Flavonoids inhibited NADPH consumption and ecdysis processes in Oncopeltus fasciatus
Correspondence Address: DOI: 10.4103/2229-5119.86259
Background: Piptadenia rigida is a source of flavonoids such as isoliquiritigenin ( 1 ), 7,3',4'-trihydroxyflavone ( 2 ) and 7,8,3',4'-tetrahydroxyflavanone ( 3 ). Flavonoids influence on the feeding behavior of insects besides the inhibition of the insect larvae growth. Nitric oxide (NO) seems to be conserved in invertebrate innate immunity and the NO synthase (NOS) activity has been implicated in insect immunity. Therefore, the NOS expression can be evaluated to determine the inhibition of NADPH consumption. Material and Methods: three natural flavonoids, isolated from P. rigida whose structures were determined by 1 H and 13 C NMR spectral data analysis, were evaluated on the Oncopeltus fasciatus control by molting processes and NADPH consumption in the insect intestine following mortality. Results: The flavonoids treatment on O. fasciatus showed 50% mortality and 50% ecdysis ( 1 ), 30% mortality and 43% ecdysis ( 2 ), and topical treatment with th 3 resulted in 43% ecdysis but did not show high toxicity at 100΅g/nymph. Intestine homogenates obtained from insects treated with flavonoids that were incubated with NADPH substrate showed percentage inhibitions of 72%, 78% and 80%, for the treatments th 3 , th1 and th 2 , respectively. Conclusion: The flavanone ( 3 ) was the most effective and least toxic to the insect, followed by 2 and then 1 . Keywords: Bioactivity, Hemiptera, molting, NOS, Piptadenia rigida
Co-evolution has developed plants with a diversity of chemical defenses against herbivorous insects. Plant derivatives have been receiving increasing research attention, and more than 2000 plant species are already known to have metabolites with insecticide properties, such as rotenone and nicotine from Pyrethrum. [1],[2],[3] Phytochemicals endowed with hormonal, anti-hormonal or toxic activity are potential agents for insect control. [4],[5] Alternatives may be found among natural sources, particularly among higher plants, which provide a number of repellent and secondary toxic metabolites. [6] Flavonoids have been shown to affect the feeding behavior of insects [7] and to inhibit the growth of insect larvae. [8] The flavonols, quercetin, rhamnetin and rutin, have been evaluated in relation to their effect on the growth parameters and food processing efficiency of the southern armyworm Spodoptera eridania C. [9] and Spodoptera litura0 F. [10] Rutin was found to have third trophic level effects on an invertebrate predator of rutin-fed Manduca sexta larvae. [11] A test on a series of naturally occurring and synthetic flavones, in relation to the growth of the navel orangeworm Amyelois transitella, showed that unsubstituted flavones had the greatest inhibitory effect. [12] Insects are known to possess efficient mechanisms for combating pathogens by building up defense responses. These mechanisms exhibit striking parallels with those of the innate immunity of vertebrates. [13] Within the innate system, constitutive and inducible components can be distinguished. The constitutive component serves as an early and continuous physiological barrier. The current data indicate that it includes various antimicrobial or digestive peptides and proteins that are constitutively expressed in the gut, pharynx, hypodermis or secretory cells. In contrast, the inducible component is believed to represent a highly efficient but costly defense, such that it is only activated after detection of pathogens or their detrimental effects. [14] Reactive oxygen radicals and nitrogen intermediates are the key immune effectors and signaling molecules in many organisms. [15] Nitric oxide (NO) also seems to be conserved in invertebrate innate immunity. [16],[17] Nitric oxide synthase (NOS) activity has been implicated in insect immunity. [16] In the present study, bioassays were performed with Oncopeltus fasciatus Dallas (1852), which occurs over a wide range, extending from Massachusetts westward over the greater part of the United States and southward to Mexico and Brazil, as a convenient model for testing the effects of flavonoids on development and mortality. We also hypothesize that NOS expression and NO production in O. fasciatus may be modulated by flavonoids. Thus, we provide new insights into the possibility of using flavonoids as natural compounds for insect control.
Extraction and Isolation of Flavonoids Roots of Piptadenia rigida Benth (Leguminosae-Mimosoideae) were collected from the Forest Garden of the Forest Institute (Instituto de Florestas, IF) of the Federal Rural University of Rio de Janeiro (Universidade Federal Rural do Rio de Janeiro, UFRRJ), Seropιdica, RJ, by Dr. A. G. de Carvalho of the Department of Forest Products, IF, UFRRJ. They were identified by Prof. Dr. Josι Aguiar Sobrinho of the Department of Environmental Sciences, IF, UFRRJ. The voucher specimen (No. JPB-21438) has been deposited in the UPR Herbarium, IB, UFRRJ. Powdered dried root material from P. rigida was extracted exhaustively with CH 2 Cl 2 and MeOH at room temperature. The solvents were removed under vacuum to yield the residues Piptadenia Rigida Roots Dicloromethane (PRRD) (19.2 g) and Piptadenia Rigida Roots Methanol (PRRM) (310.5 g), respectively. The extract PRRM was dissolved in MeOH/H 2 O (8:2) and partitioned with solvents to yield three fractions: hexane (H, 2.24 g), chloroform (C, 11.9 g) and methanol (M). Fractionation of C on a silica gel column with dichloromethane, ethyl acetate and methanol yielded D (270.0 mg), E (6.3 g) and F (5.3 g). The fraction E-8-13 was further fractionated on silica gel CC using chloroform as the initial solvent and then increasing the polarity with methanol; 50 fractions were collected and analyzed by means of silica gel thin layer chromatography (TLC) plates. The fraction E-8-13 (294.0 mg) was filtered on a Sephadex LH-20 column and the fractions were analyzed by means of silica gel TLC plates. The fractions 11-13 from this filtration produced methyl 3,4-dihydroxybenzoate and the fractions 18-21 yielded a solid material that was identified as isoliquiritigenin (1, 15.0 mg, m.p. 158-159°C) [Figure 1]. The E fraction E-27-33 was then fractionated by means of circular preparative chromatography (in a Chromatotron) using dichloromethane/methanol (8:2), and the pure fraction detected by means of the TLC plate was identified as 7, 8, 3′,4′-tetrahydroxyflavanone (3, 22.0 mg, oil) [Figure 1]. The fraction E-45-53 was then fractionated by means of preparative TLC using chloroform/ethyl acetate (1:3), and the intermediate fractions were reunited to obtain a crystalline material that was identified as 7,3′,4′-trihydroxyflavone (2, 20.0 mg, mp 213-214°C) [Figure 1]. The structures were identified by means of 1 H and 13 C [Broad Band Decoupling (BBD) and Distortionless Enhancement by Polarization Transfer (DEPT)] nuclear magnetic resonance (NMR) spectra data analysis [19a] and comparisons with data in the literature (1[18] 2, [19b],[20] and 3[21] ).
Insects The 5 th instar nymphs (same aged) of O. fasciatus used in this study were taken from a longstanding colony that has been reared and maintained in the Vector Insect Laboratory of Severino Sombra University, RJ. The insects were given water and food (sunflower seeds), and they were maintained at 24.5 ± 1°C and 68 ± 10% relative humidity (RH). Bioassay The insects were deprived of water and food (sunflower seeds) for 24 hours before treatment and were maintained under incubation in a- biochemical oxygen demand (BOD) at 24.5°C . The flavonoids 1, 2 and 3 were diluted in acetone and dissolved in 0.15 M NaCl solution at final concentrations of 1, 10 and 100 μg/μL. The substances were applied at a concentration of 1 μL to the abdominal ventral surface of each nymph. The bioassay of 10 nymphs/group of O. fasciatus was performed in triplicate experiments with regard to the effects of flavonoids. The control groups consisted of acetone and 0.15 M NaCl solution (without flavonoids) and untreated solution. Immediately after the treatment, the insects received food and water and were maintained at 24.5 ± 1°C and 68 ± 10% RH throughout the experiments. Mortality and growth development of O. fasciatus were evaluated until 18 days after the treatment period. Statistical Analyses The results were analyzed using Tukey test with a significance level of 5% [22] and analysis of variance (ANOVA), [22] and the standard deviation was calculated using the average of the experiments. Preparation of O. fasciatus Intestine Homogenates To measure NOS activity, the protocol proposed by Ghigo et al. [23] was used with some modifications. The intestines of O. fasciatus were treated with trypsin/ethylenediamine tetraacetic acid (EDTA) (0.05/0.02% v/v; Sigma Chemical Co. St. Louis, USA.), washed, resuspended at 10.0 mg/mL of protein in 2 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2; Sigma Chemical Co.) and sonicated on ice with two 10-sec bursts. The protein content was assessed spectrophotometrically (260-280 nm). Protease inhibitor buffer [0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.01% leupeptin, 0.2 mg/mL trypsin inhibitor and 1.0 mM benzamidine] was added at a final volume of 5 mL, [24] and aliquots of the homogenates were checked for NOS activity. Spectrophotometric Measurement of NOS Activity In each assay, intestine homogenate containing 200 μg/mL protein was mixed with the following reagents (all from Sigma Chemical Co.) in a 400 μL final volume: 0.2 mM Nicotinamide adenine dinucleotide phosphate -NADPH, 360 μM l-arginine, 2 μM tetrahydrobiopterin, 1.0 μM Flavine adenine dinucleotide-FAD, 1.0 μM FMN, 0.3 mM CaCl 2 , 0.2 mM dithiothreitol and 50 mM potassium phosphate buffer (pH 7.4). In some samples, the constitutive NOS inhibitor lw-nitro-l-arginine methyl ester (Sigma Chemical Co.), inducible NOS inhibitor diphenyl-iodine chloride (Sigma Chemical Co.) and flavonoids (1, 2 and 3) were added. A solution of ketone was used as a negative control and l-NAME as a positive control. NOS activity was determined in the reaction mixture by measuring the decrease in absorbance at 340 nm for 20 min continuously, as the amount of NADPH consumed during the conversion of l-arginine to l-citrulline by NOS. Three independent experiments were performed, and the data obtained using different treatments were analyzed statistically by means of the Mann-Whitney test (P < 0.05).
Flavonoids, Mortality and Ecdysial Stasis Mortality was zero for the untreated control and a maximum of 10% in the group that received the solvent (ketone in 0.15 M NaCl) [Table 1]. The treatment of O. fasciatus with 1 at a dose of 100 μg/nymph showed 50% mortality (P < 0.001) among the 5 th instar [Table 1]. The development period was not different from that of the ketone control. Complete ecdysis in the control groups required 4-13 days. The percentages of molting were 50% (P < 0.001) and 40% (P < 0.001) at doses of 10 and 100 μg/nymph, respectively. Treatment with 2 caused 30% mortality (P < 0.01) at 10 and 100 μg/nymph concentration [Table 1]. The percentages of ecdysis were only 29% (P < 0.001) and 43% (P < 0.001) at 10 and 100 μg/nymph, respectively [Table 1]. Topical treatment with 3 on the 5 th instar resulted in 25%, 40% and 43% ecdysis at concentrations ranging from 1 to 100 μg/nymph compared with controls [Table 1]. The flavanone 3 did not show high toxicity and did not interfere with the development period of O. fasciatus. The molting period did not present any significant differences between any of the flavonoid treatments.
Flavonoids and NOS Activity As shown in [Table 2], the percentage inhibition of NADPH consumption by NOS from intestine homogenates prepared 24 hours after treatment was significantly higher than in the acetone control. The NOS inhibitors l-NAME and diphenyl-iodine chloride were used to establish whether an inducible inhibition increase of NADPH consumption was derived from NOS. The NOS activity was significantly lower when l-NAME (which is a substrate competitor for NOS) was incubated with the intestinal homogenates and NADPH substrate. l-NAME inhibition using 10 mM solution resulted in 100% inhibition compared with controls. This indicates that the NADPH consumption was due to the NOS activity. The inhibitor, diphenyl-iodine chloride, at the concentration of 0.1 mM inhibited 55% of NADPH consumption (P < 0.05). Intestine homogenates obtained from insects treated with flavonoids that were incubated with NADPH substrate showed percentage inhibitions of 72% (P < 0.05), 78% (P < 0.05) and 80% (P < 0.05), for the treatments 3, 1 and 2, respectively.
The present results show that the molting of O. fasciatus was reduced through treatment with certain flavonoids that displayed nonspecific toxic effects. In fact, treatment with 1, 2 and 3 elicited different responses in this respect. The most effective and least toxic to the insect was flavanone 3, followed by flavone 2 and chalcone 1. It is possible that the high toxicity (40-50%) of 1 on insects is related to the larvicidal activity of 7-methoxyaromadendrin, a related compound from Trixis vauthieri DC. [25],[26] Flavonoid concentrations up to 100 μg/insect retarded ecdysis, and the compound 3 was the most effective and least toxic. However, these reductions in molting were not correlated with prolongation of the molting cycle. Biochemical evidence from insects suggests that flavonoids may affect the endocrine system. It has been reported that many flavonoids are able to modulate insect development and reproduction by interacting, directly or indirectly, with the steroid hormone system. [27] Apparently, these compounds inhibit transcription of the ecdysteroid gene receptor and, in some cases, reveal a synergic effect with ecdysteroids, thereby reducing cell growth. [28] This might explain why flavonoids prevent molting. It might be possible to prove this hypothesis through simultaneous treatment with ecdysone. Experiments using the flavanone 3 to decrease and reverse the molting processes by means of ecdysone therapy are now under development. It is noteworthy that in our present study, NOS expression measured as the percentage inhibition of NADPH consumption was significantly increased in all treatments with flavonoids. The blockage of intestinal NOS activity following treatment with l-NAME and diphenyl-iodine chloride indicated that the NO pathway was also inhibited by 3, 1 and 2. This could be tested in O. fasciatus in the future by using NOS RNA inhibition and a combination of quantitative real time polymerase chain reaction (Q-RT-PCR), enzyme assays and Western blotting to detect, respectively, changes in NO expression, inducible NOS activity and transcript levels. In conclusion, our findings are consonant with the hypothesis that flavonoids may be used in investigating inhibition of the molting processes among O. fasciatus, thereby disrupting the insect population. Moreover, the present study shows that NOS activity in the intestine of O. fasciatus was reduced by treatment with 3, 1 and 2. Future functional studies should address how NOS expression and NOS activity are modulated and illuminate the ultimate immunological roles of the NO thus released in this insect.
This work was supported by grants from Fundaηγo Nacional de Desenvolvimento do Ensino Superior Particular (FUNADESP), Fundaηγo Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de Desenvolvimento Tecnolσgico em Insumos para Saϊde (PDTIS/FIOCRUZ) and Conselho Nacional de Desenvolvimento Cientνfico e Tecnolσgico (CNPq). The authors thank Dr. Garcia E. S. for suggestions and for reviewing the manuscript, and Dr. Genestra M. (in memoriam).
[Figure 1]
[Table 1], [Table 2]
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