Research Article

Received: 12 April 2012 Revised: 27 July 2012 Accepted article published: 19 September 2012 Published online in Wiley Online Library: 30 October 2012

(wileyonlinelibrary.com) DOI 10.1002/ps.3409

Synergistic larvicidal effect and morphological

alterations induced by ethanolic extracts
of Annona muricata and Piper nigrum against
the dengue fever vector Aedes aegypti
Adelia Grzybowski,a,b Marcela Tiboni,a Mario AN Silva,c Rodrigo F
Chitolina,c Maurcio Passosa and Jose D Fontanaa,b

Abstract:
BACKGROUND: Phytopesticide combinations of different botanical sources are seldom reported. Annona muricata seed and
Piper nigrum fruit ethanolic extracts enriched in acetogenins and piperamides, respectively, were synergistically used as
larvicides against the dengue fever vector Aedes aegypti.

RESULTS: Individual bioassays of A. muricata and P. nigrum indicated respective LC50 values of 93.48 and 1.84 g mL-1 against
third-instar larvae. Five combinations of different proportions of plant extracts pointed to synergism between the extracts. The
best A. muricata:P. nigrum extract combination was 90:10, which showed 5.12 times the amount of synergism, as confirmed
by statistical equations and total concentration log versus combination proportions. Concerning the morphology, A. muricata
caused larvae body elongation, mainly in the abdomen, along with the appearance of a cervix. Conversely, P. nigrum induced
abdomen and whole body shortening. The morphological effects of A. muricata were prevalent in all of the combinations tested,
irrespective of its proportion in the combination.

CONCLUSION: It is suggested that the different mechanisms of action of the larvicidal actives A. muricata acetogenins and P.
nigrum piperamides explain the observed synergism. The combination of inexpensive botanicals and a low-cost organosolvent
such as ethanol leads to a simple and efficient phytolarvicidal formulation.

c 2012 Society of Chemical Industry

Keywords: acetogenins; piperamides; synergism; isobologram; phytolarvicides

1 INTRODUCTION are incorporated in the form of a -lactone. The insertion of

It is estimated that 50 million dengue infections occur annually,
mainly in the Americas, Africa, the western Mediterranean, South-
East Asia and the West Pacific. Globally, more than 2.5 billion
people live in at-risk areas for transmission of the dengue virus by
the mosquito Aedes aegypti L.1
The eradication of A. aegypti was accomplished in the Americas
during the 1960s and early 1970s, but subsequent reinfestation has
been noted, as outbreaks occurred in 1998, 2002 and 2008. More
than 1 million cases of dengue fever were reported worldwide in
2002.1 The causes of this dengue emergency are both social and
demographic, including population increases, circulation in urban
areas and the rise of slums and settlements where basic sanitation
is practically non-existent. Also important to mention are reduced
epidemiological vigilance and the progressive resistance of the
mosquito vector to several chemical insecticides.2 7 Therefore,
alternative methods of vector control have been studied, for
instance using plants that produce secondary metabolites that are
toxic to insects.
Annona muricata L. (Annonaceae), known as soursop, is a
source of acetogenins, which are derived from long-chain fatty
three tetrahydrofuran rings (THF) in the centre of the carbon
chain often occurs in this basic chemical structure, in addition
to the formation of double bonds and varied additions of
oxygen atoms in the form of hydroxyls, carboxyls, ketones
and epoxides.8 The primary mechanism of acetogenin action
occurs in the mitochondria because acetogenins are potent
inhibitors of complex I in the respiration chain. Acetogenins
inhibit the enzymes NADH:ubiquinone oxidoreductase and block
Correspondence to: Jose D Fontana, Academic Department of Chemistry and
Biology, Federal Technological University of Parana (UTFPR), CEP 81280-340,
Curitiba, PR, Brazil E-mail: anatnof2011@gmail.com
a Department of Pharmacy/Coordination for Graduation on Pharmaceutical
Sciences, Federal University of Parana (UFPR), Curitiba, PR, Brazil
b Academic Department of Chemistry and Biology/Coordination for Graduation
on Environmental Science and Technology, Federal Technological University of
Parana (UTFPR), Curitiba, PR, Brazil
c Department of Zoology, Federal University of Parana (UFPR), Curitiba, PR,
589

acids (32 or 34 carbons) when combined with 2-propanol and Brazil

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mitochondrial oxidative phosphorylation, i.e. the conversion of
ADP and inorganic phosphate into ATP.8 10 For example, extracts
from Annona squamosa display a wide range of effects on Aedes
albopictus, ranging from ovicidal to adulticidal.11
Piper nigrum L. (Piperaceae), commonly known as black
pepper, contains amides that have insecticidal potential, such
as piperine, pipercide, guineensine, pellitorine, pipgulzarine
and pipzorine.12 14 The mode of action of piperamides in
insects is through neurotoxicity. One target is the inhibition
of the enzyme cytochrome P450 from the group MDP
(methylenedioxyphenyl).15,16
Thus, combinations of acetogenins from A. muricata and
piperamides from P. nigrum in simple and low-cost ethanol extracts
were expected to form a more potent formulation against A.
aegypti. Advantage has been taken of the different mechanisms
of action of the phytolarvicides from both plants to block the time
course of mosquito morphogenetic development, thus provoking
its death.
2 MATERIALS AND METHODS
2.1 Botanical material
Seeds of ripe fruits from A. muricata (Annonaceae) were provided
by Brasfrut (Feira de Santa, Bahia State, Brazil), and the dried ripe
fruits of P. nigrum (Piperaceae) were purchased commercially at
Curitiba (Parana State, Brazil).
2.2 Plant extraction
The botanical materials were ground in a commercial Waring
blender at maximum speed for five successive 2 min cycles
and then passed through a coarse screen (12 ABNT/ASTM 10
mesh). Each comminuted plant material (250 g) was extracted
with absolute ethanol (5 vol) at the incipient boiling point and
then filtered through Whatman No. 3 paper. The residue was
washed with two volumes of warm ethanol. The solvent from
each extract and washing was removed in a rotary evaporator
(Laborota 4000; Heildolph, Germany). The stock solutions of both
crude extracts were normalised to 50 mg mL1 in ethanol and
frozen. At the time of the bioassays, the dilutions were warmed to
room temperature.
2.3 Enriched polar acetogenin preparation and reference
standard
As annonacin, the major acetogenin in A. muricata,17,18 is not a
commercially available standard for acetogenins, a crude sample
of polar acetogenins from A. muricata seeds was obtained using
acetonitrile (3 vol) under overnight extraction at 28 C in a rotatory
shaker (Gyrotory Bath Shaker Model G76).9 This acetogenin
extract was purified with active charcoal and silica gel columns
generating fraction (pAG). Piperine (97% purity) as a piperamide
standard and triolein as a triacylglycerol (TAG) standard, as well as
1,2-benzopyrone (99.0%) as the internal standard for spectrometric
quantitations, were purchased from Sigma-Aldrich, St Louis, MO.
2.4 Chromatographic and spectrometric analyses
2.4.1 Thin-layer chromatography
The chromatographic profiles of the crude ethanolic extracts of A.
muricata and P. nigrum were obtained by thin-layer chromatog-
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raphy (TLC) in 8 cm silica gel 60 chromatoplates from Merck
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using a hexane:chloroform:nitroethane:ethyl acetate:acetone:
methanol:acetonitrile:water (12 + 2 + 4 + 4 + 1 + 2 + 1.6 + 0.1, by
vol) mobile phase.9 Hot sulfuric -anisaldehyde was used as the
spray for the general constituents of both species. Specific Keddes
reagent (alkalinised 3,5-dinitrobenzoic acid) was used for aceto-
genins. Triolein and piperine standard as well as pAG were used as
the respective reference compounds for TAGs, piperamides and
acetogenins in the TLC procedure. For this technique, all samples
were applied in volumes of 1, 2, 10 or 20 L from a 10 mg mL1
stock solution. The recording of differential colours (rose-wine for
the acetogenins with Keddes reagent, purple-violet for triolein
and the TAGs, grey-green for the acetogenins and dark green for
piperine with the anisaldehyde reagent) was performed with a
Sony CyberShot camera.
Preparative TLCs were used for the purification of the major
compounds present into extracts, annonacin (from pAG) and
piperine (from the crude extract) as above, but now adopting a
progressive advancing of the mobile phase front (1/3, 2/3 and 3/3)
in 18 cm chromatoplates.
2.4.2 Nuclear magnetic resonance (13 C-NMR and 1 H-NMR)
Annonacin and piperine samples purified through preparative
TLCs were subjected to both NMR techniques. Data were acquired
in CDCl3 at 295 K on a Bruker AVANCE 400 NMR spectrometer
operating at 9.4 T, observing 1 H and 13 C at 400.13 and 100.61 MHz
respectively. The spectrometer was equipped with a 5 mm multin-
uclear direct detection probe with z-gradient. All 1 H and 13 C NMR
chemical shifts () are given in ppm, related to the TMS signal at 0.00
ppm as internal reference, and the coupling constants (J) are in Hz.
2.4.3 Liquid chromatographymass/mass spectrometry
(LC-MS/MS)
LC-MS/MS analyses were carried out on an Agilent 1200 LC system
(Wilmington, DE) consisting of a binary pump (G1311A), a degasser
(G1379B), an oven (G1316A) and manager sample CTC (Model
2777; Waters, Milford, USA). This chromatograph was coupled to a
mass spectrometer equipped with an electrospray ion (ESI) source.
The positive-ion mode was selected and the spray voltage was
set automatically using an injector (G1329A) G1315B photodiode
array detector. A LC workstation equipped with Chemistation A
10.02 software was used for data collection and acquisition.
Chromatographic separations were carried out on a Symmetry
C18 (Waters, Milford, MA), 75 4.6 mm i.d., 3.5 m column main-
tained at 25 C. The mobile phase consisted of (A) water acidified
with formic acid (0.1%) and (B) methanol, at a flow rate of 300 L
min1 in gradient elution mode: t05 min , A 50%; t540 min , A 20%;
t4060 min , A 5%; t6168 min , A 50% recycling. The mobile phase was
filtered through a 0.45 m PTFE membrane (Millipore, Molsheim,
France). and the injection volume of all samples was 20 L.
The parameters optimised in the mass spectrometer for the
analysis of LC/MS were: capillary temperature 450 C; capillary
voltage (IS) 5500 V; input potential on the cone EP 10 eV; potential
disassembly (DP) 40 eV; interface gas (CUR) 10 psi; solvation gas
(GS1) 45 psi; nebuliser gas 45 psi. High-purity nitrogen used as
CUR, GS1 and GS2 was produced by a nitrogen generator (Peak
Scientific Instruments). The spectra were acquired by scanning
from 100 to 1000 Da. Data acquisition was performed using
Analyst 1.2 software (ABI/Sciex).
The collision cell was set in motion using a product ion collision
energy (CE) of 40 eV and a potential output of the collision cell
(CXP) of 3.8 eV for the experiments of sequential MS/MS.
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Samples were analysed from stock solutions in ethanol further
diluted with methanol:water containing 0.1% formic acid (1:1).
All solutions were filtered through a 0.45 m PVDF syringe filter
before injection.
Quantification was performed in multiple reaction monitoring
(MRM) mode, maintaining the dwell time at 300 ms in the positive
ion mode. The ion transitions used were 147.3 91.0 for the
1,2-benzopyrone internal standard, 286.1 201.1 for the piperine
standard and 597.3 525.6 for the annonacin standard. Individual
compound parameters for the 1,2-benzopyrone, piperine and
annonacin standards, respectively, were: declustering potential
(DP) 36, 40 and 40 eV; entrance potential (EP) 8, 10 and 10 eV;
collision cell entrance potential (CEP) 12, 17.3 and 23.8 eV; collision
energy (CE) 31, 30 and 30 eV; cell exit potential (CXP) 4, 3.7 and
3.7 eV. The ion-source parameters for ESI positive mode were as
follows: curtain gas (CUR) 10 psi; collision gas (CAD) 5 psi; ion
spray voltage (IS) 5500 V; nebuliser gas (GS1) 45 psi; turbo gas
(GS2) 45 psi; temperature 450 C. The high-purity nitrogen and
zero-grade air that were used as the CUR, GS1, GS2 and CAD gases
were produced by a high-purity nitrogen generator from PEAK
Scientific Instruments (Chicago, IL).
The linearity was studied through an internal standardisation
method by testing the mixtures of the working standard solutions
at seven concentration levels. The extracts were diluted to obtain
a final concentration of 350 g mL1 for A. muricata and 500 ng
mL1 for P. nigrum, and each concentration level was injected in
duplicate.
Thus, the curve for each analyte was drawn using 20 L aliquots
from the stock solutions as follows: 2.5, 5, 10, 20, 40, 60 and 80 g
mL1 for annonacin (obtained by preparative TLC), and 5, 10, 25,
50, 75, 100 and 200 ng mL1 for piperine (Sigma standard). 1,2-
Benzopyrone (internal standard) was used at a concentration of
400 and 200 ng mL1 respectively for quantification of annonacin
and piperine.
For each compound, a calibration curve was generated
to confirm the linear relationship between the analyte
peak areas/internal standard peak areas versus the analyte
concentration/internal standard concentration. The slope,
intercept and regression coefficient (r) were calculated as
regression parameters by weighted (1/x) linear regression.
2.5 Insect eggs and larvae
The Rockfeller strain of A. aegypti Linneaus 1762 (Diptera:
Culicidae), originally from the CDC (Centre of Disease Control,
Porto Rico), was kindly provided by the Oswaldo Cruz Institute (Rio
de Janeiro State, Brazil). Mosquito colonies were maintained at 25
C and 80% relative humidity in the Laboratory of Medical and
Veterinary Entomology of the Department of Zoology of UFPR.
Adult female mosquitoes were fed on rat blood to stimulate egg
oviposition on filter paper. The eggs impregnated in this support
were placed in plastic trays with mineral water (Ouro Fino)
and were fed an artificial diet (Tetramin) at 28 C with a 12
h photoperiod for egg eclosion and evolution until third-instar
larvae.
2.6 Determination of larvicidal activity
The standard method for larvicidal bioassay recommended by the
World Health Organisation19 was carried out using six different
concentrations of each sample or extract. As reference sample
bioassays, concentrations of 0.0510 g mL1 for pAG and
1.2550 g mL1 for piperine were used for comparison purposes.
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Table 1. Annona muricata (Am) and Piper nigrum (Pn) individual and
combined ethanolic extracts bioassayed against Aedes aegypti
Concentration range (g mL1 )
Combinations (Am:Pn) Annona muricata Piper nigrum
Individual 10320 0.945
A (10:90) 132 0.854.5
B (30:70) 396 0.663.5
C (50:50) 5160 0.472.5
D (70:30) 7224 0.281.5
E (90:10) 9288 0.090.5
Ethanolic extracts were assayed either individually or in one of
five proportional combinations of A. muricata and P. nigrum, as
described in Table 1.
These stock solutions were prepared in ethanol and added (1
mL) to each plastic pot containing mineral water (150 mL). Twenty
healthy third-instar larvae were used in each bioassay, and each
treatment was performed in four replicates at 25 C with a 12 h
photoperiod for 24 h of exposition. Water as the bioassay medium
without and with up to 6.7 L mL1 of ethanol as a known inert
solvent to A. aegypti larvae up to 1% v/v were employed as the
negative controls,20 and 0.006 g mL1 of temephos, a synthetic
organophosphate pesticide, served as the positive control.5
The analysis of mortality and determination of lethal
concentration were done using probit analysis carried out by the
Finney probit method21 using SPSS Statistic (v.17.0.0) software.
2.7 Combination and interaction analyses
The lethal concentration (LC50 ), the broadest parameter used for
measuring the activity of drugs or extracts in bioassays, was the
basic parameter used to evaluate the effect and synergism of the
combination of A.muricata and P.nigrum extracts. For a preliminary
check for interactions and potential superadditivity or synergism,
a Cartesian isobologram graph was drawn from the individual LC50
assays, with less and more active extracts, respectively, on the x
and y axes to generate a line of simple additivity. When dealing
with the larvicidal effects of combined plant extracts, plotting the
combinations of the new LC50 pairs makes it possible to draw
a middle line of simple additivity, as well as the antagonism
and synergism above and below this middle line.22 24 Briefly,
the isobologram is a statistical tool to deduce unequivocally the
synergistic or antagonist result arising from the use or interaction
of two different substances. The individual concentrations of the
ethanolic extracts of A. muricata and P. nigrum are denoted by
z1 * and z2 *, respectively, while the concentrations in combination
are described as z1 and z2 . Using the established concentrations
alone and in combination, the numerical value of A or log (A)
is obtained, and the results are classified as follows: A = 1 or
log (A) = 0 (additivity); A < 1 or log (A) < 0 (superadditivity or
synergism); A > 1 or log (A) > 0 (subadditivity or antagonism).
The value of A can be obtained by the equation
z1 z2
A= + (1)
z1 z2
The five phytolarvicide combinations were analysed in fixed
ratios from the doseresponse curves of the individually studied
extracts. The potency ratio was obtained by R = z1 */z2 *, where
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R = z1 */z2 *, with p1 and p2 representing the proportion of A.
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muricata and P. nigrum, respectively, where p1 + p2 = 1. From
equation (1) and a fixed proportion of the extracts, it is possible to
obtain a relationship expressed in terms of the total concentration,
zt , of a mixture:

p1 p2
A = zt + (2)
z1 z2
or
Az1
zt =   (3)
p1 (1 R) + R
Total additive concentration (zadd ) values of the combination
can be obtained using equation (3) and the value A = 1. For each
combination, a t-test was used to compare the values of zt and
zadd , with P < 0.05 representing statistical significance. To estimate
and describe the increase in the synergism phenomenon, the test
was carried out on the variation in the concentrations of the less
active extract in combination proportions with the more active
extract. In Table 3, the parameter Rz is introduced, which is the ratio
between zadd (total additive concentration) and zt (total obtained
concentration).
Applying logarithms to equation (3) yields
 
log (zt ) = log (A) + log(z1 ) log p1 (1 R) + R (4)
Based on equation (4), it is possible to plot a graph relating the
log of the total additive concentration (log zadd ) to the proportion
of A. muricata (the less active phytolarvicide), resulting in a non-
linear curve representing the expected additivity.25 The next plot
used log zt to develop a curve representing the real effect of the
extract combination. A position above the former curve indicates
antagonism, and a position below the curve indicates synergism
or superadditivity. The larger the ordinal distance between the
curves, the more intense the measured effect will be.
2.8 Morphological analysis
The larvae of A. aegypti were visually examined through photos
(taken with a Sony CyberShot Smart Zoom DSC-P92) taken under
a magnifying glass (Carl Zeiss Stemi 2000 C, magnification 4.5)
to make a comparison between the different treatments. The larva
sizes were calculated from the scale included in the photographs
using Corel Photo-Paint 10 (v.10.410) and were determined with
a virtual ruler (BitRule Charten Software). The percentages of each
larval body segment were determined by comparison with the
total size of the negative control larvae (water and ethanol). The
sizes of the head, cervix (the segment between the head and the
thorax), thorax, abdomen and breathing siphon were measured.
The results were statistically analysed by a Tukey test (one-way
ANOVA) using SPSS Statistic (v.17.0.0).
3 RESULTS AND DISCUSSION
3.1 Aedes aegypti larva lethality bioassays and
chromatographic profiles of the extracts
Figure 1 shows the chromatographic profiles of the crude ethanolic
extracts from A. muricata and P. nigrum and the reference samples.
The chromatoplate on the left shows the general profile of the
constituents revealed with hot sulfuric -anisaldehyde. The crude
extract of A. muricata clearly revealed predominant purple-violet
TAG spots at RF 0.93 (which were nearly absent in the sample
of pAG owing to its previous purification on active charcoal and
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silica gel columns), and lesser amounts of the acetogenin series
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Figure 1. Thin-layer chromatography of the crude ethanolic extracts of
Annona muricata (Am), polar acetogenins (pAG), triolein, Piper nigrum (Pn)
and piperine standard. Sample spots of 20, 2, 10, 10 and 1 L were taken
from 10 mg mL1 stocks.
were displayed as a grey-greenish colour at RF 0.37 and 0.27. The
major band in pAG should be annonacin, as it is dominant in
A. muricata seeds.17,18 In the plate on the right, Keddes reagent
was used for the specific detection of unsaturated lactones. These
acetogenins showed a strong rose-wine reaction, confirming the
aforementioned interpretations. Owing to the selectivity of the
reagent, the sample overload facilitated the detection of minor
acetogenins along with annonacin. Comparing the bioactivities
of the crude ethanolic extract from A. muricata and the pAG
sample for A. aegypti larvae, LC50 values of 93.48 and 0.65 g
mL1 , respectively, were obtained. The 143-fold increase in pAG
activity is due to an enrichment in acetogenins, as observed in
the chromatographic profile. Two previous reports indicated LC50
values of 60 and 900 g mL1 for A. muricata extracts towards
fourth-instar A. aegypti larvae.26,27 Further, if the first extract from
the latter reference was initially defatted with hexane, the LC50
was found to be 74.68 g mL1 , irrespective of the stage of
the mosquito larvae.28 Therefore, the present results are similar to
previously described results.26,28 The efficient mechanism of action
of acetogenins in several cell and tissue models was explained by
specific inhibition of the mitochondrial respiration at complex
I. Therefore, inhibition of NADH:ubiquinone oxyreductase blocks
mitochondrial oxidative phosphorylation and electron transport
and results in a blockage of the conversion of ADP and Pi into
ATP.9,10,29
In Fig. 2, 13 C NMR main signal assignment for the purified
sample of annonacin isolated from the pAG fraction was
coincident with those already reported. As a further differential
feature, the present findings for the pair of carbons immediately
flanking the tetrahydrofuran ring were 74.1 and 74.0 ppm
respectively, hence matching the 74.1 and 73.9 values from
the literature.30 Furthermore, this respective pair for annonacin
A, a threo/trans/erythro-isomer for annonacin, were reported as
74.36 and 71.62, thus confirming the distinguishing power of the
spectroscopic technique employed here.
Figure 3 shows the chromatographic profile for the crude extract
of A. muricata. The mass spectrum by positive modality was
extracted from the major peak corresponding to a retention time
(RT) of 31.6 min (A). Its main peak corresponding to [M + H]+ 597.3
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Figure 2. 13 C NMR of annonacin isolated by preparative TLC.
(B) was then the source for the subsequent MS/MS spectrum,
which then generated the expected peaks of lower m/z, namely
525.6 > 543.3 > 507.5 > 561.6 > 579.5, known specifically for
annonacin, as previously described.31 The major peaks of this
subseries incidentally corresponds to the successive loss of five
water molecules, four from the hydroxyl groups and one from the
lactone ring.
Accordingly, the major chromatographic peak at 31.7 min from
sample pAG also afforded the same mass fragmentation pattern
either as MS or MS/MS.
The calibration curve for annonacin was considered to be linear
for the evaluated concentrations, with a correlation coefficient r
> 0.9898. The linear equation was y = 30.9x + 0.171. The target
seed extract from A. muricata was diluted so that the analyte
concentration fell in the calibration curve range. An annonacin
content of 2.3 g% was found on an extract dry basis. Considering
that the present extract had a yield of 20.4 g%, it follows that
the annonacin real yield in the seeds was 0.469 g%. For instance,
when A. muricata seeds were extracted with supercritical CO2 , the
whole yield for ten different acetogenins, except for annonacin,
corresponded to only 0.209 g%.32
Summarising, the positive chromogenic Kedde test and the
13
C NMR spectrum further corroborated by extensive LC-MS/MS
unequivocally indicated, for the present annonacin sample, the
presence of a mono-THF ring with flanking hydroxyl groups in a
threo/trans/threo conformation and an ,-unsaturated -lactone
ring, as already reported in the literature.33,34
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Figure 1 also shows the chromatographic profiles for the crude
extract from P. nigrum and the piperine standard. In the left plate,
which was revealed with sulfuric -anisaldehyde, violet spots
that are related to high RF (0.93) lipids (e.g. TAGs) were visible
after heating. Piperine (RF = 0.58) was the main component and
displayed a green-brown colour, and an unidentified component
at RF 0.37 also displayed the same colour as piperine.
Regarding the larvicidal activity of the crude ethanolic extract
of P. nigrum and the piperine standard, the LC50 values were 1.84
and 6.65 g mL1 . It was realised that the heterogeneous crude
extract, i.e. piperine with several other minor components, was
more active (3.6-fold) than the pure piperine standard. A previous
report suggested the presence of several other biologically
active compounds in P. nigrum, including the isobutylamides
retrofractamide A, pipercide, guineensine and pellitorine.14 The
crude ethanol extract from unripe P. nigrum fruits has been
previously tested against third-instar larvae of a pyrethroid
cypermethrin-resistant strain of A. aegypti and had an LC50
value of 0.98 g mL1 .35 The ethanolic extracts of three other
species of Piperaceae were tested against A. aegypti fourth-instar
larvae, and the LC50 results were higher than those found for P.
nigrum (P. longum 2.23 g mL1 , P. ribesoides 8.13 g mL1 and
P. sarmentosum 4.06 g mL1 ).36 The mechanism of action of
piperine, which is similar to that of other piperamides, is exerted
at the level of the enzyme cytochrome P450, with consequent
neurotoxic activity.15,16
Figure 4 shows the 1 H NMR main signal assignment for the
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purified sample of piperine isolated from the crude ethanolic
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Figure 3. Liquid chromatography/mass spectrometry of the crude ethanolic extract of Annona muricata. (A) Full scan of the extract in the positive-mode
[M + H]+ (20 L of a 700 g mL1 solution). (B) ESI mass spectrum of peak at 31.6 min. (C) MS/MS spectrum of ion at 597.3 m/z.
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Figure 4. 1 H NMR of piperine isolated by preparative TLC.
extract of P. nigrum. The particular signals seen for the hydrogens
of the short C4 doubly unsaturated alkane chain connecting the
methylenedioxyphenyl (MDP) head to the heterocyclic piperidine
tail and more particularly those linked at the second and fourth
carbons of the above-mentioned connection were = 6.76 and
6.77 ppm as a consequence of the E,E-(trans-cis) configuration,
and hence in good agreement with previously published data.37
Conversely, for instance, the isomer isopiperine, which may
be generated by the photoisomerisation of piperine, presents
respective values of = 6.50 and 6.61 ppm owing to its inverted
Z,E-(cis-trans) configuration. Thus, piperine was indeed the main
biologically active compound in the present parent ethanolic
extract, because other 1 H NMR signals displayed very close
similarity to values reported above. Furthermore, a clear 13 C NMR
spectrum (results not shown) from the same piperine sample
arising from the crude ethanolic extract from P. nigrum was of
great help in completing the spectroscopic characterisation, as
the complementary signals for the methylenedioxyphenyl head
( = 101.3 ppm for methylene dioxy; = 131.0, 105.7, 148.2, 148.1,
108.5 and 122.5 ppm respectively for carbon numbers 1 to 6 for
the phenyl group of MDP), for the piperidine N-heterocyclic ring
tail ( = 46.9, 26.7 and 24.7 ppm respectively for carbon numbers
1 , 2 and 3 ) and finally for the carbonyl ( = 165.4 ppm) very
closely matched available data,38 the difference between them
never exceeding 0.2 ppm, except for the 1 signal (0.8 ppm).
Figure 5 shows the chromatographic profile for the crude extract
of P. nigrum (A). The mass spectrum by positive modality was
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extracted from the major peak at RT = 11.9 min. The main derived
peak corresponding to [M + H]+ 286.1 was then the source for
the subsequent MS/MS spectrum, where the fragment ions of
m/z 201.1 > 135.2 > 171.1 > 143.2 correspond to piperine, as
previously described.39 A similar analysis of the chromatographic
profile from the piperine standard resulted in a dominant peak
with the same RT, the [M + H]+ 286.1 of which also generated the
same fragment profile on MS/MS.
The calibration curve for piperine was also considered to
be linear for the evaluated concentrations, with a correlation
coefficient r > 0.9989. The linear equation was y = 3.96x + 0.0406.
The fruit extract was diluted so that the analyte concentration fell
in the calibration curve range. Hence, the derived calculation led
to the expected high concentration of piperine in the P. nigrum
ethanolic extract, in the present case 31.4 g% on an extract dry
basis. Since the yield of the ethanolic extract (just incipient boiling)
was 7.06 g%, the real content of piperine found in the fruits was
2.22 g%, as opposed to reported values of 3.926.38 g% (3 h of
reflux with ethanol), depending on the black pepper source.40
3.2 Synergism in the lethality bioassays
The individual and combined lethal concentrations of both plant
extracts for A. aegypti larvae are shown in Table 2. The observed
effects were expressed as LC50 values derived from probit analysis
with a 95% confidence limit.
When the crude extracts from A. muricata and P. nigrum were
595
individually compared against A. aegypti third-instar larvae, LC50
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Figure 5. Liquid chromatography/mass spectrometry of the crude ethanolic extract of Piper nigrum. (A) Full scan of the extract in the positive-mode
[M + H]+ (20 L of a 300 g mL1 solution). (B) ESI mass spectrum of peak at 11.9 min. (C) MS/MS spectrum of ions at 286.1 m/z.

values of 93.48 and 1.84 g mL1 indicated that the latter was
50.8-fold more potent than the former. However, in spite of the
observation that all lethal concentrations of A to E are lower
than the individual ones, the natural drug interaction type can
596
Fig. 6. This graph unequivocally indicates that all phytolarvicide
combinations formulated with the ethanol extracts of A. muricata
and P. nigrum against larvae of A. aegypti behaved synergistically,
as all interpolation values from the concentration pairs (LC50 ) lay

only be confirmed through the isobologram graph shown in below the line of additivity.

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Synergistic larvicidal effect of A. muricata and P. nigrum against A. aegypti www.soci.org

Figure 7 allows a more comprehensive interpretation of the

Table 2. Individual and combined lethal concentrations (LCs) of
Annona muricata (Am) and Piper nigrum (Pn) ethanolic extracts in
various proportions against Aedes aegypti larvae
LC50 (g mL1 )a
Combinations (Am:Pn) Annona muricata Piper nigrum
Individual 93.48 (73.97109.74) 1.84 (1.751.95)
A (10:90) 3.31 (1.416.50) 1.08 (1.031.13)
B (30:70) 4.55 (0.6911.60) 0.72 (0.670.76)
C (50:50) 8.43 (0.5823.42) 0.52 (0.480.56)
D (70:30) 13.28 (2.0731.95) 0.32 (0.110.43)
E (90:10) 13.19 (1.6230.12) 0.10 (0.090.10)
a
Lethal concentration with 95% confidence limits.
Application of the pertinent equations made it possible to
establish the increasing order of lethality efficiency for the various
combinations of the two extracts.
Using the values described in Table 2 and equation (1), the
calculation of the numerical values of A and total concentrations
(zt and zadd ) was carried out with the help of equation (3),
assuming that R = 50.78 (42.3756.23), all of them leading to
the Rz parameter of interest, as shown in Table 3.
The numerical values of A for all combinations were lower
than 1, clearly confirming that there is lethal synergism or
superadditivity between the two extracts when acting against
A. aegypti larvae. When zt and zadd were compared by the paired
t-test, all combinations demonstrated statistical significance (P <
0.05), corroborating the conclusion that all assayed combinations
acted synergistically.
The analysis of the relationship between the total concentrations
of each combination (Rz = zadd /zt ) indicates how many times any
found concentration (zt ) is more efficient than the expected one
(zadd ). Accordingly, Rz values for synergistic combinations A to E
have experienced a progressive numerical increase from 1.61 to
5.12 and a corresponding decrease in numerical values of A from
0.62 to 0.20. Hence, combination E (A. muricata:P. nigrum = 90:10)
has proved to be the best formulation to kill A. aegypti larvae.
positive interaction between the combined ethanolic extracts of
A. muricata and P. nigrum with regard to their effects on killing
A. aegypti larvae. The two non-linear curves arising from the
plot of the proportion of the individually less active A. muricata
extract versus either zt (total concentrations) or zadd (additive total
concentrations) are clearly different, the former occupying the
lower part of the graph and the latter the upper part, indicating
synergism between the extracts in all tested combinations.
Furthermore, the curves differ progressively from the lowest to the
highest proportion of A. muricata in the combinations.
Figure 8 shows the morphological aspects of larvae treated
with crude ethanolic extracts from A. muricata and P. nigrum,
individually or in different combinations.
The negative control larvae, treated with water and ethanol,
displayed a wormlike appearance and a whitish-green colour,
and the body was clearly divided into head, thorax, abdomen
and breathing siphon regions. Both the head and thorax are
globose, while the abdomen has nine segments, the last of which
is differentiated into an anal lobe. The breathing siphon is located
in segment VIII.41 The negative control larvae showed an average
size of 4.1 0.36 for water and 4.2 0.85 mm for ethanol. The
larvae exposed to temephos and the crude ethanolic extract of P.
nigrum had a 12% shorter abdomen compared with the negative
controls (average size: temephos 3.4 0.41; P. nigrum 3.5 0.70
mm). On the other hand, larvae exposed to the crude ethanolic
extract of A. muricata had an elongated body and an average size of
5.3 1.05 mm. This feature was also seen in all of the combinations
of plant extracts, irrespective of the proportion of A. muricata. The
average sizes of larvae treated with combinations A to E were 5.4
0.58 mm, 5.2 0.51 mm, 5.1 0.40 mm, 5.4 0.44 mm and 5.2
0.66 mm respectively, clearly indicating that acetogenins from
the crude ethanolic extract of A. muricata are responsible for the
elongation effect. This stretching is more evident in the abdomen
and cervix segments, which also underwent darkening. Similar
changes have been reported previously in a study using 24 h
exposure to Carapa guianensis and Copaifera sp. oils.42 In contrast
to models exploring A. muricata and P. nigrum, these oils are rich
in complex terpenes and limonoids or sesqui- and diterpenes.

Figure 6. Isobologram between Annona muricata (Am) and Piper nigrum (Pn) ethanolic extracts in different proportions, showing larvicidal effects on
597

Aedes aegypti.

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Table 3. Numerical value of A and total concentration (zt and zadd ) in various proportions of ethanolic extracts of Annona muricata (Am) and Piper
nigrum (Pn) against Aedes aegypti larvae

Combination (Am:Pn) p1 a A zt (g mL1 ) zadd (g mL1 ) Rz = zadd /zt

A (10:90) 0.1 0.62 (0.610.64) 1.27 (1.181.38) 2.04 (1.932.16)b 1.61 (1.641.57)
B (30:70) 0.3 0.44 (0.390.50) 1.15 (0.971.37) 2.61 (2.472.77)b 2.27 (2.542.02)
C (50:50) 0.5 0.37 (0.280.50) 1.35 (0.961.92) 3.61 (3.413.84)b 2.68 (3.542.00)
D (70:30) 0.7 0.32 (0.090.51) 1.85 (0.503.19) 5.87 (5.526.25)b 3.17 (10.991.96)
E (90:10) 0.9 0.20 (0.070.33) 3.06 (1.065.48) 15.64 (14.4016.82)b 5.12 (13.613.07)
a Proportion of Annona muricata.
b P < 0.05 (t-test between zt and zadd ).

the appearance of a cervix (4.4% of the whole body dimension)

Figure 7. Log of total additive concentrations (zadd ) and total concentration
(zt ) versus the proportions of Annonamuricata in the different combinations
of plant extracts against Aedes aegypti larvae.
The quantitative morphological effects of the individual or
combined extracts are depicted in Fig. 9. Overall, A.muricata extract
alone led to an 18% elongation of the abdomen, while P. nigrum
extract alone caused a 12% reduction compared with the negative
controls (water and ethanol). From a morphological standpoint,
was an unforeseen and intriguing additional effect of A. muricata.
Table 4 specifies the amount of each extract required to ensure
the mortality of larvae, starting from synergistic combinations of
the two extracts as diagnostic doses, which correspond exactly
to 2 times LC99 . For practical purposes, the amount of seeds of
A. muricata or fruits of P. nigrum necessary to obtain the extracts
for each litre or ton of water is shown in grams or kilograms. It
should be noted, of course, that in all combinations of extracts
the relative amounts of each counterpart are always smaller than
those needed individually to produce the same lethal effect to
A. aegypti. Unquestionably, the experimental question described
for this study has been answered, as all tested combinations
have resulted in superadditive or synergistic effects. This table also
includes the factor of synergism, which relates the amount of plant
required for the individual and combined actions. For P. nigrum,
the synergism factor ranges from 2.27- to 22.71-fold, i.e. amounts
smaller than those for the use of the extract alone. For A. muricata,
this value ranges from 2.74 to 57.49, i.e. it is possible to reduce
further the quantities of the plant combinations. For example,
the combination of 10 and 90% A. muricata and P. nigrum, with
respect to the doseresponse curves, requires 268 g of A. muricata
seeds and only 40 g of P. nigrum fruits. It is noteworthy that, for

Figure 8. Morphological aspects of Aedes aegypti larvae. Negative controls (water and ethanol), positive control (temephos), samples treated with crude
598

ethanolic extracts of Piper nigrum (Pn) and Annona muricata (Am) and various combinations of the extracts.

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Figure 9. Measurements of Aedes aegypti larvae. Successive columns refer to negative controls (water and ethanol), positive control (temephos), samples
treated with crude ethanolic extracts of Piper nigrum (Pn) and Annona muricata (Am) and their combinations. For each group, n = 10, and measurements
were compared with negative controls = 100%.

Table 4. Diagnostic doses, quantities of plants and synergism factor of individual and mixtures of ethanolic extracts of Annona muricata and Piper
nigrum against Aedes aegypti larvae

Plant/water for
DD (g mL1 )a DD(g L1 or kg 1000 L1 )b Synergism factorc
Combination Am:Pn (%) Am Pn Am Pn Am Pn

Individual 3142.22 7.72 15.403 0.090

Am:Pn (10:90) 54.66 3.4 0.268 0.040 57.49 2.27
Am:Pn (30:70) 176.6 2.84 0.866 0.033 17.79 2.72
Am:Pn (50:50) 517.28 2.62 2.536 0.030 6.07 2.95
Am:Pn (70:30) 849.06 1.76 4.162 0.020 3.70 4.39
Am:Pn (90:10) 1146.58 0.34 5.620 0.004 2.74 22.71
a DD (diagnostic dose) = 2 LC99 .
b Amounts were calculated on the basis of extracts yielding 20.4 g% and 8.06 g% for Annona muricata and Piper nigrum respectively.
c
Synergism factor calculated by relating the quantities individually and in combination.

these experiments, the extracts were combined and extraction
conditions were optimised by semi-purification of the extracts,
making it possible to reduce further the required quantities of
each plant. These results showed that the use of extracts in
combination is a good alternative for dengue vector mosquito
control.
In summary, the present results support the innovative work
initially proposed because all combinations of ethanol extracts of
A. muricata seeds and P. nigrum fruits exhibit synergistic effects on
the lethality for A. aegypti larvae, the main vector for dengue and
yellow fevers.
4 CONCLUSION
The analysis of the interactions of all combinations of crude
ethanolic extracts from A. muricata and P. nigrum showed
synergism or superadditivity against A. aegypti mosquito larvae as
a result of the different biochemical mechanisms of action of the
phytolarvicides present in each species: acetogenins from A. muri-
cata inhibit mitochondrial energy generation (ATP), and piperine
P450 enzyme inhibitors. Accordingly, annonacin as the dominant
acetogenin and piperine as the major piperamide were charac-
terised as such and further quantified in the respective extracts
both by spectroscopic and mass spectrometric techniques.
Therefore, taking advantage of the intense synergistic effects, it
is possible to reduce the concentration of the individual extracts
required to produce mortality in A. aegypti larvae and thus
more easily control the vector of dengue and yellow fevers,
which persist as serious public health problems. From a strictly
economical standpoint, the company providing the A. muricata
seeds processes an average of 500 t of soursoup pulp per crop-
year, meaning about 35 t of seeds whose only destination is
soil fertilisation, thus representing a zero cost starting material.
Additionally, dried black pepper fruits are an abundant and low-
cost spice source. Moreover, ethanol is, at least in the United
States and Brazil, the least expensive environmentally friendly
organosolvent. These three statements show that the combination
of ethanolic extracts of A. muricata and P. nigrum is an attractive,
inexpensive synergistic phytolarvicide for the control of A. aegypti
larvae, especially considering that preventive actions on eggs and
599

and related amides from P. nigrum act as neurotoxic cytochrome flying female adults are less practical. Given that science and

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 www.soci.org
particularly technology need also to address simpler solutions for
people mostly affected by dengue as a recurrent disease, such as
those in the Southern Hemisphere, the present synergism from
common and widespread plants is to be looked at as a valid
proposition for basic sanitation in Latin America and in African and
Asian countries.
ACKNOWLEDGEMENTS
The authors thank the National Research Council for Scientific
and Technological Development (CNPq), the Coordination for
the Improvement of Higher Education Personnel (CAPES) and
the Parana State Secretary for Superior Education, Science and
Technology (SETI-PR) for financial support, and Brasfrut for
provision of A. muricata seeds. Special thanks to Prof. Dr Francinete
Ramos Campos and Prof. Dr Andersson Barison for their tireless
help in performing the LC-MS/MS and NMR techniques. Part of the
synergistic methodology described herein has been submitted as
a Brazilian patent request (INPI, PI 11057866, 7 November 2011).
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