 10 September 2008
(www.interscience.com) DOI 10.1002/ps.1643
Isobutyl amides – potent compounds for controlling Diatraea saccharalis†‡ ´ Hosana M Debonsi,a∗ Jose´ E Miranda,b Afonso T Murata,c Sergio A de d e f Bortoli, Massuo J Kato, Vanderlan S Bolzani and Maysa Furlanf Abstract BACKGROUND: A dichloromethane-methanol extract of the seeds of Piper tuberculatum Jacq. (Piperaceae) and two isobutyl amides, 4,5-dihydropiperlonguminine (1) and pellitorine (2), which were isolated by chromatographic methods, were assayed for their lethality against the sugarcane borer Diatraea saccharalis F. (Lepidoptera: Pyralidae). RESULTS: Bioassays were carried out with fourth-instar caterpillars through topical application of test solutions to the dorsal surface of the prothorax, and dose–response correlations were determined. Significant insect mortalities were observed 24, 48 and 72 h after treatment at concentrations of ≥100 µg insect−1 . The LD50 and LD90 values for compound 1 were 92.83 and 176.50 µg insect−1 , and for compound 2 they were 91.19 and 184.56 µg insect−1 . CONCLUSION: According to the LD50 and LD90 for compounds 1 and 2, it can be inferred that the values reflect an acute lethal response to both compounds, based on interaction(s) of the toxicants with a primary target or series of targets. Thus, the amides were demonstrated to have potential value in the control of the sugarcane borer. c 2008 Society of Chemical Industry Keywords: Piper tuberculatum; Diatraea saccharalis; sugarcane borer; isobutyl amides
1
INTRODUCTION
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these substances have demonstrated potent effects on, for example, cowpea weevil (Callosobruchus maculatus F.), rice weevil (Sitophilus orizae L.), adzuki bean weevil (C. chinensis L.), houseflies (Musca domestica L.) and mosquito (Aedes atropalpus Coq.), as well as leaf-cutting ants (Atta sexdens rubropilosa Forel).7 – 12
∗
Correspondence to: Hosana M Debonsi, Departamento de F´ısica e Qu´ımica, Faculdade de Ciˆencias Farmacˆeuticas de Ribeir˜ao Preto, Universidade de S˜ao Paulo 14040903, Ribeir˜ao Preto, S˜ao Paulo, Brazil. E-mail:
[email protected]
† Part of this study was presented at the 2001 Annual Meeting of the Phytochemical Society of North America, Oklahoma City, OK, 4–8 August 2001. ‡ ‘Composic¸a˜ o inseticida, carrapaticida, moluscicida e/ou fungicida e uso’, AG, USP Inovac¸a˜ o/592/2007, Brazil Patent PI 0.703.109-2 (2007). a Faculdade de Ciˆencias Farmacˆeuticas de Ribeir˜ao Preto, Universidade de S˜ao Paulo 14040903, Ribeir˜ao Preto, S˜ao Paulo, Brazil b Empresa Brasileira de Pesquisa Agropecu´aria, Centro Nacional de Pesquisa de Algod˜ao, Santa Genoveva, 74001-970, Goiˆania, Goi´as, Brazil c CEFET Cuiab´a, BR 634 Km 329, Serra de S˜ao Vicente, Santo Antˆonio do Leverger, 78.106-000, Cuiab´a, Mato Grosso, Brazil d Faculdade de Ciˆencias Agr´arias e Veterin´arias, Universidade Estadual Paulista, 14884-900, Jaboticabal, S˜ao Paulo, Brazil e Instituto de Qu´ımica, Universidade de S˜ao Paulo, 05599-970, S˜ao Paulo, SP, Brazil f Instituto de Qu´ımica, Universidade Estadual Paulista, 14800-900, Araraquara, S˜ao Paulo, Brazil
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The indiscriminate use of insecticides in agriculture causes significant damage to the ecosystem, as the toxic components affect not only the target pests but also non-target organisms, and long-term exposure to these toxins could clearly be deleterious to the health of farm workers, local inhabitants and, ultimately, consumers.1 – 3 Furthermore, the continued application of a limited range of chemical products can lead to the development of pest resistance, and the ecological perturbations produced could give rise to a sudden increase in secondary pests. Integrated pest management attempts to solve these problems through the use of alternative strategies for pest control, including biological methods and the use of toxins that are more environmentally acceptable. In this context, natural toxins derived from plant sources could provide interesting replacements for the conventional and, typically, more persistent synthetic insecticides. Many plant allelochemicals, including pyrethroids, rotenones, limonoids and amides, are poisonous to herbivorous insects but are characterized by short residual effects and relatively low toxicities to mammals.4 The science of natural products has advanced significantly in recent times, and these compounds are being utilized as crop protection agents.5 Among the natural products synthesized by plants, there are a number of species of the genus Piper (family Piperaceae) that contain amides and lignans that exhibit growthreducing properties and significant toxicities towards insects. The amides that have been isolated from such sources are typically of the isobutyl, pyridone and pyridine type, and include piperine, piperettine, trichostachine, peepuloidin and piplartine.6 Moreover,
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O O
N H
O
4,5-Dihydropiperlonguminine (1) O N H Pellitorine (2) Figure 1. Chemical structures of the isobutyl amides 4,5dihydropiperlonguminine (1) and pellitorine (2) isolated from the seeds of Piper tuberculatum.
These amides show antifungal activities against Leucoagaricus gongylophorus Singer, the symbiotic fungus of leaf-cutting ants, and phytopathogenic fungi Cladosporium cladosporioides (Fr.) de Vries and C. sphaerospermum Penzig.6,12 – 14 The sugarcane borer, Diatraea saccharalis F., is an important pest in sugarcane plantations of Brazil, causing loss of yield not only by direct damage to the plants but also by allowing the ingress of fungi at the injury sites. Considering the potent insect toxicity exhibited by amides derived from species of Piper, it was of interest to determine if the isobutyl amides 4,5-dihydropiperlonguminine (1) and pellitorine (2) (Fig. 1), isolated from the seeds of Piper tuberculatum Jacq., might have potential value in the control of the sugarcane borer.
2
MATERIALS AND METHODS
2.1 Instrumentation Analytical HPLC was performed on a Varian (S˜ao Paulo, SP, Brazil) ProStar LC/PDA chromatograph equipped with a Supelcosil (S˜ao Paulo, SP, Brazil) C18 reverse-phase column (250 × 4.6 mm ID; 5 µm) eluted isocratically with methanol + water (4 + 1 by volume) at a flowrate of 1.0 mL min−1 . Preparative HPLC was performed on a Varian PrepStar chromatograph using a Supelcosil C18 reversephase column (250 × 21.2 mm ID; 10 µm) eluted isocratically with methanol + water (55 + 45 by volume) at a flowrate of 12.0 mL min−1 . HPLC detection was at 254 nm, and all solvents were redistilled prior to use. 2.2 Plant material Seeds of P. tuberculatum were collected in 1998 at the campus of ˆ the Instituto Nacional de Pesquisa da Amazonia (INPA), ManausAmazonas, Brazil, and identified by Dr Guillermo ED Paredes (Universidad Nacional Pedro Ruiz Gallo, Peru). A voucher specimen (reference code Kato-163) is deposited at the herbarium of the Instituto de Biociˆencias, S˜ao Paulo, SP, Brazil.
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2.3 Isolation of amides Immediately after harvesting, the collected plant material was dried at room temperature and powdered. Seeds (24.33 g) were extracted with dichloromethane + methanol (2 + 1 by volume; 2 × 0.6 L) for 24 h at room temperature. The resulting seed extract was concentrated under vacuum to yield 2.87 g of a green, semi-solid residue. Part of the crude seed extract (2.00 g) was
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HM Debonsi et al.
fractionated by column chromatography over silica gel (40 g; Merck 230–400 mesh) with a polarity gradient formed from mixtures of hexanes and ethyl acetate to yield 36 fractions (15.0 mL each). Fractions 8 (0.187 g) and 13 (0.122 g) were submitted for preparative RP-HPLC to afford pellitorine (2; 0.122 g, 6.10% yield) from fraction 8 and 4,5-dihydropiperlonguminine (1; 0.089 g, 4.45% yield) from fraction 13, each in a high degree of purity (ca 95%). HPLC analyses of the extracts of the other parts of the plant (i.e. leaves and stems) showed that the amides 1 and 2 were present in all tissues; their presence has also been reported in seedlings of P. tuberculatum cultivated in vivo and in vitro.15 Compound 1 crystallized from hot aqueous methanol solution as white needles (melting point 101.3–101.9 ◦ C). 1 H and 13 C NMR data were in agreement with those previously described for 4,5-dihydropiperlonguminine.6,10 Compound 2 crystallized from chloroform as pale needles (melting point 86.7–88.0 ◦ C), and the 1 H and 13 C NMR data were identical to those previously recorded for pellitorine.16 2.4 Insect bioassays Caterpillars of the sugarcane borer, D. saccharalis (Lepidoptera: Pyralidae), were collected from a sugarcane field in Jaboticabal, SP, Brazil, and reared in the laboratory under controlled conditions (25 ± 2 ◦ C, 70 ± 10% relative humidity, 14 : 10 h light : dark photoperiod). Larvae were maintained in individual plastic cups (30 mL) containing 6 mL of an artificial diet.17 Bioassays were carried out on fourth-instar caterpillars, 11–14 days old and weighing between 195.00 and 215.00 mg. Ethanol solutions containing 0.01, 0.10, 1.00, 10.00, 100.00 and 1000.00 µg µL−1 of the seed extract of P. tuberculatum, and solutions of 30.00, 40.00, 50.00, 60.00, 70.00, 80.00, 90.00, 100.00, 110.00, 130.00, 150.00, 190.00, 200.00 and 250.00 µg µL−1 of the pure amides 1 and 2 were prepared. Test solutions were applied topically to the dorsal surface of the prothorax18 of each caterpillar using a 100.0 µL Hamilton syringe with a 27-gauge needle installed in a Burkhard (Rickmansworth, Hertfordshire, UK) model 900-x electric microapplicator and calibrated to apply 2.0 µL to each insect. Test solutions were each applied to 40 larvae, and for each test a control group of larvae was treated with ethanol (2.0 µL) alone. Larval mortalities were recorded at 24, 48 and 72 h after treatment with the test solutions. The larvae were considered dead if they displayed no observable response to a mechanical stimulus, i.e. short-term pressure applied with a spatula. Mortality rates were compared between treatments, and dose–response correlations were determined by probit analysis19 carried out using Polo PC computer program (USDA Forest Service, CA) software. Separation of the 95% fiducial limits at the lethal doses was used to indicate significant difference (P < 0.05). A positive control was performed by application of various doses of a widely used commercial insecticide, parathion-methyl (Folidol), the LD50 and LD90 of which were determined. The dose range employed was based on the recommended field dose of the insecticide and sequential dilutions.
3
RESULTS AND DISCUSSION
3.1 Activity of the seed extract of Piper tuberculatum against Diatraea saccharalis In insect toxicology it has been established that mortality is proportional to the logarithm of the stimulus.20 The mortality rates of the sugarcane borer, D. saccharalis, when exposed to the dichloromethane + methanol extract of seeds of P. tuberculatum,
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Mortality (%)
Table 1. Percentage mortalities of larvae of Diatraea saccharalis following topical treatment with different amounts of seed extract of Piper tuberculatum Percentage mortality following exposure to extract for Extract applied (µg insect−1 )
24 h
48 h
72 h
0 0 0 2 8 46
0 2 0 6 14 60
4 4 4 8 22 66
0 0.01 0.10 1.00 10.00 100.00
100 90 80 70 60 50 40 30 20 10 0 30
60 90 pellitorine
120
150 180 210 240 4,5-dihydropiperlonguminine
Dose applied (µg per insect) Figure 3. Dose–response of sugarcane borer (Diatraea saccharalis) larvae exposed to 4,5-dihydropiperlonguminine (1) and pellitorine (2).
8
5 4 3 2 1 0 0
1
2
3 24 h
4
5 6 Log dose 48 h
7
8
9
10
250 200 150 100 50 0
ranged from 0 to 95% following topical application of doses between 0.50 and 5000.00 µg extract insect−1 for 24, 48 and 72 h. Doses of less than 10.00 µg extract insect−1 exhibited only slight effects on insect mortality, but a dose of 100.00 µg extract insect−1 resulted in the death of >50% of the larvae after 48 h of exposure (Table 1). The dose–response correlation obtained using a linear regression model to fit the probit data to the log of the amount of seed extract topically applied is shown in Fig. 2. The resultant regression lines for the 24, 48 and 72 h mortality values appear to be very similar, showing a relatively fast intoxication process on the insects when exposed to P. tuberculatum seed extracts. The LD90 (Table 2) value predicted by probit analysis from results obtained after 72 h of exposure to the seed extract was 640.66 µg extract insect−1 .
200 150 100 50 0 24 48 72 96 Time after treatment (h)
Figure 4. LD90 values as a function of time of exposure of sugarcane borer (Diatraea saccharalis) larvae to (a) 4,5-dihydropiperlonguminine (1) and (b) pellitorine (2) (bars indicate 95% fiducial limits).
for each compound, and in both cases the LD90 values approached an equilibrium point at 48 h. For both amides, the small variations in LD90 values determined between 24 and 72 h after topical application suggest an immediate toxic action on contact with the insect. The application of higher doses of each compound was generally followed by a decrease in movement of the insect, together with the cessation of feeding. Moreover, typical symptoms of intoxication, as described by Marchini et al.,21 including spasmodic movements, regurgitation and fecal elimination, were observed, confirming the hypothesis that these compounds give rise to acute toxicity to the sugarcane borer. 3.3 Dose–response correlations of isobutyl amides 1 and 2 against Diatraea saccharalis Because of the high insect mortality rates following 48 and 72 h of treatment with the test compounds over the dosage range indicated, the LD50 and LD90 values were obtained from the 24 h mortality data by plotting log dose versus probit mortality (Fig. 5). While the sugarcane borer appeared to be more susceptible to pellitorine (compound 2) than to 4,5-dihydropiperlonguminine (compound 1), probit analysis based on the 24 h treatment data (Table 3) revealed that the predicted LD50 and LD90 values were not statistically different for the two compounds. The predicted values from the probit analysis were submitted to the likelihood ratio test in order to verify the hypothesis that the slopes and intercepts were the same.22
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3.2 Activities of isobutyl amides 1 and 2 against Diatraea saccharalis Increased amounts of compounds 1 and 2 applied topically to the sugarcane borer resulted in increased mortality (Fig. 3). The rates of mortality ranged from 0 to 90% following applications of doses of 35–210 µg 1 insect−1 and 40–190 µg 2 insect−1 . The responses of the sugarcane borer larvae to amides 1 and 2, plotted as a function of time of exposure, are presented in Fig. 4. The plots show lower LD90 values for 2 than for 1 following 48 and 72 h of exposure to the test compounds. Although the trends in LD90 values of the two amides were opposite with respect to time, there were no statistical differences between the observed values
250
0 0 24 48 72 96 Time after treatment (h)
72 h
Figure 2. Regression lines obtained from the dose–response of sugarcane borer (Diatraea saccharalis) larvae exposed to a seed extract of Piper tuberculatum for 24, 48 and 72 h.
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(b) 300 Dose applied (µg per insect)
Probit mortality
6
Dose applied (µg per insect)
(a) 300
7
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HM Debonsi et al.
Table 2. Components of the probit analysis of the mortality rate of Diatraea saccharalis exposed to seed extract of Piper tuberculatum Time after treatment (h)
Probit slope (± SE)
LD50 (95% CL) (µg insect−1 )
LD90 (95% CL) (µg insect−1 )
t-ratio
Heterogeneity χ 2 (df), significancea
24 48 72
1.09 (±0.22) 1.22 (±0.33) 1.16 (±0.25)
135.23 71.11 50.15
2012.20 799.42 640.66
5.03 3.68 4.64
0.99, ns 1.04, ns 0.36, ns
2.02
8.20
10.09
32.08, ns
Positive control (72) a
Significance level: ns = not significant (P < 0.05).
Table 3. Components of the probit analysis of the mortality rate of Diatraea saccharalis exposed to the isobutyl amides 4,5-dihydropiperlonguminine (1) and pellitorine (2) for 24 h
Compound
Probit slope (± SE)
LD50 (95% CL) (µg insect−1 )
LD90 (95% CL) (µg insect−1 )
t-ratio
Heterogeneity χ 2 (df), significancea
1 2
4.59 (±0.61) 5.37 (±0.58)
92.83 (85.04–103.09) 91.19 (80.95–102.89)
176.50 (148.11–234.81) 184.56 (133.03–215.94)
7.55 9.32
0.18, ns 1.27, ns
Significance level: ns = not significant (P > 0.05).
Probit mortality
a
8 7 6 5 4 3 2 1 0 2.0
REFERENCES y = 5.3723x - 10.53
y = 4.5926x - 9.0369
2.2 pellitorine
2.4
2.6 2.8 Log dose 4,5-dihydropiperlonguminine
3.0
Figure 5. Regression lines obtained from the dose–response of sugarcane borer Diatraea saccharalis larvae exposed to 4.5-dihydropiperlonguminine (1) and pellitorine (2) for 24 h [χ 2 (df) 6.43 (10)].
4
CONCLUSION
In conclusion, the seed extract of Piper tuberculatum is highly toxic to the sugarcane borer Diatraea saccharalis when applied topically at concentrations of 100.0 µg insect−1 and higher. The values of LD50 and LD90 obtained for compound 1 were 92.83 and 176.50 µg insect−1 , and for compound 2 they were 91.19 and 184.56 µg insect−1 , reflecting an acute lethal response to both compounds, based on interaction(s) of the toxicants with a primary target or series of targets. In this way, both isobutyl amides were demonstrated to have potential value in the control of the sugarcane borer.
ACKNOWLEDGEMENTS
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This work was funded by grants from CNPq/PADCT and supported by the State of S˜ao Paulo Research Foundation (FAPESP) within BIOTA/FAPESP – The Biodiversity Virtual Institute Programme (www.biotasp.org.br). MF, MJK and VSB are grateful to CNPq for research fellowships; HMD, JEM and ATM acknowledge FAPESP for providing scholarships.
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