Journal of Natural Pharmaceuticals, Volume 2, Issue 3, July-September, 2011 133

Address for
correspondence:
Prof. Dr. Marise M. O.
Cabral, Vectors Insect
Laboratory /CECETEN,
Education, Research
and Extension Unit
Prof. Antonio Orlando
Izolani, Severino Sombra
University, Av. Exp.
Oswaldo de Almeida
Ramos, 280, Vassouras,
27700-000, RJ, Brazil.
E-mail:
mmaleck@oi.com.br
Laboratrio de Insetos
Vetores, Universidade
Severino Sombra, Avenida
Expedicionrio Oswaldo de
Almeida Ramos, Vassouras,
RJ,
1
Departamento de
Qumica-ICE, Universidade
Federal Rural do Rio de
Janeiro, Instituto de Cincias
Exatas, Seropdica, RJ,
2
Laboratrio de Bioqumica de
Tripanosomatdeos, Instituto
Oswaldo Cruz, Fundao
Oswaldo Cruz, Av. Brasil, Rio
de Janeiro, RJ, Brazil
Flavonoids inhibited NADPH consumption and ecdysis processes
in Oncopeltus fasciatus
Juliana Oliveira Abreu Narciso, Marco Antonio Soares de Souza, Mario Geraldo de Carvalho
1
,
Mrio Sergio da Rocha Gomes
1
, Marcelo Genestra
2
, Marise Maleck de Oliveira Cabral
ABSTRACT
Background: Piptadenia rigida is a source of avonoids such as isoliquiritigenin (1), 7,3,4-trihydroxyavone
(2) and 7,8,3,4-tetrahydroxyavanone (3). Flavonoids inuence 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 avonoids, 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 avonoids treatment on O. fasciatus
showed 50% mortality and 50% ecdysis (1), 30% mortality and 43% ecdysis (2), and topical treatment with
3 resulted in 43% ecdysis but did not show high toxicity at 100g/nymph. Intestine homogenates obtained
from insects treated with avonoids that were incubated with NADPH substrate showed percentage inhibitions
of 72%, 78% and 80%, for the treatments 3, 1 and 2, respectively. Conclusion: The avanone (3) was the
most effective and least toxic to the insect, followed by 2 and then 1.
Key words: Bioactivity, Hemiptera, molting, NOS, Piptadenia rigida
INTRODUCTION
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.
[1-3]
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 avonols,
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
litura 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 avones, 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
Original Article
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Website: www.jnatpharm.org
DOI: 10.4103/2229-5119.86259
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Journal of Natural Pharmaceuticals, Volume 2, Issue 3, July-September, 2011 134
represent a highly efcient 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 avonoids on development and mortality. We
also hypothesize that NOS expression and NO production
in O. fasciatus may be modulated by avonoids. Thus,
we provide new insights into the possib ility of using
avonoids as natural compounds for insect control.
MATERIALS AND METHODS
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), Seropdica,
RJ, by Dr. A. G. de Carvalho of the Department of
Forest Products, IF, UFRRJ. They were identied by
Prof. Dr. Jos 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 ltered on a Sephadex LH-20
column and the fractions were analyzed by means of
silica gel TLC plates. The fractions 1113 from this
ltration produced methyl 3,4-dihydroxybenzoate and
the fractions 1821 yielded a solid material that was
identied as iso liquiritigenin (1, 15.0 mg, m.p. 158159C)
[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 identied as 7,8,3,4-tetrahydroxyavanone
(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 213214C) [Figure 1]. The structures
were identied 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 (sunower seeds),
and they were maintained at 24.5 1C and 68 10%
relative humidity (RH).
Bioassay
The insects were deprived of water and food (sunower
seeds) for 24 hours before treatment and were maintained
under incubation in a biochemical oxygen demand
(BOD) at 24.5C . The avonoids 1, 2 and 3 were diluted
in acetone and dissolved in 0.15 M NaCl solution at nal
concentrations of 1, 10 and 100 g/L. The substances
were applied at a concentration of 1 L 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 avonoids. The
control groups consisted of acetone and 0.15 M NaCl
solution (without avonoids) and untreated solution.
Immediately after the treatment, the insects received
Figure 1: Structure of isoliquirigenin (1), 7,3,4-trihydroxyavone (2) and
7,8,3,4-tetrahydroxyavanone (3)
Narciso, et al.: Flavonoids inhibit NADPH and ecdysis on Oncopeltus fasciatus
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Journal of Natural Pharmaceuticals, Volume 2, Issue 3, July-September, 2011 135
food and water and were maintained at 24.5 1C 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 modications. 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 (260280 nm). Protease
inhibitor buffer [0.1 mM phenylmethylsulfonyl uoride
(PMSF), 0.01% leupeptin, 0.2 mg/mL trypsin inhibitor
and 1.0 mM benzamidine] was added at a nal 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 g/
mL protein was mixed with the following reagents (all
from Sigma Chemical Co.) in a 400 L nal volume:
0.2 mM Nicotinamide adenine dinucleotide phosphate
-NADPH, 360 M L-arginine, 2 M tetrahydrobiopterin,
1.0 M Flavine adenine dinucleotide-FAD, 1.0 M
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 L

-nitro-L-arginine methyl
ester (Sigma Chemical Co.), inducible NOS inhibitor
diphenyl-iodine chloride (Sigma Chemical Co.) and
avonoids (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
MannWhitney test (P < 0.05).
RESULTS
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 g/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 413 days. The percentages of molting
were 50% (P < 0.001) and 40% (P < 0.001) at doses of
10 and 100 g/nymph, respectively. Treatment with 2
caused 30% mortality (P < 0.01) at 10 and 100 g/nymph
concentration [Table 1]. The percentages of ecdysis were
only 29% (P < 0.001) and 43% (P < 0.001) at 10 and 100
g/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 g/nymph
compared with controls [Table 1]. The avanone 3 did
not show high toxicity and did not interfere with the
development period of O. fasciatus. The molting period
did not present any signicant differences between any
of the avonoid treatments.
Flavonoids and NOS Activity
As shown in Table 2, the percentage inhibition of NADPH
consumption by NOS from intestine homogenates
Table 1: Number of days taken for development and ecdysis, and
mortality percentage, for Oncopeltus fasciatus topically treated with
isoliquirigenin (1), trihydroxyavone (2) and tetrahydroxyavanone
(3) at 1, 10 and 100 g (concentraons/nymph)
Ecdysis 5
th
instar adult (days) Mortality
X SD % X SD Range %
1
Control 10 1
a
100 8.4 3.8
a
413 0
Ketone 9 1
a,b
90 7.7 2.1
a
511 10
1 g 7 1
b
70 5.7 1.1
b
58 30
10 g 3 1
c
50*** 5.3 0.5
b
56 40**
100 g
2 1
c
40*** 10 4.1
a
513 50***
2
Control 10 1
a
60 8.4 3.8
a
413 0
Ketone 9 1
a,b
90 7.7 2.1
a
511 10
1 g 7 1
b
100 5.2 0.7
a
46 40**
10 g 2 0.5
c
29*** 8 4.2
a
511 30
100 g 3 1
c
43*** 11.6 1.1
b
1113 30
3
Control 10 1
a
100 8.4 3.8
a
413 0
Ketone 9 1
a
90 7.7 2.1
a
511 10
1 g 2 1
b
25*** 6.5 4.9
a
311 11
10 g 4 1
b
40*** 8 4.6
a
413 0
100 g 3 1
b
43*** 6.3 4
a
411 22
Topical treatment of O. fasciatus with 1, 2 and 3 at 1, 10 and 100 g (concentraons/
nymph). Means followed by the same leer (a = a, b = b and c = c) did not dier
amongst themselves and those followed by dierent leers (a b; a c; b c) had
signicant dierence (P > 0.05) when the Tukey test was used. Values are mean
standard deviaon (X SD): average of three replicates of 10 nymphs (5
th
instar) for
each group. Signicance levels are represented as ***P < 0.001 and **P < 0.01 versus
ketone control, Tukey test.
Narciso, et al.: Flavonoids inhibit NADPH and ecdysis on Oncopeltus fasciatus
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Journal of Natural Pharmaceuticals, Volume 2, Issue 3, July-September, 2011 136
prepared 24 hours after treatment was signicantly
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 signicantly 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 avonoids 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.
DISCUSSION
The present results show that the molting of
O. fasciatus was reduced through treatment with certain
avonoids that displayed nonspecic 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 avanone 3, followed by avone 2 and
chalcone 1. It is possible that the high toxicity (4050%)
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
g/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 avonoids 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
avonoids prevent molting. It might be possible to prove
this hypothesis through simultaneous treatment with
ecdysone. Experiments using the avanone 3 to decrease
and reverse the molting processes by means of ecdysone
therapy are now under development.
It is notewort hy that in our present study, NOS
expression measured as the percentage inhibition of
NADPH consumption was signicantly increased in all
treatments with avonoids. 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 avonoids 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.
ACKNOWLEDGMENTS
This work was supported by grants from Fundao Nacional de
Desenvolvimento do Ensino Superior Particular (FUNADESP),
Fundao Carlos Chagas Filho de Amparo a Pesquisa do Estado
do Rio de Janeiro (FAPERJ), Programa de Desenvolvimento
Tecnolgico em Insumos para Sade (PDTIS/FIOCRUZ) and
Conselho Nacional de Desenvolvimento Cientco e Tecnolgico
(CNPq). The authors thank Dr. Garcia E. S. for suggestions
and for reviewing the manuscript, and Dr. Genestra M. (in
memoriam).
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Narciso, et al.: Flavonoids inhibit NADPH and ecdysis on Oncopeltus fasciatus
Table 2: Percentage inhibion of NADPH consumpon by NOS in the
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Narciso, et al.: Flavonoids inhibit NADPH and ecdysis on Oncopeltus fasciatus
Cite this article as: Abreu Narciso JO, Soares de Souza MA, Geraldo de
Carvalho M, Gomes MS, Genestra M, Cabral MM. Flavonoids inhibited
NADPH consumption and ecdysis processes in Oncopeltus fasciatus. J Nat
Pharm 2011;2:133-7.
Source of Support: Grants from Fundao Nacional de Desenvolvimento
do Ensino Superior Particular (FUNADESP), Fundao Carlos Chagas Filho
de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Programa de
Desenvolvimento Tecnolgico em Insumos para Sade (PDTIS/FIOCRUZ)
and Conselho Nacional de Desenvolvimento Cientco e Tecnolgico (CNPq).
Conict of Interest: None declared.
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This is simple HTML version for faster download on mobiles (if viewed on desktop, it will be automatically redirected to full HTML version)
E-Pub for hand-held devices
EPUB is an open e-book standard recommended by The International Digital Publishing Forum which is designed for reflowable content i.e. the
text display can be optimized for a particular display device.
Click on [EPub] from Table of Contents page.
There are various e-Pub readers such as for Windows: Digital Editions, OS X: Calibre/Bookworm, iPhone/iPod Touch/iPad: Stanza, and Linux:
Calibre/Bookworm.
E-Book for desktop
One can also see the entire issue as printed here in a flip book version on desktops.
Links are available from Current Issue as well as Archives pages.
Click on View as eBook
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