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Larvicidal Activity against Aedes aegypti and Chemical Characterization of the Inflorescences of Tagetes patula


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The crude acetone extract (CAE) of defatted inflorescences of Tagetes patula was partitioned into five semipurified 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 34 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 semipurified fractions (750, 500, 300, 100, and 50 mg/L) were assayed for larvicidal activity against Aedes aegypti , with mortality rate expressed as percentage. All fractions except AQF showed insecticidal activity after 24 h exposure of larvae to the highest concentration. However, EAF showed the highest activity with more than 50% reduction in larval population at 50 mg/L. The insecticidal activity observed with EAF might have been due to the higher concentration of patuletin present in this fraction.
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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´
ao em Ciˆ
encias Farmacˆ
euticas, Department of Pharmacy, Laboratory of Pharmaceutical Biology (Palato),
Universidade Estadual de Maring´
a, Avenida Colombo 5790, Maring´
a, PR, Brazil
2Programa de P´
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
orio de Produtos Naturais e Espectrometria de Massas (LAPNEM), Universidade Federal de Mato Grosso do Sul,
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;
Received 30 June 2017; Accepted 13 November 2017; Published 7 December 2017
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 inorescences of Tagetes pat u l a was partitioned into ve semipuried 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 semipuried 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 aer  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,
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
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Volume 2017, Article ID 9602368, 8 pages
Evidence-Based Complementary and Alternative Medicine
diubenzuron, 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 nonspecic 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
“cravo-de-defunto,” or “bot˜
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 inorescences 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-inammatory [], hepatoprotective
[], insecticidal [], nematicidal [, ], larvicidal [],
antibacterial [], antiviral [], and antifungal [].
Accordingly, the aim of this work was to isolate and
identify compounds from the semipuried n-butanol frac-
tion of T. patula by reversed-phase column chromatography
and high-speed countercurrent chromatography (HSCCC)
and to evaluate the chemical prole of the crude extract
and semipuried 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
semipuried fractions was evaluated against Ae. aegypti.
2. Materials and Methods
2.1. Plant Material. e inorescences 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´
(HUEM) under number , and the identication was
provided by Professor Dr. Jimi Nakajima at the Institute
T : Eluent systems used for HSCCC to obtain subfractions.
Subfraction Eluent systems (v/v)
hexane : ethyl acetate : methanol : water ( :  : . : )
Gradient elution with n-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, 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 Semipuried Fractions.
e milled inorescences (. kg) were defatted with n-
hexane by dynamic maceration for three days, with sub-
sequent drying of the inorescences at room temperature.
Aerwards, acetone was used as extraction solvent at a
proportion of % (w/v) in an Ultra-Turrax(UTCKT,
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 []. Briey,  g CAE was resuspended in  L of
methanol : water ( : , v/v) and partitioned with dierent
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-
.–. mm; Macherey Nagel) according to Degani et al.
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 polytetrauoroethylene (PTFE)
column (. mm i.d., total volume capacity of  mL),
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
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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 13CandMHzfor1H), 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
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 MassLynxsoware
(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 identication was performed analyzing the
information of the product ion spectra in comparison to a
previously published dataset.
2.7. Identication 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
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 prole was the
following: initial % B, –min % B, – min % B,
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 . soware. e column used was a fused
silica capillary column (Beckman-Coulter) with dimensions
of . cm total length, . cm eective 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 buer (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 Tpwere used as standards for peak identication. 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
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
semipuried 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
 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 ± 2C. Mortality readings were
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
(2) (3)
(5) (6) (7)
5 1015202530350
Time (min)
(1) (4)
(8) (9)
5 1015202530350
Time (min)
(1) (2)
(6) (7)
(10) (11)
5 1015202530350
Time (min)
(5) (6) (7)
5 1015202530350
Time (min)
5 1015202530350
Time (min)
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 identication of the constituents is given in Table .
was performed by NMR, HPLC-IES-MS/MS and LC-DAD-
MS and resulted in the identication 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 [–], Tpand Tpwere
identied as the avonols patuletin and patuletin--O-𝛽-
glycoside (patulitrin), respectively, which was conrmed 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 identied
data, which were compared with the literature data. From
the samples, eleven compounds were detected and identied
(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 identied
by addition of the isolated substances of this work. Peak  was
identied as Tpand 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 ecient, being
almost four times faster. In addition, in the CE method,
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 aer  h
exposure of the larvae to a concentration of  mg/L, with
exception of AQF.
Aer  h ( days) of exposure, the following mortality
rates were observed at a concentration of  mg/L for the
dierent 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 eective
against Ae. aegypti. us, CAE and all fractions evaluated,
Evidence-Based Complementary and Alternative Medicine
T : Identication 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)
() . 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. — .
, , ,
, , ,
() .
,  C41H50N6O10 . . , , ,
, , 
RT: retention time; MF: molecular formula; error lower than  ppm; st conrmed by authentic standard.
4 5
F : CE-UV electropherogram of the n-butanol fraction of Ta g e tes pat u l a . Experimental conditions:  mmol/L borate buer at pH .
with  mmol/L Me-𝛽-CD; uncoated fused-silica capillary column, . cm ( cm eective length) × 𝜇m i.d.;  kV; C; hydrodynamic
injection . psi ×s;detectionatnm;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
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 dierent 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 dier according to Tukey test (𝑝 = 0.01).
Time (hours)
Proportion of living individuals
Fraction Ethyl acetate
Fraction part greasy
Control (distilled water)
Control (DMSO)
100 150500
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
4. Conclusion
Among the semipuried fractions obtained from CAE of
the inorescences of T. pa t ula,BFshowedahigheryield
of the avonoids patuletin and patuletin--O-𝛽-glycoside
LC-DAD-MS analysis of CAE and the fractions DF, EAF,
BF, and AQF conrmed 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
patuletin in this fraction.
Conflicts of Interest
e authors thank the Brazilian agencies Coordenac¸˜
ao de
Aperfeic¸oamento de Pessoal de N´
ıvel Superior (CAPES),
Conselho Nacional de Desenvolvimento Cient´
ıco e Tec-
ogico (CNPq), Instituto Nacional de Ciˆ
encia e Tecnolo-
gia para Inovac¸˜
ao Farmacˆ
eutica (INCT if), and Fundac¸˜
aria and Programa de P´
ao em Ciˆ
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 inorescences of
Tagetes patu la. Peaks: () Tp(patulitrin) and () Tp
(patuletin). (Supplementary Materials)
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... However, the chemicals that make up the plants determine the usefulness of representatives of this genus (marigolds) as sources of various classes of secondary metabolites that are used in the pharmaceutical and food industries (Giri et al., 2011;Chkhikvishvili et al., 2016). In addition, these substances can also be used as the main component of agents with biocidal activity (Mares et al, 2004;Mulabagal and Tsay, 2004;Faizi et al., 2011;Politi et al., 2016;Ayub et al., 2017;Krzyzaniak et al., 2017;Mir et al., 2019). For example, marigolds are known to be a source of thiophenes, which are a group of heterocyclic sulfur compounds, the most common of which is α-Tertienyl (Ketel, 1986). ...
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The hairy root cultures are promising sources of secondary metabolites of plants, including rare and endangered species. They possess genetic and biochemical stability, unlimited growth rate in free-hormone medium, short doubling times, high biosynthetic activity and ecological purity of plant raw materials. The hairy root cultures of Tagetes patula L. can be used to produce biologically active substances with biocidal activity. The study aimed to determine the virulent strain of Agrobacterium rhizogenes and the most effective period of co-cultivation of T. patula leaf explants with an agrobacterium to induce actively growing hairy root cultures. We used 3 strains (A-4b, 8196RT and 15834). The time of infection ranged from 3 to 33 hours in increments of 3 hours. We found that 24 h is the best time of infection to induce hairy roots with the highest transformation efficiency (92%). The wild strain A. rhizogenes 15834 turned out to be the most virulent when infected leaf explants of spreading marigold. This strain provided the maximum transformation effect, reaching 85.4%. We have identified 5 actively growing clones of hairy roots with intensive branching, the growth indices of which were 64-75. In the future, they will be transferred to a liquid medium for biomass accumulation and scaling.
... Five compounds were isolated from the cytotoxic fractions of R. abyssinicus. The spectroscopic data of isolated compounds agree well with those reported for chrysophanol (1) [21], physcion (2) [22], emodin (3) [23], citreorosein (4) [24] and β-sitosterol (5) [25]. ...
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Rumex abyssinicus showed strong cytotoxicity against HeLa cells (IC50 = 22.25 μg/mL) and weak cytotoxicity against PC3 and BJ cells with percent inhibition of 58.6, 25.8 and 29.7% at 30.0 μg/mL. It showed moderate antifungal activity against Aspergillus niger with a percent growth inhibition of 55.5% at 3000 µg/mL. It also strongly inhibited oxidative burst with IC50 value of 24.8 μg/mL. DCM (100%) and DCM: EtOAc (1:1) fractions showed strong cytotoxicity against HeLa cells, whilst pet ether: DCM (1:1) fraction showed strong cytotoxicity against PC3 cells with IC50 values of 29.3, 26.3 and 24.3 μg/mL, respectively. Moreover, the DCM: EtOAc (1:1) fraction inhibited ROS production with IC50 value of 18.8 μg/mL. Cytotoxic fractions afforded chrysophanol (1), physicon (2), emodin (3), citreorosein (4) and β-sitosterol (5). Among the isolated compounds, emodin (3) showed strong cytotoxicity against HeLa cells, whilst chrysphanol (1) and physicon (2) showed strong cytotoxicity against PC3 cells with IC50 values of 8.94, 22.5, and 28.5 µM, respectively. In addition, emodin (3) and citreorosein (4) showed strong inhibition against ROS production with an IC50 value of 16.2 and 38.2 μg/mL. The findings of this study suggest R. abyssinicus as a good candidate for cancer and inflammation management. KEY WORDS: Polygonaceae, Rumex abyssinicu Jacq., Cytotoxic, Antifungal, Anti-Inflammatory, Reactive oxygen species Bull. Chem. Soc. Ethiop. 2022, 36(4), 879-892. DOI:
... International agencies are encouraging the development and application of different botanical formulations, such as extracts and essential oils, for the control of epidemiologically important insects, as such formulations cause less damage to nontarget organisms than do conventional insecticides (Kumar et al. 2020;Goodarzi et al. 2019;Bilal et al. 2017). Other advantages of using these botanical insecticides include obtaining them from renewable resources (Krzyzaniak et al. 2017) and producing them in the form of complex combinations, in which bioactive compounds act synergistically to reduce the possibility of insecticide resistance (Pavela et al. 2019;Ravi et al. 2018). ...
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Mosquitoes (Diptera: Culicidae) are insect vectors of epidemiologically important arboviruses owing to their behavior, physiology, morphology, and proximity to humans, which require incisive strategies to contain their spread. The failure of current arbovirus management plans and lack of fully effective treatments suggest that vector control by botanical insecticides could be an effective and safe strategy. Botanical insecticides are obtained from renewable sources and have complex chemical compositions, different modes of action, and selective toxicity for target organisms. In this review, we present the main control strategies for insects belonging to the genera Aedes, Culex, and Anopheles and discuss the possibility of using botanical insecticides in the integrated management of vectors. Numerous botanical insecticide formulations are presented, and their potential modes of action during the immature stages include damage to the egg exocorionic network and abnormal disruption of embryos, which result from deficiencies in egg chitinization, impairment of larval morphology, and inhibition or differential expression of enzymes, promoting changes in the digestive tract epithelium and reduced larval mobility, and impairment of external surfaces or the respiratory system of pupae, altering pupal swimming patterns. In adult insects, botanical insecticides can promote incomplete ecdysis, in addition to dysfunction of olfactory receptors, food traffic, and reproductive function. Thus, broad-spectrum botanical insecticides can be used to control the different stages of insect development. The contributions of nanotechnology to vector control should be further explored to enhance the insecticidal activity and stability of botanical insecticides under different conditions.
... Then, the dry residue was extracted with butyl alcohol and methylene chloride to give butyl alcohol and methylene chloride layers. The butyl alcohol layer was subjected to silica gel column chromatography using CH 2 Acid Hydrolysis of Compound 2. Acid hydrolysis was performed using 0.1 mol/L sulfuric acid at 100 °C for 1 hour. The mixture was neutralized with silver carbonate and extracted with ethyl acetate. ...
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Two novel flavonoids (1, 2) and 3 known compounds (3-5) were isolated from the flowers and whole plant of Tagetes patula L., and their structures were elucidated by means of ultra-high performance liquid chromatography with electrospray ionization, coupled to quadrupole-time-of-flight/mass spectrometry, 1 H and 13 C-nuclear magnetic resonance (NMR), as well as 2-dimensional-NMR (heteronuclear single quantum correlation and heteronuclear multiple bond correlation) and chemical methods. In addition, all the compounds were examined for their neuroprotective action on the injury of SH-SY5Y cells induced by glutamate, indicating that the protective effect of these compounds on glutamate-induced SH-SY5Y cell was marigold biflavone > patuletin > quercetin > kaempferol-3-O-β-d-glucoside > patuletin-3-O-α-l-arabinopyranoside. Thus, it could be concluded that flavonoids played a key role in the neuroprotective action of T. patula.
... Further studies can be carried out towards chemical fractionation, in search of a new bioactive compound (Munhoz et al., 2014). Sub ethyl acetate fraction (patuletin enriched) of acetone extract of the T. patula flowers showed more potent larvicidal action against Aedes aegypti (Krzyzaniak et al., 2017). The previous larvicidal action might be due to the presence of flavonoid but the work of Rajasekaran et al. linked it to thiophenes produced in callus cultures of T. patula, that showed larvicidal effect against mosquito larvae (Rajasekaran et al., 2003). ...
... Biomonitored phytochemical research aims to identify which substances are responsible for specific biological activities [37][38][39]. Biomonitored research involves studies across several knowledge areas to identify the bioactive compounds of interest [40]. ...
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Helicteres velutina K. Schum (Sterculiaceae), a member of Malvaceae sensu lato, is a Brazilian endemic plant that has been used by the indigenous tribe Pankarare as an insect repellent. A previous study has reported the isolation of terpenoids, flavonoids and pheophytins, in addition to the larvicidal activity of crude H. velutina extracts derived from the aerial components (leaves, branches/twigs, and flowers). The present study reports the biomonitoring of the effects of fractions and isolated compounds derived from H. velutina against A. aegypti fourth instar larvae. A crude ethanol extract was submitted to liquid–liquid extraction with hexane, dichloromethane, ethyl acetate and n-butanol to obtain their respective fractions. Larvicidal evaluations of the fractions were performed, and the hexane and dichloromethane fractions exhibited greater activities than the other fractions, with LC50 (50% lethal concentration) values of 3.88 and 5.80 mg/mL, respectively. The phytochemical study of these fractions resulted in the isolation and identification of 17 compounds. The molecules were subjected to a virtual screening protocol, and five molecules presented potential larvicidal activity after analyses of their applicability domains. When molecular docking was analysed, only three of these compounds showed an ability to bind with sterol carrier protein-2 (1PZ4), a protein found in the larval intestine. The compounds tiliroside and 7,4′-di-O-methyl-8-O-sulphate flavone showed in vitro larvicidal activity, with LC50 values of 0.275 mg/mL after 72 h and 0.182 mg/mL after 24 h of exposure, respectively. This is the first study to demonstrate the larvicidal activity of sulphated flavonoids against A. aegypti. Our results showed that the presence of the OSO3H group attached to C-8 of the flavonoid was crucial to the larvicidal activity. This research supports the traditional use of H. velutina as an alternative insecticide for the control of A. aegypti, which is a vector for severe arboviruses, such as dengue and chikungunya.
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Mosquito-borne diseases caused by infected mosquitoes like dengue fever, Zika fever, Chikungunya and malaria are spread to humans through biting causing disease epidemics all over the world. This indicates the necessity for new mosquito-borne disease control strategies in Saudi Arabia and internationally. This study evaluated the potential of local bacteria isolated from the soil of the Rahat region of Makkah, Saudi Arabia for the biocontrol of Aedes aegypti larvae, a major agent for the transmission of dengue fever. Soil samples were collected from the Rahat region, Makkah for the isolation of bacteria. The bacteria were evaluated for larvicidal activity against the larvae of A. aegypti. The bacteria that show toxicity to the larvae were identified using morphological and molecular characteristics. Four different strains of the bacteria with a toxicity towards A. aegypti larvae were isolated and identified. Two of the isolates (Brevibacillus centrosporus N8 and Cytobacillus species N7) caused 100% mortality in 24 h while the other two isolates (Escherichia coli N3 and Escherichia coli N4) caused at least 70% mortality. The findings of this study revealed the larvicidal activity of soil bacteria from the Rahat region, Makkah and could be a potential candidate for the biocontrol of mosquito vectors.
This study aimed to characterize T. erecta and T. patula extracts, develop a technique of encapsulation, evaluate insecticidal activity of extracts, fractions and liposomes containing fractions. Extracts were characterized by HPLC-MS/MS. Liposomes were prepared using Dipalmitoylphosphatidylcholine (DPPC) and were characterized by Dynamic Light Scattering (DLS) measuring average size, polydispersion index and zeta potential. Insecticidal evaluation was performed applying extracts on S. frugiperda, and fractions and liposomes on S. zeamais. Characterization of extracts showed several flavonoids in both extracts and characterization by DLS showed formation of liposomes. In the evaluation of insecticidal activity on S. frugiperda, both extracts presented interference in larval viability. In the evaluation of insecticidal activity on S. zeamais, T. erecta Hex/AcOEt (50:50) fraction at 50 mg mL⁻¹ presented 98.33% mortality at the 60th hour, and T. patula Hex/AcOEt (50:50) fraction at 50 mg mL⁻¹ presented 100% mortality at the 48th hour, being these the greatest activities of each species at the lowest concentration and time. Fraction incorporated into liposomes did not present insecticidal activity (T. erecta fraction = 6.67%; T. patula fraction = 0.00%), demonstrating encapsulation efficacy. Therefore, evaluated extracts presented insectidical activity on the experimental models. Besides, liposomes containing fractions were formed and they interfered in insecticidal activity. Thus, these results provide information of T. erecta and T. patula extracts and fractions, and suggest a possible application in agricultural crops, as well as the use of liposomes technology to encapsulate bioactive compounds with insecticidal activity, supporting new studies.
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Background: Derris elliptica extracts have a high larvicidal potential against the laboratory strain of Aedes aegypti larvae, but the effect on offspring larvae of pyrethroid-resistant strains of the species is lack understood. This study aimed to determine the larvicidal activity of the ethyl acetate extract of tuba root against the third-instar larvae of the Cypermethrin-resistant Ae. aegypti offspring. Methods: The experimental study occupied four levels of ethyl acetate extract of D. elliptica namely 10, 25, 50, and 100 ppm, and each level was four times replicated. As many as twenty of healthy third-instar larvae, offspring of Cy­permethrin-resistant Ae. aegypti were subjected to each experiment group. Larval mortality rate and lethal concentration 50% subject (LC50) were calculated after 24 and 48 hours of exposure time. Results: Mortality of larvae increased directly proportional to the increase of extract concentration. Larval mortality rates after 24 and 48 hours of exposure were 40–67.5% and 62.5–97.5%, and LC50 were 34.945 and 6.461ppm, respec­tively. Conclusion: The ethyl acetate extract of D. elliptica has the high effectiveness larvicidal potential against the third-instar larvae, offspring of the Cypermethrin-resistant Ae. aegypti. Isolation of the specific compound is necessarily done to obtain the active ingredient for larvicide formulation.
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Abstract Importance of medicinal plants to health care has been great and herbal preparations are being produced at industrial scale particularly in developing countries. The plant products obtained have a long history of use in therapeutics, aromatherapy and food depending on the chemical constituents and their bioactivity. In the recent past, marigolds have received a great attention in scientific research, because of their multiple use and also the information available about their phytochemistry and bioactivity. Tagetes species commonly known as marigold is native to Mexico, being used for medicinal and ornamental purposes. The plant is useful due to its unique phytoconstituents for a range of diseases and disorders and is reportedly effective against piles, kidney troubles, muscular pain, ulcers and wound healing and the flowers are helpful in fever, stomach and liver complaints and also in eye diseases. In India, marigold is also extensively used on religious and social occasions such as in the beautification of mandaps and pooja places; offerings at temples; marriage decorations and landscape planning due to variable size and colour of its flower. Present review is an effort to bring together the different strategies developed for the growth and cultivation of marigold, its ecophysiological and remediation relevance under a variety of environmental conditions and possible allelopathic potential. It includes reports on pharmacological aspects like antibacterial, antifungal, larvicidal, hepatoprotective, insecticidal, mosquitocidal, nematicidal, wound healing, antioxidant, anticancer and antidiabetic properties/ activity of Tagetes .
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Background The tick Rhipicephalus sanguineus is the species with the largest worldwide distribution and is proven to be involved in the transmission of pathogens such as Babesia canis, Ehrlichia canis, Coxiella burnetii, Rickettsia ricketsii, Rickettsia conorii, among others. Studies have demonstrated acquisition of resistance to some of the active principles used in commercial formulations of acaricides. Tagetes patula (Asteraceae) is a plant with highlighted economic and commercial importance due to the production of secondary metabolites with insecticide and acaricide potential, mainly flavonoids, thiophenes and terpenes. Methods The in vitro acaricide action of the ethanolic 70% extract from aerial parts of T. patula, obtained by percolation, was evaluated against larvae and engorged adult females of Rhipicephalus sanguineus by immersion test for 5 minutes. The chemical characterization of this extract was done by liquid chromatography coupled with mass spectrometry (LC-MS), using direct injection of sample. Results Despite T. patula not proving lethal to adults in any of the concentrations tested, at 50.0 mg/mL oviposition rate decreased by 21.5% and eliminated 99.78% of the larvae. Also it was determined that the best results were obtained with 5 minutes of immersion. From the chromatographic analysis twelve O-glycosylated flavonoids were identified. Conclusions This is the first report on the acaricidal activity of T. patula extract against Rh. sanguineus. If we consider the application of the product in the environment, we could completely eliminate the larval stage of development of the ixodid Rh. sanguineus.
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The objective of present study was to evaluate the variation in phenolic profile, β-carotene, flavonoid contents, antioxidant and antimicrobial properties of Tagetes eracta and Tagetes patula (T. erecta and T. patula) through different in vitro assays.,Antioxidant activity was determined through 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and inhibition of linoleic acid peroxidation assays and antibacterial and antifungal activities studied using the disc diffusion and resazurin microtiter-plate assays against bacterial and fungal strains. Moreover, Total phenolics (TP), total carotenoids (TC) and total flavonoids (TF) were also determined. Highest (TP 35.8 mg GAE/g) and TF (16.9 mg CE/g) contents were found in methanolic extract of T. patula. T. erecta extract showed higher TC contents (6.45 mg/g) than T. patula extract (6.32 mg/g). T. erecta exhibited the highest DPPH radical-scavenging activity (IC50 ) (5.73 μg/mL) and inhibition of linoleic acid peroxidation (80.1%). RP-HPLC revealed the presence of caffeic acid, sinapic acid and ferulic acid in Tagetes extracts, m-coumaric acid in T. erecta whereas chlorogenic acid in T. patula extract only. Both extracts possessed promising antimicrobial activity compared to the ciprofloxacin and flumequine (+ve controls) against Bacillus subtilis and Alternaria alternate. Both extract were rich source of polyphenols exhibiting excellent biological activities. This article is protected by copyright. All rights reserved.
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The flowers of French marigold ( Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μ M H 2 O 2 . Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10) in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers.
Naftoquinonas são metabólitos secundários produzidos por algas, fungos, plantas e animais, caracterizadas por apresentarem múltiplas atividades biológicas. Em Angiospermae as naftoquinonas são encontradas em diversas famílias com destaque para Bignoniaceae, Ebenaceae, Plumbaginaceae, Verbenaceae, dentre outras. O perfil químico da família Bignoniaceae distingue-se pela ocorrência predominante de terpenóides e quinonas, além de alcalóides, flavonóides e derivados não nitrogenados de cadeia longa, entre outros. Esse trabalho visa apresentar as atividades biológicas descritas na literatura para as naftoquinonas isoladas de espécies da família Bignoniaceae, em particular dos gêneros Handroanthus, Paratecoma, Tabebuia e Tecoma.
The methanol spectra of flavones and flavonols exhibit two major absorption peaks in the region 240 – 400 nm¹. These two peaks are commonly referred to as Band I (usually 300 – 380 nm, Table V-1 records the λmaxvalues for Band I for all flavones and flavonols examined in the present investigation), and Band II (usually 240 – 280 nm). Band I is considered to be associated with absorption due to the B-ring cinnamoyl system, and Band II with absorption involving the A-ring benzoyl system (see III) [1].