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Research Article
Larvicidal Activity against Aedes aegypti and Chemical
Characterization of the Inflorescences of Tagetes patula
Letícia Maria Krzyzaniak,1Tânia Mara Antonelli-Ushirobira,1
Gean Panizzon,1Ana Luiza Sereia,1José Roberto Pinto de Souza,2
João Antonio Cyrino Zequi,3Cláudio Roberto Novello,4
Gisely Cristiny Lopes,1Daniela Cristina de Medeiros,1Denise Brentan Silva,5
Eneri Vieira de Souza Leite-Mello,6and João Carlos Palazzo de Mello1
1Programa de P´
os-Graduac¸˜
ao em Ciˆ
encias Farmacˆ
euticas, Department of Pharmacy, Laboratory of Pharmaceutical Biology (Palato),
Universidade Estadual de Maring´
a, Avenida Colombo 5790, Maring´
a, PR, Brazil
2Programa de P´
os-Graduac¸˜
ao em Agronomia, Department of Agronomy, Universidade Estadual de Londrina,
Rodovia Celso Garcia Cid, Km 380, s/n, Londrina, PR, Brazil
3Department of Animal and Plant Biology, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid,
Km 380, s/n, Londrina, PR, Brazil
4Academic Department of Chemistry and Biology, Universidade Tecnol´
ogica Federal do Paran´
a, Linha Santa B´
arbara, s/n,
Francisco Beltr˜
ao, PR, Brazil
5Laborat´
orio de Produtos Naturais e Espectrometria de Massas (LAPNEM), Universidade Federal de Mato Grosso do Sul,
AvenidaCostaeSilva,s/n,CampoGrande,MS,Brazil
6Department of Morphological Sciences, Universidade Estadual de Maring´
a, Avenida Colombo 5790, Maring´
a, PR, Brazil
Correspondence should be addressed to Jo˜
ao Carlos Palazzo de Mello; mello@uem.br
Received 30 June 2017; Accepted 13 November 2017; Published 7 December 2017
A
cademicEditor:GheeT.Tan
Copyright © Let´
ıcia Maria Krzyzaniak et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.
e crude acetone extract (CAE) of defatted inorescences of Tagetes pat u l a was partitioned into ve semipuried fractions: n-
hexane (HF), dichloromethane (DF), ethyl acetate (EAF), n-butanol (BF), and aqueous (AQF). BF was fractionated by reversed-
phase polyamide column chromatography, obtaining subfractions, which were subjected to HSCCC, where patuletin and
patulitrin were isolated. CAE and the fractions BF, EAF, DF, and AQF were analyzed by LC-DAD-MS, and patuletin and patulitrin
were determined as the major substances in EAF and BF, respectively. BF was also analyzed by HPLC and capillary electrophoresis
(CE), and patulitrin was again determined to be the main substance in this fraction. CAE and the semipuried fractions (, ,
, , and mg/L) were assayed for larvicidal activity against Aedes aegypti, with mortality rate expressed as percentage. All
fractions except AQF showed insecticidal activity aer h exposure of larvae to the highest concentration. However, EAF showed
the highest activity with more than % reduction in larval population at mg/L. e insecticidal activity observed with EAF
might have been due to the higher concentration of patuletin present in this fraction.
1. Introduction
Aedes aegypti (Linnaeus, ) is an anthropophilic and
domicile mosquito, and it is the main vector for dengue
viruses in the Americas. is mosquito puts half of the world’s
population at risk with a -fold increase in incidence in
the past years in more than endemic countries [, ].
According to data from the World Health Organization,
thenumberofpeopleaectedwithdengueinwas.
million, with , people hospitalized per year [].
Ae. aeg ypti also carries chikungunya, zika, and yellow
fever urban viruses; so its monitoring and control are
necessary. Vector control in Brazil currently occurs with
the use of growth regulators of immature stages, such as
Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2017, Article ID 9602368, 8 pages
https://doi.org/10.1155/2017/9602368
Evidence-Based Complementary and Alternative Medicine
diubenzuron, and the control of adult mosquitoes with
alpha-cypermethrin, deltamethrin, malathion, and others
according to recommendations of the WHO Pesticide Evalu-
ation Scheme [], which are nonspecic products that select
resistant insects due to their great genetic plasticity [], with
consequent environmental contamination [].
ere is currently a great deal of interest in alterna-
tive methods and selective principles for the control of
mosquitoes with less environmental damage []. In this sense,
substances extracted from plants present a great perspective
for the control of Ae. aegypti.
e substances of natural origin have some advantages:
they are obtained from renewable resources, and the selection
of resistant forms occurs at a slower rate than with synthetic
insecticides [, ]. Another advantage is that they show low
or no toxicity to mammals and bees [].
Among the plants with bioactive substances, there is
Tagetes patula L., popularly known as “cravo-francˆ
es,”
“cravo-de-defunto,” or “bot˜
oes-de-solteir˜
ao” []. T. pat u l a
belongs to the family Asteraceae, which is one of the oldest
groups of higher plants [], with approximately genera
and species in Brazil [], and its avonoids patuletin
and patulitrin are considered important taxonomic markers
[].
Its inorescences have been used in folk medicine for
antiseptic, diuretic, blood purifying, and insect repellent
purposes. Its leaves have been used for renal problems and
muscle pain and its roots and seeds used as purgatives [].
Some studies on the chemical composition of T. patula up to
now indicate that the owers and leaves are rich in terpenes
[, ], alkaloids [], thiophenes [], and avonoids [–
]. is plant has shown the following activities: anti-
hypertensive [], anti-inammatory [], hepatoprotective
[], insecticidal [], nematicidal [, ], larvicidal [],
antibacterial [], antiviral [], and antifungal [].
Accordingly, the aim of this work was to isolate and
identify compounds from the semipuried n-butanol frac-
tion of T. patula by reversed-phase column chromatography
and high-speed countercurrent chromatography (HSCCC)
and to evaluate the chemical prole of the crude extract
and semipuried fractions using high performance liquid
chromatography (HPLC), capillary electrophoresis (CE), and
liquid chromatography-mass spectrometry (LC-DAD-MS).
In addition, the larvicidal activity of the crude extract and
semipuried fractions was evaluated against Ae. aegypti.
2. Materials and Methods
2.1. Plant Material. e inorescences of T. pat u la were
collected in November in the Garden of Medicinal
Plants of the Universidade Estadual de Londrina, Londrina,
Brazil, where they were organically grown. e plant material
was collected under a permit from IBAMA-SISBIO, number
-, May , , authentication code , under
the responsibility of J. C. P. Mello. An exsiccate is deposited
at the Herbarium of the Universidade Estadual de Maring´
a
(HUEM) under number , and the identication was
provided by Professor Dr. Jimi Nakajima at the Institute
T : Eluent systems used for HSCCC to obtain subfractions.
Subfraction Eluent systems (v/v)
FB
hexane : ethyl acetate : methanol : water ( : : . : )
Gradient elution with n-butanol:
–mL–mLn-butanol
–mL– mLn-butanol
–mL–mLn-butanol
– mL – mL n-butanol
FB hexane : ethyl acetate : methanol : water ( : : : )
FB hexane : ethyl acetate : methanol : water ( : : . : )
of Biology of the Universidade Federal de Uberlˆ
andia,
Uberlˆ
andia, Brazil. e owers were dried in a convection
oven at ∘C for h. e dried plant material was macerated
using a hammer mill (Tigre ASN-).
2.2. Preparation of Crude Extract and Semipuried Fractions.
e milled inorescences (. kg) were defatted with n-
hexane by dynamic maceration for three days, with sub-
sequent drying of the inorescences at room temperature.
Aerwards, acetone was used as extraction solvent at a
proportion of % (w/v) in an Ultra-Turrax(UTCKT,
Ika Works) for min and then subjected to maceration for
h. Next, turbo-extraction was performed for min, with
intervals of min (𝑡<40
∘C). e extract was ltered,
concentrated under reduced pressure, frozen, and lyophilized
(Alpha –, Christ) to give the crude acetone extract
(CAE, .%). CAE was fractionated according to Filho and
Yunes []. Briey, g CAE was resuspended in L of
methanol : water ( : , v/v) and partitioned with dierent
solvent volume ratios. e yields were n-hexane (HF) .%,
dichloromethane (DF) .%, ethyl acetate (EAF) .%, n-
butanol (BF) .%, and aqueous (AQF) .%.
2.3. Reversed-Phase Column Chromatography of n-Butanol
Fraction. BF (. g) was separated by column chromatog-
raphy(CC)withapolyamidecolumn(CCKorngrobe,
.–. mm; Macherey Nagel) according to Degani et al.
[],andthemobilephasewas%methanolorwateror
a combination thereof, providing subfractions (BF–).
e subfractions BF ( mg) and BF ( mg) precipitated
during the organic solvent removal process and were ana-
lyzed by nuclear magnetic resonance (NMR), MS, and HPLC.
2.4. High-Speed Countercurrent Chromatography (HSCCC).
e subfractions BF, BF, and BF were rechro-
matographed by HSCCC using a PC Itochromatograph
(model ) equipped with a polytetrauoroethylene (PTFE)
column (. mm i.d., total volume capacity of mL),
-𝜇Lsampleloop,rpm,anddoublepistonsolvent
pump (Waters model ), using a ow-rate of . mL/min.
e organic phase (hexane : ethyl acetate : methanol : water;
Table ) was used as the mobile phase, and water was the
stationary phase. Only in HSCCC of BF was a gradient
system with n-butanol also used. BF, BF, and BF
Evidence-Based Complementary and Alternative Medicine
yielded , , and subfractions, respectively. e subfrac-
tions BF. ( mg), BF. ( mg), BF. (. mg), and
BF. ( mg) were selected and analyzed by NMR and MS.
2.5. NMR Analysis. e subfractions BF, BF, BF.,
BF., BF., and BF. were analyzed by NMR
spectroscopic methods D (1Hand13C) and D (1H/1H-
COSY and HMBC), with a Varian Mercury Plus ( MHz
for 13CandMHzfor1H), using deuterated solvents and
TMS as internal reference. e spectra of the subfractions
were related to the compounds Tp (BF., ., and
.) and Tp (BF, , and .), which were analyzed
andcomparedtoliteraturedata.
Patuletin (Tp1)1H-NMR (𝐶𝐷3𝑂𝐷, 300 MHz)..(H-),
. (d, J. Hz, H-), . (d, J. Hz, H-), . (dd,
J. Hz; . Hz, H-), . (OCH3-). 13C-NMR (CD3OD,
MHz): . (C-), . (C-), . (C-), . (C-),
. (C-), . (C-), . (C-), . (C-), . (C-),
. (C-), . (C-), . (C-), . (C-), . (C-
), . (C-), . (CH-).
Patulitrin (Tp2)1H-NMR (𝐶𝐷3𝑂𝐷, 300 MHz). . (s) (H-
), . (d, J. Hz, H-), . (d, J. Hz, H-), . (dd, J
. Hz; . Hz, H-), . (d; J. , H - ), . (d; J,, H-)
. (m) (H-), . (m) (H- ), . (m) (H- ), . (m)
(H-), . (s) CH3O-. 13C-NMR (CD3OD, MHz): .
(C-), . (C-), . (C-), . (C-), . (C-), .
(C-), . (C-), . (C-), . (C-), . (C-), .
(C-), . (C), . (C-), . (C-), . (C-), .
(C-), . (C-), . (C-), . (C-), . (C-), .
(C-), . (CH3O-).
2.6. HPLC-ESI-MS/MS Analysis. Fractions and subfractions
were analyzed with a Waters HPLC system coupled with a
triple quadrupole mass spectrometer (Micromass, Quattro
microAPI) equipped with a Z-electrospray ionization
(ESI) source (Waters) and processed by MassLynxsoware
(version ., Waters). Chromatographic conditions were as
follows: column was a Symmetry C- (. 𝜇m, ×. mm,
Waters); mobile phase was water with .% formic acid (v/v)
(solvent A) and acetonitrile with .% formic acid (v/v)
(solvent B). e gradient system employed was as follows:
– min % B; min % B; min %; and – min % B.
e ow rate was . mL/min and the injection volume 𝜇L.
A sample containing ng/mL of the isolated substances
was injected, and identication was performed analyzing the
information of the product ion spectra in comparison to a
previously published dataset.
2.7. Identication of the Constituents by LC-DAD-MS. e
analyses of CAE and the fractions DF, EAF, BF, and AQF were
performed on UFLC Shimadzu Prominence chromatograph
coupledtoadiodearraydetector(DAD)andMicrOTOF-
Q III mass spectrometer (Bruker Daltonics). A Kinetex C-
chromatographic column (. 𝜇m, ×. mm, Phe-
nomenex) was used. Acetonitrile (solvent B) and deionized
water (solvent A), both with .% formic acid (v/v), were
used as mobile phase. e gradient elution prole was the
following: initial % B, –min % B, – min % B,
and–min%B.enegativeandpositiveionmodes
were carried out, and nitrogen was applied as a nebulizer gas
( bar) and dry gas ( L/min).
2.8. Capillary Electrophoresis (CE). CE for BF analysis was
carried out using a Beckman P/ACEMDQ electrophoresis
system equipped with a lter-based UV/Vis detector and
Karatversion . soware. e column used was a fused
silica capillary column (Beckman-Coulter) with dimensions
of . cm total length, . cm eective length, 𝜇mo.d.,
and 𝜇mi.d.esamplewasinjectedhydrodynamically
at . psi for s, kV, and the electropherogram was
recorded at nm. e cartridge coolant of the CE was
set with a thermostat at ∘C. e background electrolyte
consisted of mmol/L borate buer (pH .) containing
mmol/L methyl-𝛽-cyclodextrin (Me-𝛽-CD). e sample
solution ( 𝜇g/mL) was prepared by dissolving mg BF
in mL of % methanol and was eluted through the
solid-phase extraction (SPE) cartridge (Strata C-E, Phe-
nomenex), preconditioned with methanol and water. Tp
and Tp were used as standards for peak identication. All
solutions were ltered with . 𝜇m Millipore lters.
2.9. Evaluation of Larvicidal Activity. CAE, AQF, EAF, HF,
BF, DF, and the fatty waste obtained in the preparation of the
crude extract were tested for larvicidal activity.
Immature forms of Ae. aeg ypti were obtained from the
insectary of the Malaria and Dengue Laboratory, Instituto
Nacional de Pesquisas da Amazˆ
onia (INPA), Manaus, Brazil.
e insectary began with the collection of eggs in the
eld by using traps (egg traps). All the procedures for the
maintenance of mosquitoes and the use of animals for blood
meal were authorized by the Animal Experiment Ethics
Committee (CEUA/INPA /). e bioassay methods
were according to Lacey [], and WHO [, ], with
modications.
Fourth instar larvae were used for all experiments. ree
replicates with immature forms and mL of distilled
water per container were assayed. e crude extract and
semipuried fractions were diluted in dimethyl sulfoxide
(DMSO) at an initial concentration of , mg/L in a total
volume of mL. e samples were solubilized using an
ultrasonic bath for min. To determine mortality rates in
percent, lethal concentrations (LC50 and LC90), and their
limits,veconcentrationswereused:,,,,and
mg/L. DMSO solution at mg/L and distilled water
were used as controls. e assay was performed using a
photoperiod of / h, at 26 ± 2∘C. Mortality readings were
performedat,,,,andh.
Statistical package Spss Inc. was used for the calcu-
lation of the survival curve of the fractions and the lethal time
of Ae. aeg ypti for EAF.
3. Results and Discussion
3.1. Structural Analysis. e structural analysis of subfrac-
tions BF, BF, BF., BF., BF., and BF.
Evidence-Based Complementary and Alternative Medicine
0.0
0.5
1.0
1.5
Intens.
(mAU)
(2) (3)
(4)
(5) (6) (7)
(8)
(9)
5 1015202530350
Time (min)
×104
(a)
Intens.
(mAU)
500
1000
(1) (4)
(8) (9)
(10)
(11)
5 1015202530350
Time (min)
(b)
Intens.
(mAU)
0
1000
2000
(1) (2)
(3)
(4)
(5)
(6) (7)
(8)
(9)
(10) (11)
5 1015202530350
Time (min)
(c)
Intens.
(mAU)
0
1
2
(2)
(4)
(5) (6) (7)
(8)
(9)
5 1015202530350
Time (min)
×104
(d)
Intens.
(mAU)
0
2000
4000
(2)
(4)
(8)
5 1015202530350
Time (min)
(e)
F : Chromatogram at to nm of the crude acetone extract of Tag etes pat u l a (a) and its fractions obtained with dichloromethane
(b), ethyl acetate (c), n-butanol (d), and water (e). e identication of the constituents is given in Table .
was performed by NMR, HPLC-IES-MS/MS and LC-DAD-
MS and resulted in the identication of the compounds Tp
and Tp.
Tp (BF., BF., and BF.) was obtained
as a yellow powder. e mass spectrum obtained by ESI
showed an intense ion peak at 𝑚/𝑧 , corresponding to the
protonated ion and fragment ions of 𝑚/𝑧 and . e
UV spectrum of Tp revealed two absorption maxima in the
region of nm (band I) and nm (band II), compatible
with the UV spectrum of avonols.
Tp (BF, BF, and BF.) was also obtained as a
yellow powder. Its mass spectrum showed an intense ion of
𝑚/𝑧 and fragment ions at 𝑚/𝑧 and . e UV
spectrum of Tp revealed maxima at nm (band I) and
(band II), which was also compatible with avonols.
On the basis of 1Hand13C-NMR data obtained and
comparison with literature values [–], Tp and Tp were
identied as the avonols patuletin and patuletin--O-𝛽-
glycoside (patulitrin), respectively, which was conrmed by
HMBC, HSQC, and COSY data.
CA E a n d A Q F, E A F, D F, a n d B F f r o m T. p atula were ana-
lyzed by LC-DAD-MS, and the compounds were identied
onthebasisofUVandaccuratemassandfragmentation
data, which were compared with the literature data. From
the samples, eleven compounds were detected and identied
(Figure , Table ). e higher peak intensity was compound
(patulitrin) for BF and AQF, compounds , (patulitrin
isomer), and (patuletin) for EAF, compound for DF, and
compounds and for CAE (Figure ).
3.2. CE Fingerprint of BF of T. patula. In this work, BF of T.
patula was evaluated by CE. e major peaks were identied
by addition of the isolated substances of this work. Peak was
identied as Tp and peak as Tp (Figure ). is ngerprint
shows that the major substance was Tp,andthesameprole
was observed by LC-DAD-MS analysis (Figure ).
Some studies with T. pa t ula have been performed using
thin layer chromatography (TLC), HPLC, and HPLC-MS
[, ]. However, CE was employed here for the rst time
to identify the compounds obtained from T. pa tula.
Comparing the HPLC and CE methods developed for
evaluation of BF of T. pat u l a , CE was more ecient, being
almost four times faster. In addition, in the CE method,
organicsolventsarenotusedtoseparatetheanalytes,and
the volume of electrolytic solution used for analyses is small,
making the technique less costly and polluting [–].
3.3. Larvicidal Activity. All the fractions of T. pa tula evalu-
ated showed insecticidal activity against Ae. aegypti aer h
exposure of the larvae to a concentration of mg/L, with
exception of AQF.
Aer h ( days) of exposure, the following mortality
rates were observed at a concentration of mg/L for the
dierent samples evaluated: CAE (.%), AQF (.%), EAF
(.%), HF (.%), BF (.%), DF (.%), and fatty waste
(.%). No deaths occurred during a four-day observation
period for the DMSO control, but at the end of the h day,
mortality was .%. e distilled water control did not cause
any mortality during the whole experiment period (Table ).
EAF and fatty waste showed the best time-dependent
results (Figure ). e lethal time for percent mor-
tality (LT50)ofAe. aegypti with EAF was . h (range:
.–. h).
Komalasmisra et al. [] demonstrated that plants with a
LC50 lower than mg/L for larvicidal activity are eective
against Ae. aegypti. us, CAE and all fractions evaluated,
Evidence-Based Complementary and Alternative Medicine
T : Identication of the constituents from Tag etes pat u l a by LC-DAD-MS.
Peak RT (min) Compound UV (nm) MF Negative mode (𝑚/𝑧)Positivemode(𝑚/𝑧)
MS (∗)MS/MS MS (∗)MS/MS
() . NI
() . Quercetagetin
O-hexoside , C21H20O13 . , , . , , ,
,
() . Ellagic acidst , C14H6O8. , , . , , ,
() . Patulitrinst , C22H22O13 . , , ,
, , . ,,,
() . Patulitrin isomer , C22H22 O13 . , , . ,
() . Isorhamnetin
O-hexoside , C22H22O12 . , , ,
, . ,
() . Kaempferol , C15H10O6. . , ,
() . Patuletinst , C16H12O8. , , ,
, . , ,
() . O-Methyl
kaempferol , C16H12O7. , , ,
, . , , ,
,
() . Tri coum a roy l
spermidine , C34H37N3O6. — .
, , ,
, , ,
() .
Coumaroyl
spermidine
derivative
, C41H50N6O10 . — . , , ,
, ,
RT: retention time; MF: molecular formula; ∗error lower than ppm; st conrmed by authentic standard.
（3CO
（3CO
HO
HO
HO
1
23
4
5
6
1
23
4
5
6
1
2
3
4 5
6
0.07
0.08
0.09
0.10
0.11
(mAu)
0.07
0.08
0.09
0.10
0.11
234567891
(Minutes)
O
O
OH
OH
OH
OH
O
O
OH
OH
2
3
4
5
6
789
10
A
B
2
3
4
5
6
789
10
O
O
OH
OH
OH
OH
B
(1)
(2)
C
AC
F : CE-UV electropherogram of the n-butanol fraction of Ta g e tes pat u l a . Experimental conditions: mmol/L borate buer at pH .
with mmol/L Me-𝛽-CD; uncoated fused-silica capillary column, . cm ( cm eective length) × 𝜇m i.d.; kV; ∘C; hydrodynamic
injection . psi ×s;detectionatnm;BF:𝜇g/mL. Peaks: () Tp (patulitrin); () Tp (patuletin).
with the exception of AQF, showed notable larvicidal activity
in the present study. Among the fractions analyzed, EAF was
the most promising, where a concentration as low as mg/L
reduced the larval population by more than half, and where
mg/Lcausedthedeathofalllarvaewithinh.
Faizi et al. [] carried out a nematicidal study with
owers of T. patula, which were rst subjected to a defat-
ting process with petroleum ether and then extracted with
methanol, nally resulting in aqueous, dichloromethane,
ethyl acetate, and butanol fractions. In that study, EAF had
a higher concentration of patuletin, while the BF showed a
lower amount of this substance. e authors reported that
patuletin is generally more potent than patulitrin in other
biological assays, such as antimicrobial and antioxidant.
Comparing our results of larvicidal activity against Ae.
aegypti with those for nematicidal activity against Heterodera
zeae reported by Faizi et al. [], it is observed that, in both
studies, the fraction with better activity was that with a higher
concentration of patuletin and lower level of patulitrin. us,
it is suggested that the larvicidal activity observed in EAF may
Evidence-Based Complementary and Alternative Medicine
T : Percentage of mortality of Aedes aegypti larvae exposed to dierent fractions of Tag e t es pat u l a under laboratory conditions at
mg/L, for h.
Sample h h h h h
Crude acetone extract . . . .B
Fatty waste . . . .B
Aqueous fraction .B∗
Ethyl acetate fraction . . . . .A
n-Hexane fraction . . . . .B
n-Butanol fraction . . . .B
Dichloromethane fraction . . . . .B
DMSO .B
Distilled water B
∗Numbers followed by same letters in a column do not dier according to Tukey test (𝑝 = 0.01).
Time (hours)
Proportion of living individuals
40%
93.4%
100%
Fraction Ethyl acetate
Fraction part greasy
Control (distilled water)
Control (DMSO)
100 150500
0
0.2
0.4
0.6
0.8
1.0
F : Survival rates (log-rank test) of the immature stages of
Ae. aeg ypti exposedtocontrols(distilledwaterandDMSO),ethyl
acetate fraction, and fatty waste of Tagetes p a t u la at mg/L, for
h (𝑝 < 0.0001; variance: .; chi-square: .).
be due to the higher concentration of patuletin seen in this
fraction.
4. Conclusion
Among the semipuried fractions obtained from CAE of
the inorescences of T. pa t ula,BFshowedahigheryield
of the avonoids patuletin and patuletin--O-𝛽-glycoside
(patulitrin).
LC-DAD-MS analysis of CAE and the fractions DF, EAF,
BF, and AQF conrmed that the main substance in EAF was
patuletin and patulitrin in BF.
EAF showed the highest larvicidal activity against Ae.
aegypti with more than % decrease in larval population
at a concentration of mg/L. is high insecticidal activity
observedinEAFmaybeduetothehigherconcentrationof
patuletin in this fraction.
Conflicts of Interest
eauthorshavedeclarednoconictsofinterest.
Acknowledgments
e authors thank the Brazilian agencies Coordenac¸˜
ao de
Aperfeic¸oamento de Pessoal de N´
ıvel Superior (CAPES),
Conselho Nacional de Desenvolvimento Cient´
ıco e Tec-
nol´
ogico (CNPq), Instituto Nacional de Ciˆ
encia e Tecnolo-
gia para Inovac¸˜
ao Farmacˆ
eutica (INCT if), and Fundac¸˜
ao
Arauc´
aria and Programa de P´
os-Graduac¸˜
ao em Ciˆ
encias
Farmacˆ
euticas for their nancial support. Dr. A. Leyva (USA)
helped with English editing of the manuscript.
Supplementary Materials
Figure S. High performance liquid chromatography nger-
print of CAE (A) and BF (B) from the inorescences of
Tagetes patu la. Peaks: () Tp (patulitrin) and () Tp
(patuletin). (Supplementary Materials)
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