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Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 153-169, March 2021

Crude extract of the tropical tree Gallesia integrifolia (Phytolaccaceae)

for the control of Aedes aegypti (Diptera: Culicidae) larvae

Wanessa de Campos Bortolucci

, Herika Line Marko de Oliveira

, Leiluana Roque Oliva

José Eduardo Gonçalves

2,3

, Ranulfo Piau Júnior

, Carla Maria Mariano Fernandez

Nelson Barros Colauto

, Giani Andrea Linde

& Zilda Cristiani Gazim

1. Universidade Paranaense, Umuarama-PR, Brazil; wanessa.bortolucci@edu.unipar.br, herika.marko@edu.unipar.br,

carlamfernandez@prof.unipar.br, nbc@prof.unipar.br, gianilinde@prof.unipar.br, cristianigazim@prof.unipar.br

2. UniCesumar, Maringá-PR, Brazil; leiluana.oliva@gmail.com, jose.goncalves@unicesumar.edu.br

3. Cesumar Institute of Science, Technology and Innovation - ICETI, UniCesumar, Maringá-PR, Brazil.

4. Graduate Program in Animal Science with emphasis on Bioactive Products. Universidade Paranaense, Umuarama-PR,

Brazil; piau@prof.unipar.br

* Correspondence

Received 26-III-2020. Corrected 12-X-2020. Accepted 11-XI-2020.

ABSTRACT. Introduction: Phytoinsecticides are alternatives to control insects in different stages, Gallesia

integrifolia (Spreng.) Harms, Phytolacaceae family, popularly known as pau d’alho, garlic tree, and guararema

in Brazil, is known due to its strong alliaceous odor because of the presence of sulfur molecules in the plant.

This species presents biological activity and potential insecticide effect that is still unexploited. Objective: This

study aimed to evaluate the biological activity of the ethanolic crude extract from G. integrifolia leaves, flowers,

and fruits on the control of Aedes aegypti third-stage larvae and pupae. Methods: The botanical material was

collected in city Umuarama, Paraná, Brazil at the coordinates (23º46’16” S & 53º19’38” WO), and altitude of

442 m, the fruits of G. integrifolia were collected in May and the leaves and flowers in December 2017. The

crude extracts of G. integrifolia leaves, flowers, and fruits were prepared by dynamic maceration technique. The

chemical composition of the extracts was determined by gas chromatography coupled to a mass spectrometry.

The insecticidal activity of the crude extracts of G. integrifolia were carried out on larvae and pupae of A.

aegypti in concentrations between 0.001 to 25 000 mg/mL, and afterwards the lethal concentrations that kill 50

% (LC

) and 99.9 % (LC

99.9

) were determined by probit analysis. Anticholinesterase activity was determined

by bioautographic method at concentrations from 0.000095 to 50 mg/mL. Results: The yield of G. integrifolia

crude extracts were 8.2, 9.1, and 17.3 % for flowers, fruits, and leaves, respectively. The chemical composition

of G. integrifolia extracts was characterized by presence of fatty acid esters, phytosterols, vitamins, oxygenated

diterpenes and organosulfur compounds. The flower extract presented the high amount of sulfur compounds

(20.2 %) such as disulfide, bis (2-sulfhydryl ethyl) (11.9 %), 2,3,5-trithiahexane (6.2 %), 1,2,4-trithiolane (1.1

%), and 2,4-dithiapentane (1.1 %). Regarding the insecticidal activity, flower extract showed highly active with

99.9

of 0.032 mg/mL and LC

99.9

of 0.969 mg/mL on A. aegypti larvae and pupae, respectively, and the highest

inhibition of acetylcholinesterase enzyme (0.00019 mg/mL) ex situ. The flower extract presented anticholines-

terase and larvicide activity, respectively, 12.8 % and 35.6 % greater than the control temephos. Conclusions:

This study opens new perspectives on the use of extracts from G. integrifolia as a bioinsecticide alternative for

the control of A. aegypti larvae and pupae.

Key words: anticholinesterase; disulfide; bis(2-sulfhydryl ethyl); pau d’alho; semiochemical; sulfur compounds.

de Campos Bortolucci, W., Marko de Oliveira, H.L., Roque Oliva, L., Gonçalves,

J.E., Piau Júnior, R., Mariano Fernandez, C.M., Barros Colauto, N., Linde, G.A.,

& Gazim, Z.C. (2021). Crude extract of the tropical tree Gallesia integrifolia

(Phytolaccaceae) for the control of Aedes aegypti (Diptera: Culicidae) larvae. Revista

de Biología Tropical, 69(1), 153-169. DOI 10.15517/rbt.v69i1.41225

ISSN Printed: 0034-7744 ISSN digital: 2215-2075

DOI 10.15517/rbt.v69i1.41225

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Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 153-169, March 2021

Aedes aegypti (Linnaues, 1762) is the

main vector of arboviruses such as dengue,

chikungunya fever, Zika virus and yellow fever

(Fofana, Beugré, Yao-Acapovi, & Lendzele,

2019). The combat to disseminate this mosqui-

to is a challenge in tropical countries, mainly in

South America, and a problem to health due to

the high morbidity and mortality rates (Pavela,

2015; Benelli & Mehlhorn, 2016; González et

al., 2019). In Brazil, in 2019, there were 1 544

987 recorded cases of dengue with 782 deaths,

besides 132 205 cases of chikungunya with 92

deaths, and 10 768 cases of Zika virus with

three deaths (Ministério da Saúde, 2020).

Synthetic chemical insecticides from

organophosphate class are utilized against A.

aegypti, mainly temephos, an organothiophos-

phate insecticide (Benelli et al., 2017). How-

ever, organothiophosphates are reported to

have genotoxic action on human hepatic cells

(Benitez-Trinidad et al., 2015) and neural dam-

ages in rats (Laurentino et al., 2019). Besides, the

broad action spectrum of this insecticide causes

non-specific deaths of insects (Braga & Valle,

2007; Čolović & Krstić, 2013; Mrdaković et

al., 2016), bees, butterflies, and other pollinat-

ing insects (Morais, Bautista, & Viana, 2000).

Moreover, the increased concentration of this

insecticide in Brazilian cities indicates that

there is a resistance process to this chemical

to control A. aegypti (Jonny, Silva, Fantinatti,

& Silva, 2015). An alternative to organophos-

phates for the control of A. aegypti are the non-

cumulative synthetic pyrethroids which are,

however, toxic to aquatic arthropods, fish, and

bees, eliminating essential insects that act on

the biological control of insects, besides caus-

ing adverse effect to human health due to long

exposure to it (Camargo et al., 1998; Bellinato

et al., 2016).

Another alternative to organophosphates

would be phytoinsecticides such as essential

oils that in general have a quicker degra-

dation and/or volatilization, causing smaller

environmental damage, mainly to pollinating

insects. For instance, to control Varroa destruc-

tor that parasites bees, essential oils of neem

(Azadirachta indica), lemon (Citrus limon),

and eucalyptus (Eucalyptus citriodora, current

name Corymbia citriodora) are utilized without

killing the beehive (Bakar et al., 2017). How-

ever, some phytoinsecticides can have a similar

action to organophosphates such as the garlic

essential oil (Allium tuberosum and Allium

sativum) used to control larvae and/or repel

mosquitoes such as Aedes spp., Anopheles

spp., and Culex spp. (Denloye, Makanjuola,

& Babalola, 2003; Trongtokit, Rongsriyam,

Komalamisra, & Apiwathnasorn, 2005; Rai-

mundo et al., 2018).

Gallesia integrifolia (Spreng.) Harms,

Phytolacaceae family, popular names pau

d’alho, garlic tree, and guararema in Brazil,

is known due to its strong alliaceous odor

because of the presence of sulfur molecules in

the plant, despite not belonging to the Alliaceae

family (Barbosa, Teixeira, & Demuner, 1997;

Sambuichi, Mielke, & Pereira, 2009). It has

several synonyms such as Gallesia gorazema,

Gallesia integrifolia var. ovata, Gallesia ovata,

Gallesia scorododendrum, and Thouinia inte-

grifolia (Hassler, 2018). This plant has been

reported since 1821 for common treatments of

orchitis, verminosis, and rheumatism (Corrêa-

Filho, 1984; Akisue, Akisue, & Oliveira, 1986),

and the essential oil has been reported to act

against bovine tick (Rhipicephalus microplus)

(Raimundo et al., 2017) and to have antifungal

activity (Raimundo et al., 2018).

The aqueous crude extract of G. integri-

folia leaves are used in organic agriculture to

control Botrytis cinerea during pre- and post-

harvest of grapes in viniculture (Silva et al.,

2017). Hydroalcoholic extracts of G. integri-

folia leaves were effective to control eggs and

larvae of bovine ticks (Rhipicephalus micro-

plus) (Dias, Tanure, & Bertonceli, 2018). The

hydroethanolic extract of G. integrifolia inner

bark had bacteriostatic activity and no toxicity

to epithelial cells of Chinese hamster ovary

(Arunachalam et al., 2016). The dichlorometh-

ane and methanolic extracts of G. integrifolia

bark had antifungal activity (Freixa et al.,

1998). Moreover, dichloromethane extract of G.

integrifolia roots has antinociceptive and anti-

inflammatory activities in vivo with mice and

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antiviral activity against herpes simplex virus

(Silva et al., 2013). In addition, riverside dwell-

ers usually burn G. integrifolia dried leaves to

repel mosquitoes from the genus Anopheles,

vector of malaria, showing its potential biocide

activity (Pérez, 2002). However, despite these

reports, there are no studies on the extract from

G. integrifolia leaves to control insect, mainly

A. aegypti. Thus, this study aimed to evaluate

the biological activity of the ethanolic crude

extracts from G. integrifolia leaves, flowers,

and fruits to control A. aegypti third-stage lar-

vae and pupae to develop an alternative for the

control of this insect.

MATERIALS AND METHODS

Plant material: fruits, leaves, and flowers

of G. integrifolia were harvested in Umuarama

at the coordinates (23º46’16” S & 53º19’38”

WO) and altitude of 442 m. Leaves and flow-

ers were harvested in the morning in December

2017 and fruits were harvested in the morn-

ing in May 2017. The plant was identified

and a sample was deposited in the Herbarium

of Western Paraná State University, Cascavel

Campus, Center of Biological Sciences and

Health, under the number 1 716. This spe-

cies was registered in the National System

for the Management of Genetic Heritage and

Associated Traditional Knowledge (SisGen,

acronym in Portuguese) under the registration

number A0A10E1.

Crude extract preparation: leaves, flow-

ers, and fruits of G. integrifolia (500 g) were

pulverized to 850 μm with a knife mill (Willye

TE-650) and three extracts were obtained after

dynamic maceration with solvent (ethanol 96

ºGL) depletion. The extracts were filtered and

the solvent was removed in a rotating evapora-

tor (Tecnal TE-210) at 40 °C. The yield of each

crude extract (dry basis) was calculated in trip-

licate by the mass of the obtained crude extract

(g) divided by the mass of the vegetal matter

(leaves, flowers, or fruits) (g), multiplied by

100 and expressed in percentage (Ministério da

Saúde, 2010).

Chemical characterization of the crude

extract: for the chemical identification of the

compounds, the crude extracts from G. inte-

grifolia leaves, flowers, and fruits (100 mg)

were analyzed by gas chromatography (Agilent

19091S-433) coupled to a mass spectropho-

tometer (Agilent 19091J-433) (CG-MS). An

HP-5MS UI 5 % analytical column (30 m ×

0.25 mm × 0.25 μm) was utilized with an ini-

tial temperature of 60 °C, and kept for 3 min.

Then, a ramp of 5 °C/min and the temperature

was increased to 300 °C and kept for 10 min

and, finally, to 310 °C with a ramp of 10 °C/

min for 10 min. Helium was utilized as the car-

rier gas at the linear speed of 1 mL/min until

300 °C and pressure release of 56 kPa. The

injector temperature was 300 °C; the injection

volume was 2 μL; the injection was in split

mode (20:1). The transfer line was kept at 285

°C and the ionization source and quadrupole

at 230 °C and 150 °C, respectively. The MS

detection system was utilized in scan mode, in

the range of mass/load ratio (m/z) of 40-550

with 3 min solvent delay. The compounds were

identified by comparing their mass spectra with

the ones from Wiley 275 libraries, and compar-

ing their retention indices (RI) obtained by a

homologous series of n-alkane standards (C

) (Adams, 2017).

Insecticidal activity: Aedes aegypti third-

stage larvae and pupae were obtained from

the Department of Sanitary Surveillance of

Umuarama, Paraná, Brazil. The extracts were

diluted from 0.001 to 25 000 mg/mL in an

aqueous solution containing 20 mL/L polysor-

bate-80. Ten A. aegypti larvae were placed in

25 mL flasks with 1.0 mL extract solution at

different concentrations and kept in the dark at

room temperature for 24 h (Costa et al., 2005;

Fernandes, Souza Freitas, da Costa, & da Silva,

2005; Bonato, Cavalca, & Lolis, 2010). Larvae

without movement and response to stimuli

were considered dead (Bonato et al., 2010).

The same procedure was used for A. aegypti

pupae. The negative control was 20 mL/L poly-

sorbate-80 aqueous solution and the positive

control was the organothiophosphate temephos

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at the concentration ranging from 0.009 to 500

mg/mL (Macoris, Camargo, Silva, Takaku,

& Andrighetti, 1995; Camargo et al., 1998).

Lethal concentrations (LC

and LC

99.9

) were

calculated by the probit analysis.

Anticholinesterase activity: the anticho-

linesterase activity was determined by the

bioautographic method according to Marston,

Kissling, & Hostettmann (2002) with modi-

fications (Yang et al., 2009). The methanolic

solutions of the leaf, flower, and fruit extracts,

and the positive control temephos were tested

at concentrations varying from 50 to 0.000095

mg/mL (in methanol). The samples were plot-

ted in aluminum TLC (thin-layer chromatog-

raphy) plates (10 × 10 cm; 0.2 mm-thick 60

F254 silica gel), dried and sprayed with an

acetylcholinesterase enzyme (500 U/mL) solu-

tion in a buffer solution of tris (hydroxymethyl)

aminomethane hydrochloridrate (0.05 M, pH

7.8), sprayed with an α-naphthyl acetate solu-

tion (0.15 %) and kept at 37 °C for 20 min.

After that, the chromo plates were sprayed with

fast blue B salt colorimetric reagent (0.05 %)

resulting in a purple color surface. The anticho-

linesterase activity of each G. integrifolia crude

extract was determined by the emergence of

white stains after 10 min, showing the inhibito-

ry action of the evaluated concentration on the

enzyme activity by contrasting with the purple

color of the colorimetric reagent. Temephos

was used at the same concentrations of the

crude extracts as a positive control.

Statistical analysis: the experimental

design was completely randomized and all

assays carried out in triplicate. Data were sub-

mitted to analysis of variance (ANOVA) and

compared utilizing SPSS statistics 22 program

by Duncan test (P ≤ 0.05). Lethal concentra-

tions (LC

and LC

99.9

) and confidence inter-

vals were calculated by probit analysis.

RESULTS

The yield of the crude extracts from G.

integrifolia flowers, fruits, and leaves was 8.2,

9.1, and 17.3 %, respectively, with the greatest

yield for the leaf extract. The chemical identifi-

cation of the extracts showed 20 chemical com-

pounds in the flowers, 22 in the fruits, and 17

in the leaves, considering only the compounds

in the relative area greater than 0.5 % (Table 1).

The major compounds of the ethanolic extract

from G. integrifolia flowers were vitamin E

(18.0 %) and disulfide, ethyl iso-allocholate

(10.6 %), from fruits were vitamin E (20.9 %),

linolenic acid methyl ester (14.0 %), disulfide,

bis(2-sulfhydryl ethyl) (11.9 %), and phytol

(10.2 %), whereas from leaves were phytol

(30.9 %), linolenic acid methyl ester (30.5 %),

and methyl palmitate (10.9 %).

TABLE 1

Chemical composition obtained by gas chromatography-mass spectrometry of the extracts

from Gallesia integrifolia leaf, flower, and fruit

Peak

Compound

calc

Relative area (%)

m/z Structure

Leaves Flowers Fruits

1 2,4-dithiapentane 808 - 1.06 2.12 108.01 C

a,b,c

2 1,2,4-trithiolane 810 0.86 1.10 1.76 124.23 C

a,b,c

3 R-limonene 847 - 0.91 - 139.98 C

a,b,c

4 2,3,5-trithiahexane 885 - 6.17 4.67 140.27 C

a,b,c

5 Disulfide, bis(2-sulfhydryl ethyl) 935 - 11.91 - 185.96 C

a,b,c

6 Cedr-8-en-13-ol 936 - 0.93 - 220.18 C

O a,b,c

7 n-hexadecanoic acid 937 - 5.57 1.04 256.24 C

a,b,c

8 Palmitoleic acid, methyl ester 938 1.57 - - 268.24 C

a,b,c

9 Methyl palmitate 949 10.91 4.69 5.05 270.26 C

a,b,c

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TABLE 1 (Continued)

Peak

Compound

calc

Relative area (%)

m/z Structure

Leaves Flowers Fruits

10 Linoleic acid 973 5.99 - - 280.45 C

a,b,c

11 Ethyl palmitate 1 099 1.43 - 2.11 284.48 C

a,b,c

12 Stearic acid 1 100 1.05 - - 284.28 C

a,b,c

13 Linolenic acid methyl ester 1 101 30.53 - 4.54 292.24 C

a,b,c

14 10,13-octadecadienoic acid, Methyl ester 1 103 - 6.07 5.04 294.26 C

a,b,c

15 Methyl linoleate 1 107 - 5.17 - 294.47 C

a,b,c

18 Phytol 1 137 30.92 10.24 6.50 296.31 C

O a,b,c

19 Linoleic acid ethyl ester 1 171 3.87 14.00 2.28 308.27 C

a,b,c

20 Ethyl octadecanoate 1 174 - 0.55 - 312.30 C

a,b,c

22 α-monoolein 1 262 - - 2.61 354.61 C

a,b,c

24 Isohumulone 1 367 4.95 - - 356.29 C

a,b,c

25 n.i. 1 430 0.60 0.74 0.30 - - a,b,c

26 Stigmasterol 1 478 1.57 - 7.38 412.70 C

O a,b,c

27 β-sitosterol 1 479 0.64 - 5.59 414.38 C

O a,b,c

28 γ-sitosterol 1 506 2.61 - 2.14 414.38 C

O a,b,c

29 β-tocopherol 1 559 0.56 - - 416.36 C

a,b,c

30 γ-tocopherol 1 618 0.73 - - 416.36 C

a,b,c

32 Lupeol 1 671 - - 6.84 426.38 C

O a,b,c

33 α-amyrin 1 716 - - 1.97 426.38 C

O a,b,c

35 Vitamin E 1 761 - 20.86 18.04 430.38 C

a,b,c

36 Ethyl iso-allocholate 1 780 - - 10.60 436.63 C

a,b,c

37 Betulin 1 782 1.11 - 1.10 442.38 C

a,b,c

38 Inotodiol 1 792 - 2.63 - 442.38 C

a,b,c

40 Lupeol acetate 1 847 - - 2.60 468.39 C

a,b,c

41 Cycloartenol acetate 1 914 - 0.99 4.88 468.76 C

a,b,c

42 13,14-epoxyoleanan-3-ol, acetate 1 916 - 1.18 - 470.37 C

a,b,c

44 Barringtogenol C 2 174 - 3.53 - 490.36 C

a,b,c

45 3-O-Acetyl-6-methoxy-cycloartenol 2 194 - 1.68 0.77 498.40 C

a,b,c

Total identified 99.30 99.24 99.63

Fatty acids 7.04 5.57 1.04

Sulfur compounds 0.86 20.24 8.55

Monoterpene hydrocarbons - 0.91 -

Oxygenated sesquiterpene - 0.93 -

Oxygenated diterpene 30.92 10.24 6.50

Triterpenes 1.11 6.16 12.52

Fatty acid esters 48.31 29.93 19.02

Vitamin precursor 1.29 - -

Phytoesterol 4.82 0.99 19.99

Vitamins - 20.86 18.04

Alpha acids 4.95 - -

Steroidal compound - - 10.60

Others - 3.41 3.37

Compounds listed in order of elution in column HP-5MS;

RI = Identification based on retention index (RI) using

a homologous series of n-alkane C

-C

on Agilent HP-5MS column;

MS = identification based on comparison of

mass spectra using Wiley 275 libraries; Relative area (%) = percentage of the area occupied by the compounds in the

chromatogram; m/z = mass values; n.i. = not identified; (-) = absent.

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The projection of the main classes of

compounds by principal component analysis

indicates that factor 1 represents 68 % of the

variability of the classes found in the extracts

of the leaves, flowers, and fruits of G. integri-

folia (Fig. 1). In factor 2, the extracts of leaves

and flowers showed a positive correlation, this

can be explained because both presented as

majority class fatty acid esters (48.2 and 29.9

%, respectively) (Table 1, Fig. 1). However, the

extracts of leaves and fruits presented negative

correlation in factor 2, the fruit extract showed

high concentrations of phytosterols (18.0 %)

and vitamins (20.0 %), while in the leaves

extract was identified lower concentrations

of phytosterols (4.8 %) and did not identi-

fied vitamins (Table 1, Fig. 1). In general, the

class projection indicates variation between the

chemical composition of extracts, mainly those

related to the esters of fatty acids, oxygenated

diterpenes, vitamins, phytosterols, and sulfur

compounds (Fig. 1).

A class of predominant compounds was

the esters of fatty acids in flowers (29.9 %),

fruits, (19.0 %) and leaves (48.3 %). The vita-

mins were observed in the flowers (20.9 %) and

fruits (18.0 %). The sulfur compounds of the

characteristic alliaceous odor of this plant were

predominant in the flowers (20.2 %), followed

by fruits (8.6 %) and a smaller concentration in

the leaves (0.9 %) (Table 1, Fig. 1). The major

compounds in the crude extract in the flowers

were ethyl ester of linoleic acid (14 %) (Fig.

2) and disulfide, bis (2-sulfhydryl ethyl) (11.9

%) (Fig. 3); in the fruits, they were ethyl iso-

allocholate (10.6 %) and stigmasterol (7.4 %);

and in the leaves, they were phytol (30.9 %)

(Fig. 4), linoleic acid methyl ester (30.5 %) and

methyl palmitate (10.9 %). Some major com-

pounds were found only in one part of the plant

such as linoleic acid (6.0 %) and isohumulone

(5.0 %) just in the leaves; disulfide, bis(2-sulf-

hydryl ethyl) (11.9 %) and methyl linoleate (5.2

%) were found only in the flowers; and lupeol

(6.8 %) and ethyl iso-allocholate (10.6 %) were

present just in the fruits (Table 1).

The main biomolecules of organosulfate

class present in the extract from G. integrifolia

flower were disulfide, bis(2-sulfhydryl ethyl)

) (Fig. 2, Table 1), methyl (methyl-

sulfinyl) methyl sulfide (C

), 1,2,4-trithio-

lane (C

), and 2,4-dithiapentane (C

)

(Table 1) with compounds with four, three, and

two sulfur atoms, respectively.

Besides the chemical composition, the

biological potential of three G. integrifolia

extracts was evaluated against A. aegypti third-

stage larvae and pupae. Overall, all the tested

Fig. 1. Biplot of principal component analysis scores and loadings for the gas chromatography and mass spectrometry

representing the projection of chemical classes of the crude extract from leaves, flowers, and fruits of Gallesia integrifolia.

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Fig. 2. Mass spectrum of linoleic acid ethyl ester of the extract from Gallesia integrifolia flower (14.0 %) and fruit (2.3 %)

obtained by gas chromatography-mass spectrometry.

Fig. 3. Mass spectrum of disulfide, bis(2-sulfhydryl ethyl) of the extract from Gallesia integrifolia flower (11.9 %) obtained

by gas chromatography-mass spectrometry.

Fig. 4. Mass spectrum of phytol of extract from Gallesia integrifolia leaves (30.9 %) obtained by gas chromatography-mass

spectrometry.

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samples were more efficient for the larvae as

well as for the pupae than the positive control.

The best lethal concentrations were for the

larvae when compared to pupae, as seen in

the extract of G. integrifolia flowers where

the LC

99.9

for larvae was 0.032 mg/mL, and

for pupae was LC

99.9

of 0.969 mg/mL, which

is 3 % more effective than in larvae when

compared to pupae. Followed by the raw fruit

extract the LC

99.9

for larvae was 0.124 mg/

mL, and for pupae was LC

99.9

of 6.086 mg/

mL. However, the extract from G. integrifolia

leaves was 5 % more effective for larvae when

compared to pupae (Table 2).

To help elucidate the action mechanism

of these crude extracts, acetylcholinesterase

enzyme (AChE) was evaluated using the crude

extracts from G. integrifolia flowers, fruits, and

leaves and temephos (commercial organophos-

phate) as a positive control (Table 3).

For the inhibiting activity test of acetyl-

cholinesterase enzyme, the crude flower extract

presented inhibitory effect at the concentration

of 0.00019 mg/mL, followed by the positive

TABLE 2

Lethal concentration (LC

and LC

99.9

) and confidence interval (CI) of Gallesia integrifolia extracts from flowers, fruits,

and leaves against Aedes aegypti larvae and pupae by probit analysis

G. integrifolia extract of

Larvae Pupae

(mg/mL) [CI] LC

99.9

(mg/mL) [CI] LC

99.9

(mg/mL) [CI]

Leaves

0.094 ± 0.001

[0.091-0.093]

0.278 ± 0.001

[0.279-0.282]

2.177 ± 0.177

[2.000-2.354]

14.040 ± 0.581

[13.459-14.622]

Flowers

0.006 ± 0.001

[0.006-0.006]

0.032 ± 0.001

[0.030-0.032]

0.036 ± 0.004

[0.035-0.037]

0.969 ± 0.026

[0.968-0.970]

Fruits

0.025 ± 0.002

[0.023-0.027]

0.124 ± 0.003

[0.120-0.126]

1.037 ± 0.080

[0.840-1.152]

6.086 ± 0.039

[5.704-6.307]

Temephos (control)

0.398 ± 0.050

[0.348-0.448]

1.140 ± 0.060

[1.080-1.200]

234.370 ± 22.090

[212.280-256.460]

443.640 ± 14.870

[428.770-458.510]

= lethal concentration that kills 50 % of A. aegypti larvae and pupae populations; LC

99.9

= lethal concentration that kills

99.9 % of A. aegypti larvae and pupae populations; CI = confidence interval; Positive control = commercial organophosphate

temephos; Equal letters in the same column indicate that there is no significant difference among treatments by Duncan’s

test (P ≤ 0.05).

TABLE 3

Inhibiting activity of acetylcholinesterase enzyme at different concentrations of extracts from

Gallesia integrifolia flowers, fruits, and leaves by bioautographic method

Concentration

(mg/mL)

Inhibition of acetylcholinesterase enzyme

Leaf Flower Fruit PC Concentration (mg/mL) Leaf Flower Fruit PC

50 +++ +++ +++ +++ 0.0488 - + + ++

25 ++ +++ +++ +++ 0.0244 - + + +

12.5 + ++ ++ +++ 0.0122 - + - +

6.25 + ++ ++ +++ 0.0061 - + - +

3.125 + + ++ +++ 0.0030 - + - +

1.5625 + + ++ +++ 0.0015 - + - +

0.7812 + + + ++ 0.00076 - + - +

0.3906 + + + ++ 0.00038 - + - +

0.1953 - + + ++ 0.00019 - + - -

0.0976 - + + ++ 0.00009 - - - -

Concentration = mg/mL; PC = positive control (commercial organophosphate temephos); (+++) = strong inhibition of

acetylcholinesterase enzyme; (++) = moderate inhibition; (+) = weak inhibition; (-) = absence of inhibition.

161

Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 153-169, March 2021

control at 0.00038 mg/mL, by the fruit extract

(0.0244 mg/mL) and the leaf extract (0.3906

mg/mL). When compared to the ex situ test

(LC

99.9

) with A. aegypti larvae, the extracts

from flowers, fruits, and leaves were 16.8, 0.5,

and 0.07 %, respectively, less effective than in

the bioautographic test in TLC.

DISCUSSION

The chemical identification of crude

extracts of G. integrifolia flowers, fruits, and

leaves was carried out by CG-MS and it was

verified the presence of esters of fatty acids

as the predominant class in the three extracts:

leaf extract (48.3 %), flower extract (29.9

%), and fruit extract (19 %). The amount and

distribution of fatty acids varied in the several

species of the plants, besides being affected by

seasonal influences (Kozlowski & Pallardy,

1996). The fatty acids found in the leaves are

responsible for controlling water loss to the

environment during gas exchanges, and protect

the plant against the nutrient loss due to the

high incidence of ultraviolet rays and intense

rainfall (Kozlowski & Pallardy, 1996); in the

flowers, as well as in the leaves, the fatty acids

are part of the surface impermeabilization,

avoiding water loss, and part of the pollination

process as they are in the chemical composi-

tion of flower nectar (Levin, McCue, & Davi-

dowitz, 2017) whereas, in the fruits and seeds,

these acids act as a barrier to moist diffusion,

as a form of protection, because it is a reserve

organ to the embryo (Esau, 1986; Kunst &

Samuels, 2009). In general, fatty acids act on

the cellular structure of vegetables, growth,

nutrition, senescence, and protection against

phytopathogens and the environment (Meï et

al., 2015; Li, Xu, Li-Beisson, & Philippar,

2016; Silva et al., 2016).

The linoleic acid ethyl ester found in the

extract from flowers and fruits (Fig. 2) is a

long-chain polar compound with antibacterial

and anti-inflammatory activities utilized in the

cosmetic industry (Jelenko, Wheeler, Ander-

son, Callaway, & McKinley, 1975; Park et al.,

2014). The linoleic acid ethyl ester as well as

the linolenic acid methyl ester are present in

the dark green leaves because they are part of

the apolar lipid fraction of plants (Simopoulos,

2002). There are no reports on the activity of

these molecules against A. aegypti, but fatty

acid methyl ester had LC

99.9

of 0.17 mg/mL

in Culex quinquefasciatus larvae (Silva et al.,

2016), indicating insecticide effect against lar-

vae of this insect.

Ethyl iso-allocholate found in the fruits

(10.6 %) does not present cytotoxicity in

zebrafish and can induce apoptosis through cas-

pases signaling pathway, causing morphologi-

cal alterations in the cell membranes (Thakur

& Ahirwar, 2019). According to Cooper, Thi,

Chamberlain, Pio, & Lowenberger (2007), this

signaling is also possibly responsible for A.

aegypti mortality because it acts as a biochemi-

cal cascade, triggering a proteolytic effect.

Another compound with biological potential

found in G. integrifolia is methyl palmitate

(leaves 10.9, flowers 4.7, and fruits 5.0 %), an

antagonist for muscarinic receptors with broad

toxicity against phytophagous mites such as

Tetranychus cinnabarinus (Boisduval) at 10

mg/mL (Wang et al., 2009).

Moreover, phytol found at high concentra-

tion in the extract from G. integrifolia leaves

(Fig. 4) chemically corresponds to a branched

long-chain aliphatic alcohol that gives hydro-

phobic characteristic to chlorophyll molecules;

however, when it is broken by chlorophyllase,

phytol is converted into phytanic acid with

an important biological effect on thermogenic

activities and inhibitor of teratogenic effects

of retinol (Marquez, 2003). Phytol also has

anti-inflammatory activity by releasing his-

tamine (26.9 %), serotine, and bradykinin

(49.9 %), and prostaglandin (68 %) compared

to control (diclofenac 5 mg/kg) (Phatangare,

Deshmukh, Murade, Hase, & Gaje, 2017).

Phytol has been reported as an inhibitor of

proteins and enzymes from bacteria (Ghaneian,

Ehrampoush, Jebali, Hekmatimoghaddam, &

Mahmoudi, 2015). The compound stigmasterol

(Table 1) obtained from G. integrifolia fruit

extract has been related to the inhibition of ace-

tylcholinesterase enzyme in human embryonic

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Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 153-169, March 2021

kidney cells (HEK293), that is the same mecha-

nism of action of organophosphate insecticides

(Alout et al., 2012; Gade et al., 2017).

Vitamin E (Fig. 5) was identified at a high

concentration in the extracts of G. integrifolia

flower (20.9 %) and fruit (18.0 %). Vitamin E,

which consists of tocopherols and tocotrienols,

is found in plants giving them photoprotec-

tive and antioxidant characteristics (Havaux,

Eymery, Porfirova, Rey, & Dormann, 2005).

Vitamin E was not detected in the extract

from G. integrifolia leaves but the presence of

its precursors, β-tocopherol and γ-tocopherol,

was identified.

Sulfur compounds were predominant in G.

integrifolia flower extract (20.2 %), followed

by fruits (8.6 %) and in a small concentration

in the leaf extract (0.9 %) (Table 1). The major

chemical compounds were disulfide, bis(2-

sulfhydryl ethyl) (flowers 11.9 %) (Fig. 3).

This biomolecule presents antioxidant potential

due to its high potential to eliminate free radi-

cals (Murugesan, Pandiyan, Saravanakumar,

Moodley, & Mackraj, 2019). Other present

compounds were 2,3,5-trithiahexane (flowers

6.2 and fruits 4.7 %), 1,2,4-trithiolane (flowers

1.1, fruits 1.8, and leaves 0.9 %) and 2,4-dithia-

pentane (flowers 1.1 and fruits 2.1 %). All

these biomolecules can be related to the char-

acteristic alliaceous odor of the plant, mainly

during flowering to attract specific pollinators

(Kishimoto, Maeda, Haketa, & Okubo, 2014).

When the insecticidal activity against A.

aegypti was analyzed, the values of LC

99.9

for

the control of A. aegypti was 0.032 mg/mL for

larvae and 0.969 mg/mL for pupae, using the

extract from G. integrifolia flower (Table 2),

whereas in the positive control (temephos) at

99.9

was 1.140 mg/mL for larvae and 443.64

mg/mL for pupae. However, the extract from

G. integrifolia flowers was 3.6 % more effec-

tive against A. aegypti larvae than the positive

control. This indicates that this crude extract

may be an alternative to be used in the con-

trol of this insect. Nevertheless, these values

are much over the values of 0.0000031 mg/

mL for temephos against Rockefeller strain

larvae cited by Jonny et al. (2015). According

to Cheng, Chang, Chang, Tsai, & Chen (2003),

compounds that presented LC

< 0.100 mg/

mL are considered active as larvicides on A.

aegypti, and highly active with LC

< 0.05

mg/mL. The crude extract of G. integrifolia

leaves showed active (LC

= 0.094 mg/mL)

on A. aegypti larvae and the crude extracts of

flowers (LC

= 0.006 mg/mL) and fruits (LC

= 0.025 mg/mL) of G. integrifolia demonstrat-

ing highly effective in controlling the larvae

of A. aegypti (Kiran, Bhavani, Devi, Rao, &

Reddy, 2006).

The lethal concentrations found for lar-

vae were lower than the ones for pupae, as

it occurred in the extract from G. integrifo-

lia fruit in which the LC

99.9

for larvae was

Fig. 5. Mass spectrum of vitamin E identified in the extract from Gallesia integrifolia flower (20.9 %) and fruit (18.0 %)

obtained by gas chromatography-mass spectrometry.

163

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0.124 mg/mL, and LC

99.9

of 6.086 mg/mL for

pupae, which was 4.9 % more effective for

A. aegypti larvae than pupae. Moreover, the

extract from G. integrifolia leaves was 5 %

more effective for larvae when compared to

pupae (Table 2). The intoxication pathways of

the product are distinct for larvae and pupae

because in the pupal stage, there is no food

intake and, therefore, there is no compound

intake. However, in this stage, the compounds

can act on the cutaneous surface of the pupae

and cause death by protein denaturation, enzy-

matic inhibition, and/or disintegration of the

cell membrane (Consoli & Oliveira, 1994;

Regnault, 1997; Carvalho et al., 2003; Cavalca,

Lolis, Reis, & Nonato, 2010). In the larval

stage, the culicid is more susceptible, because,

besides the contact by the external membranes,

when ingesting food, it can intake compounds

found in the water, increasing its potential of

action (Procópio et al., 2015).

Extracts of G. integrifolia presented insec-

ticide activity for the holometabolic phase of

A. aegypti when compared to the positive con-

trol (commercial organophosphate temephos).

When analyzing LC

99.9

of A. aegypti larvae,

it was verified that the best results were for

the extract from G. integrifolia flower, fol-

lowed by the extract from fruits, and then from

leaves, that was 35.6-, 9.2-, and 4.1-fold more

effective, respectively, than the positive control

(Table 2). The greatest insecticide activity (P ≤

0.05) was for the extract from G. integrifolia

flower with LC

99.9

of 0.032 mg/mL for larvae

and 0.969 mg/mL for pupae, which is 3.9-fold

more effective than the extract from G. integri-

folia fruit and 8.7-fold more effective than the

extract from G. integrifolia leaves for larvae.

99.9

of the crude ethanolic extract from

G. integrifolia flowers is 3.2-fold more effec-

tive to control A. aegypti larvae than Lippia

alba essential oil, 3.7-fold more effective than

Ocimum gratissimum essential oil, 4.3-fold

more than Cymbopogon citratus essential oil,

and 9-fold more effective than Eucalyptus

citriodora (current name Corymbia citriodora)

essential oil (Cavalcanti, Morais, Lima, & San-

tana, 2004; Vera et al., 2014). This indicates

the larvicide potential of the extract from G.

integrifolia flowers as a phytoinsecticide. In

addition, the extract from G. integrifolia flow-

ers has 20.2 % of sulfur compounds that can

be related to this greater insecticide activity

efficiency and the sulfur compounds were also

verified in the extract from G. integrifolia fruits

(8.5 %) and leaves (0.9 %) (Table 1).

The major biomolecules of the organosul-

furates class found in the extract from G. inte-

grifolia flower were disulfide, bis(2-sulfhydryl

ethyl) (C

) (Fig. 3), methyl (methylsul-

finyl) methyl sulfide (C

), 1,2,4-trithio-

lane (C

), and 2,4-dithiapentane (C

)

(Table 1) with compounds containing four,

three, or two sulfur atoms. Garlic essential

oil (Allium tuberosum and Allium sativum)

has been utilized to control insect larvae and/

or as a repellent of mosquitoes such as Aedes

spp., Anopheles spp., and Culex spp. due to the

presence of sulfur atoms in their composition

(Denloye et al., 2003; Trongtokit et al., 2005).

Liu, Liu, Zhou, & Liu (2014) observed that

Allium macrostemon essential oil with 98.1 %

of sulfur compounds presented LC

99.9

of 0.139

mg/mL and the methyl propyl disulfide isolates

had LC

99.9

of 0.151 mg/mL, whereas dimethyl

trisulfide presented LC

99.9

of 0.058 mg/mL

against Aedes albopictus. The concentrations

of the extract from G. integrifolia flowers

were 1.5-fold more efficient than A. tuberosum

essential oil and 2-fold more efficient than allyl

methyl trisulfide (C

) (Liu, Liu, Chen,

Zhou, & Liu, 2015).

Another plant with sulfur compounds in

the essential oil is Petiveria alliacea that

has 35.3 % of dibenzyl disulfide (C

)

(Zoghbi, Andrade, & Maia, 2002; Kerdudo et

al., 2015) and insecticide activity against A.

aegypti larvae with LC

99.9

of 0.023 mg/mL

(Hartmann, Silva, Walter, & Jeremias, 2018),

similarly to what was found in the extract from

G. integrifolia flowers (LC

99.9

of 0.036 mg/

mL). Pseudocalymma alliaceum (current name

Mansoa alliacea) presents, in the composition

of leaf essential oil, sulfur molecules such as

11.8 % diallyl sulphide (C

S), 50 % diallyl

disulphide (C

), and 10.4 % trisulfide,

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Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 153-169, March 2021

di-2-propenyl (C

), and insecticide activ-

ity with LC

of 0.267 mg/mL and LC

99.9

of 0.547 mg/mL against C. quinquefasciatus

pupae (Echegoyen et al., 2014). This suggests

that the insecticide activity may be related to

the sulfur molecules in the chemical composi-

tion of the essential oil from these plants.

The biological activity of sulfur com-

pounds against A. aegypti is still not well

understood. However, sulfur compounds can

form disulfide bridges (covalent bonds) with

amino acids and proteins that are important

to keep the protein structure and the catalytic

functions of enzymes; however, exogenous

sulfur compounds can cause the destabilization

of protein quaternary structure and enzymatic

inactivation (Belitz, Grosch, & Schieberle,

2009; Berkmen, 2012). The greater the number

of sulfur atoms in a molecule, then the greater

the number of polysulfide bridges (-Sn-) are

present in the chemical structures of the com-

pound. Consequently, the water solubility will

be smaller, increasing the chemical affinity

of the molecule with the structure of the cell

wall and membranes, mainly consisting of

ergosterol and chitin which in microbial cells

promote the membrane rupture and cell imbal-

ance (Cahagnier, 1988; Peacock & Goosey,

1989; Levinson, 2016). This suggests that these

results can be related to the cell wall perme-

ability, physical, and chemical characteristics

of solubility, and molecular absorption in lipo-

philic and hydrophilic media, inherent to the

test in A. aegypti larvae and pupae (Benson,

2005; Brain, Green, & Apia, 2007). In addition,

Kumar (2015) reported that sulfur compounds

in A. sativum such as allicin (C

O) act

by inhibiting acetylcholinesterase enzyme up

to the concentration of 0.05 mg/mL because

they present a greater chemical affinity by

anion sites of cholinesterase (Mahfouz, Met-

calf, & Fukuto, 1969). These data corroborate

our studies because the presence of sulfur

atoms in the flower (20.2 %) and fruit (8.6 %)

extract of G. integrifolia can be related to the

best results of the acetylcholinesterase enzyme

inhibition in relation to the leaf extract (0.9

%). This indicates that the extracts from G.

integrifolia strongly inhibit the acetylcholines-

terase enzyme, mainly when correlated to the

presence of sulfur compounds. However, more

studies are necessary to identify the active

compounds with this biological activity.

The yield of crude extract from G. inte-

grifolia flowers, fruits, and leaves was 8.2,

9.1, and 17.3 %, respectively. The major com-

pounds of the ethanolic crude extract from G.

integrifolia leaves are phytol (30.9 %), lino-

lenic acid methyl ester (30.5 %), and methyl

palmitate (10.9 %), from flowers are vitamin

E (20.9 %), linolenic acid methyl ester (14 %),

disulfide, bis(2-sulfhydryl ethyl) (11.9 %), and

phytol (10.2 %), and from fruits are vitamin

E (18 %) and ethyl iso-allocholate (10.6 %).

Only extracts from fruits and flowers present a

high concentration of vitamin E whereas only

flower extract presents a high concentration

of disulfide, bis(2-sulfhydryl ethyl). There are

organosulfurates compounds such as 1,2,4-tri-

thiolane in flowers (1.1), fruits (1.8), and leaves

(0.9 %), 2,3,5-trithiahexane in flowers (6.2 %)

and fruits (4.7 %), 2,4-dithiapentane in flow-

ers (1.1 %) and fruits (2.1 %), and disulfide,

bis (2-sulfidril ethyl) in flowers (11.9 %). The

flower extract has greater larvicidal activity

on A. aegypti LC

99.9

of 0.032 mg/mL and the

smallest concentration for acetylcholinester-

ase enzyme, which is of 0.00019 mg/mL. The

flower extract is 35.6-fold more efficient for

A. aegypti larvae than the positive control, and

12.8 % more efficient for the anticholinesterase

activity test. The crude extracts from G. integ-

rifolia fruits, leaves and, mainly flowers are a

potential alternative bioinsecticides to control

A. aegypti larvae and pupae.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

165

Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 153-169, March 2021

ACKNOWLEDGMENTS

The authors thank Universidade

Paranaense, Fundação Araucária, Coordenação

de Aperfeiçoamento de Pessoal de Nível Supe-

rior – Brazil (CAPES) -finance code 001-, Con-

selho Nacional de Desenvolvimento Científico

e Tecnológico (CNPq) for the fellowship and

financial support.

RESUMEN

Extracto crudo del árbol tropical Gallesia inte-

grifolia (Phytolaccaceae) para el control de larvas de

Aedes aegypti (Diptera: Culicidae). Introducción: los

fitoinsecticidas son alternativas para el control de insec-

tos en diferentes etapas, Gallesia integrifolia (Spreng.)

Harms, familia Phytolacaceae, conocida popularmente

como pau d’alho, árbol de ajo y guararema en Brasil, es

conocida por su fuerte olor aliáceo debido a la presencia

de moléculas de azufre en la planta. Esta especie presenta

actividad biológica y potencial efecto insecticida que aún

no está explotado. Objetivo: El objetivo de este estudio fue

evaluar la actividad biológica del extracto crudo etanólico

de las hojas, flores y frutos de G. integrifolia en el control

de las larvas y pupas de la tercera etapa de Aedes aegypti.

Métodos: El material botánico fue recolectado en la ciudad

de Umuarama, Paraná, Brasil (23º46’16” S & 53º19’38”

W), a una altitud de 442 m y los frutos de G. integrifolia

fueron recolectados en mayo de 2017 y las hojas y flores

en diciembre del mismo año. Los extractos crudos de hojas,

flores y frutos de G. integrifolia se prepararon por técnica

de maceración dinámica. La composición química de los

extractos se determinó por cromatografía de gases acoplada

a espectrometría de masas. La actividad insecticida de los

extractos crudos de G. integrifolia fue evaluada en larvas

y pupas de A. aegypti en concentraciones entre 0.001 a 25

000 mg/mL, y las concentraciones letales que matan 50 %

(CL

) y 99.9 % (CL

99.9

) de larvas fueron determinados

por análisis de probit. La actividad anticolinesterasa fue

evaluada por método bioautográfico en concentraciones

de 0.000095 a 50 mg/mL. Resultados: El rendimiento

de los extractos crudos de G. integrifolia fue de 8.2, 9.1

y 17.3 % para flores, frutos y hojas, respectivamente. La

composición química de los extractos de G. integrifolia

se caracterizó por la presencia de ésteres de ácidos grasos,

fitosteroles, vitaminas, diterpenos oxigenados y compues-

tos organosulfurados. El extracto de las flores presentó

alta cantidad de compuestos de azufre (20.2 %) como

disulfuro, bis(2-sulfhidril etilo) (11.9 %), 2,3,5-tritiahexano

(6.2 %), 1,2,4-tritiolano (1.1 %) y 2,4-ditiapentano (1.1

%). En relación con la actividad insecticida, el extracto de

las flores mostró una gran actividad con CL

99.9

de 0.032

mg/mL y CL

99.9

de 0.969 mg/mL en larvas y pupas de A.

aegypti, respectivamente, y la inhibición más alta de la

enzima acetilcolinesterasa (0.00019 mg/mL) ex situ. El

extracto de las flores presentó actividad anticolinesterasa

y larvicida, 12.8 y 35.6 %, respectivamente, mayor que el

control temephos. Conclusiones: Este estudio abre nuevas

perspectivas sobre el uso de extractos de G. integrifolia

como alternativa bioinsecticida para el control de larvas y

pupas de A. aegypti.

Palabras clave: anticolinesterasa; disulfuro; bis(2-sul-

fhidril etilo); pau d’alho; semioquímico; compuestos de

azufre.

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