Article Type: Full Paper

Chemical composition and in vitro anthelmintic activity of extracts of

Accepted Article
Tagetes patula against a multidrug-resistant isolate of Haemonchus
contortus
Flávio Augusto Sanches Politi*,a Amauri Alves Souza Júnior,a Rafaela Regina Fantatto,b Rosemeire
Cristina Linhari Rodrigues Pietro,b Waldomiro Barioni Júnior,c Márcio Dias Rabelo,c Humberto
Ribeiro Bizzo,d Ana Carolina de Souza Chagas,c and Maysa Furlan,a
a
Institute of Chemistry, UNESP - Univ Estadual Paulista, Department of Organic Chemistry, Rua Professor Francisco Degni 55, Bairro
Quitandinha, CEP 14800-060, Araraquara (SP), Brazil, e-mail: flaviopoliti@hotmail.com
b
School of Pharmaceutical Sciences, UNESP - Univ Estadual Paulista, Department of Drugs and Medicines, Rodovia Araraquara-Jaú Km 01
s/n, Bairro Campos Ville, CEP 14800-903, Araraquara (SP), Brazil
c
Empresa Brasileira de Pesquisa Agropecuária, Embrapa Pecuária Sudeste, Rodovia Washington Luiz Km 234 s/n, Fazenda Canchim, CEP
13560-970, São Carlos (SP), Brazil
d
Empresa Brasileira de Pesquisa Agropecuária, Embrapa Agroindústria de Alimentos, Avenida das Américas 29501, Bairro Guaratiba CEP
23020-470, Rio de Janeiro (RJ), Brazil

Abstract. Sheep breeding has suffered economic losses due to parasitism by gastrointestinal nematodes, particularly Haemonchus contortus.

The use of natural products, specifically Tagetes patula, has been suggested as an alternative method of combatting this issue. Chemical

analyses of the extracts of this species described in the literature report the presence of important classes of secondary metabolites such as

thiophenes, flavonoids, alkaloids and benzofurans, some of which were identified and isolated in this study. The aim of this work was to test

the effect of the essential oil (EO) and the ethanolic extract of the aerial parts (Tp EtOH) of T. patula on eggs and larvae of H. contortus,

through an egg hatch test (EHT) and a larval development test (LDT). In the EHT, the EO showed 100% inhibition at 0.75 mg.mL-1 (LC50 =

0.0780 mg.mL-1), and the TpEtOH showed 100% inhibition at 100 mg.mL-1 (LC50 = 12.8 mg.mL-1). In the LDT, the EO showed 100%

inhibition at 0.375 mg.mL-1 (LC50 = 0.0400 mg.mL-1), and the TpEtOH showed 100% inhibition at 1.56 mg mL-1 (LC50 = 0.340 mg.mL-1).

Compared to available literature data, the results presented here suggest that the crude extracts of T. patula have substantial potential for

controlling this nematode by interrupting its life cycle and/or preventing it from reaching the infective stage.

Keywords: Tagetes patula • Haemonchus contortus • terpenoids • thiophenes • natural products

Introduction

Sheep breeding is a traditional zootechnical industry that is constantly expanding to support the needs of humans through animal proteins

such as meat and milk as well as by supplying natural raw materials (wool, skins, fur sheep and leather). According to the Food and

Agriculture Organization of the United Nations (FAO) [1], the size of sheep flocks worldwide increased significantly between 2000 and

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 2013 from 1059.1 million heads in 2000 to 1172.8 million in 2013; the world production of sheep meat has increased from 7829.1 thousand

tons in 2000 to 8960.3 thousand tons in 2014; and the production of sheep milk has increased from 8159.9 thousand tons in 2000 to 10429.2

thousand tons in 2014. Brazil has approximately 17.3 million head of sheep and a goat flock of approximately 9.3 million head, placing it
Accepted Article
eighteenth in flock size globally [2]. According to the Brazilian Institute of Geography and Statistics [3], between 1997 and 2007, there was

an increase of approximately 11.7% in the total number of sheep heads. The flock size in the Southeast region grew substantially, increasing

its effective flock by 79%. At the same time, the sheep industry has suffered huge economic losses due to parasitism by gastrointestinal

nematodes [4, 5]. Among the parasites, Haemonchus contortus (Rudolphi 1803) Cobb 1898 is the most important because of its wide

distribution and high prevalence in flocks worldwide [6]. These hematophagous parasites can cause acute haemorrhagic anaemia in the

infected animals followed by the formation of submandibular edema, damage to gastric functions and hypoproteinemia [7, 8]. The main way

to control this parasitosis is through the use of synthetic anthelmintics. However, their indiscriminate use has resulted in some of the parasites

being resistant to the currently available products. This nematode develops resistance quickly probably due to its high biotic potential [9], it

also has great genetic variability, and it hosts an allele that causes decreased susceptibility to certain drugs [10]. Moreover, the breeders

commonly make operational errors in the control of this worm, such as under dosing of treatments, errors in frequency and continued use of

treatments and rapid rotation of the active principle employed [11]. A brief review was published by Kaplan and Vidyashankar [12] and

reported the existence of anthelmintic resistance in many regions, including United States, Brazil, South Africa, Oceania and European

countries. In the southern United States, greater than 90% of all the goat farms tested had worms resistant to two of the three drug classes

(ivermectin and albendazole) and approximately 30% of farms had worms resistant to all three drug classes (ivermectin, albendazole and

levamisole) [13]. In Brazil, there are reports from the 60’s on the ineffectiveness of chemicals in combating nematodes of sheep and goats

[14]. In Santa Catarina, resistance to ivermectin, levamisole, closantel, albendazole [15] and moxidectin [16] has already been reported. In

Paraná, beyond the resistance to the anthelmintics mentioned above, the ineffectiveness of associated compounds such as

closantel/oxfendazole has also been revealed [17]. There have also been reports of resistance to three groups of antiparasitics, namely,

benzimidazoles, levamisoles and ivermectin, in the states of Ceará and São Paulo [18, 19]. Recently, Albuquerque et al. [20] verified the

development of a resistant strain of H. contortus in lambs under suppressive or selective treatment regimens using monepantel – a compound

belonging to a new class of anthelmintic molecules described as amino-acetonitrile derivatives (AADs). After 3 months, the efficacy of the

anthelmintic was reduced to only 76%. Lamb et al. [21] confirmed the existence of a H. contortus strain that is highly resistant to a new

broad-spectrum anthelmintic in Australia; treatment efficacies ranged from 21.3% (monepantel) to 93.8% (derquantel/abamectin

combination). Furthermore, resistance to the multi-combination anthelmintic containing four active ingredients was also verified (52.5%).

This current situation involving parasites that are highly resistant to the available chemicals has influenced the course of scientific research in

the pharmaceutical field, and the search for alternatives aimed at minimizing these problems has been proposed. This includes the search for

methods for integrated control and/or selective control of parasitic diseases, immunization of infected animals, biological control and use of

botanical insecticides derived from plant extracts. Although a wide variety of plant species with therapeutic potential have been well

described, research on their use in the control of animal parasites is incipient [22]. The use of plants as phytotherapeutics or as a source of

prototype substances to synthesise in the laboratory is a scientific niche of great interest to the major pharmaceutical companies. Among the

advantages of phytotherapeutics that justify their use are the synergistic effects of their components, which act on different molecular targets

simultaneously, and that they have few or no side effects and are sustainable [23].

Tagetes patula L. (Asteraceae), popularly known as dwarf marigold or French marigold, is an annual plant that is 20-30 cm high. It is native

to North America and is widely disseminated around the world. It is easy to cultivate and propagate because it produces flowers and seeds all

year and has high germination rates. Due to its rich phytochemical composition, its high biocidal potential has been described in the

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 literature; it exhibits nematicidal, fungicidal and insecticidal activities against various species [24, 25]. Regarding the phytochemical

composition, Politi et al. [26] reported the substantial presence of flavonoids in the ethanolic extract of the aerial parts of T. patula; however,

other authors have reported the presence of thiophenes, steroids and terpenoids [27]. Thus, the objective of this study was to determine, for
Accepted Article
the first time, the in vitro anthelmintic potential of the crude extracts of this plant species against the eggs and larvae of a multi-resistant

isolate of H. contortus. The verification of a prominent biocidal effect of a complex plant-derived material is a necessary first step in the

search for active compounds, and from the results described herein, additional steps can and must be taken in order to find a novel treatment

for parasitic nematodes.

Results and Discussion

Steam distillation of the dried plant materials resulted in a yield of 2.5 µL.g-1 of a yellowish oil with a density of 0.73 g.mL-1. GC-MS

analysis of the EO of T. patula revealed 43 compounds representing 92.6% of the total signals detected (Figure 1, Table 1). The main

constituents were piperitenone (23.5%) and piperitone (20.1%). Piperitone is also a major compound in the essential oils of Cymbopogon

martini and Mentha pulegium, and it is related to their formidable anthelmintic actions against the Indian earthworm Pheritima posthuma

[28] and Echinococcus granulosus [29], respectively. Other typical compounds found in the genus Tagetes such as (Z)-tagetone (4.7%), (E)-

tagetone (2.3%) and spathulenol (1.7%) also appear in high levels. Compounds such as (Z)-β-ocimene (1.1%) and dihydro tagetone (0.4%)

are present in lower levels; these two compounds were isolated by Adekunle et al. [30] from Tagetes minuta essential oil and were reported

to have suitable anthelmintic activities against the root-knot nematode Meloidogyne incognita.

The crude ethanolic extract of T. patula (TpEtOH) showed an efficacy of 100% up to a concentration of 100 mg.mL-1 in the EHT and 1.56

mg.mL-1 in the LDT (Table 2). The essential oil of the aerial parts of T. patula (EO) was 100% effective up to 0.75 mg.mL-1 in the EHT and

up to 0.375 mg.mL-1 in the LDT (Table 3). With the exception of the EO at 0.003 mg.mL-1 in the LDT, the efficacies of all tested

concentrations in both tests were significantly different than the negative controls (p ≤ 0.05). At concentrations below 6.25 mg.mL-1 in the

EHT and 0.18 mg.mL-1 in the LDT, there were no statistically significant differences (p ≤ 0.05) in the measured inhibition values, indicating

that at low concentrations, the dose-effect for the TpEtOH does not change. The same is true for the EO at concentrations below 0.047 mg.mL-1

in the EHT and below 0.023 mg.mL-1 in the LDT. Regarding the negative controls used, all the controls showed inhibition percentages below

10%, validating the tests (Tables 2 and 3). Table 4 shows the LC 50 and LC90 values obtained by probit analysis (using Finney’s method) in

both assays.

The TpEtOH employed in the in vitro tests against H. contortus showed very promising anthelmintic potential, with much higher efficacies

than what have been found in other studies. For comparison, Cala et al. [31] tested the hexane extract of Melia azedarach and the methanol

extract of Trichilia claussenii, both of which are species in the Meliaceae family, and they found LC 50 and LC90 values greater than those

described in this study (EHT: LC50 = 572.2 mg.mL-1 and LC90 = 1137.8 mg.mL-1 for M. azedarach, LC50 = 263.8 mg.mL-1 and LC90 = 522.5

mg.mL-1 for T. claussenii; LDT: LC50 = 0.7 mg.mL-1 and LC90 = 60.8 mg.mL-1 for M. azedarach, LC50 = 1.1 mg.mL-1 and LC90 = 26.4

mg.mL-1 for T. claussenii). In another study, using aqueous extract of Ananas comosu (Bromeliaceae), Domingues et al. [32] also found

higher LC50 and LC90 values (EHT: LC50 = 31.0 mg.mL-1, LC90 = 81 mg.mL-1; LDT: LC50 = 1.73 mg.mL-1 and LC90 = 7.3 mg.mL-1).

Regarding the EO, it also showed great potential as an anthelmintic with results as good as or better than those of other compounds described

in the literature. For example, Katiki et al. [33] tested the essential oils of Cymbopogon martinii (Poaceae), C. shoenanthus (Poaceae) and

Mentha piperita (Lamiaceae) and found the following LC50 values in the in vitro assays: LC50 = 0.13 mg.mL-1 for C. martinii, LC50 = 0.04

mg.mL-1 for C. shoenanthus and LC50 = 0.026 mg.mL-1 for M. piperita (EHT); LC50 = 0.15 mg.mL-1 for C. martinii, LC50 = 0.06 mg.mL-1

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 for C. shoenanthus and LC50 = 0.26 mg.mL-1 for M. piperita (LDT). Macedo et al. [34] tested the essential oil of Tagetes minuta and found

LC50 = 0.53 mg.mL-1 (EHT) and LC50 = 1.67 mg.mL-1 (LDT).

To date, the chemical analyses of different extracts of T. patula reported in the literature have confirmed the presence of important classes of
Accepted Article
secondary metabolites, such as flavonoids [24, 26, 35-36], alkaloids [37], thiophenes [24, 27, 38] and benzofurans [39-42]. The latter two

classes are important indicators of the anthelmintic potential of the extract [43-46] and have been proven to be involved in the control of

nematodes. For example, González et al. [47] synthesized a series of analogues of 4-(4-fluorophenyl)-2-methylthio-thiophene-3-carbonitrile

and evaluated their in vitro and in vivo anthelmintic activities against H. contortus. They found that some compounds showed great

anthelmintic potential; however, their in vitro efficacies did not translate into significant in vivo activities. Lee et al. [48] proved that flavones

induce embryonic and larval lethality in Caenorhabditis elegans, C. brissage and Bursaphelenchus xylophilus nematodes. The biochemical

and pharmacological activities of flavonoids have been attributed to their anti-oxidative and free radical scavenging properties [49]. However,

the molecular basis for the biocidal effect against helminths needs to be elucidated. In this regard, it is worth mentioning that phenolic

compounds with catechol moieties are readily oxidized into ortho-quinones, which can inhibit the development of these parasites [50].

In the present study, a combination of column chromatography (silica gel) and HPLC (C18) was used to purify the ethanolic extract from

aerial parts of T. patula and allowed the isolation of two benzofurans, 1-(2-prop-1-en-2-yl-2,3-dihydro-1-benzofuran-5-yl) ethanone (1) and

1-(6-hydroxy-2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran-5-yl) ethanone (2), and one benzopyran, 6-acetyl-2,2-dimethylchroman-4-one (3).

The thiophene 5-(4-hydroxybut-1-ynyl)-2,2'-bithiophene (4) was also obtained by chromatographic separations, and this compound is being

reported for the first time in the species (Figure 1). Previous reports have indicated that the presence of thiophenes may be related to the

formation of singlet oxygen, which enables the formation of highly reactive free radicals [51]. The authors sought to identify these

compounds to better clarify the rich phytochemical composition of the ethanolic extract of T. patula. However, they did not intend to use

these compounds in biological assays; first, because of the difficulty in isolating a sufficient amount of purified compound to perform the in

vitro assays, and second, because the crude matrix has already been shown to have excellent activity against eggs and larvae of H. contortus.

Therefore, it is more advantageous to consider the application of the crude extract in future formulations.

Despite reports about the presence of terpenes in the extracts, especially in Tagetes erecta [52-54], these compounds are much more

abundant in the essential oil of the genus and are widely found in the T. patula sample used in the present study (Table 1). Terpenes amplify

the effects of other toxins; they act as solvents and facilitate the passage of these substances through membranes [55], allowing different

compounds to simultaneously act upon different molecular targets. Fang et al. [56] described the role specifically played by α-terpineol,

linalool, 1,8-cineole and nerolidol in the transdermal transport of therapeutically active agents, including catechins and theophylline, derived

from plant species. In some cases, essential oils have shown nuclear and cytoplasmic mutagenicity by acting against the mitochondria and

respiratory system [57].

Differences in the LC50 and LC90 values found in the tests may be attributed to the sensitivity of each life stage of the worm. The eggs are

stronger than L1 larvae due to their robust wrapping that comprises three layers: an outer layer composed of yolk; an intermediate layer

composed of chitin; and a basal portion formed by lipopolysaccharides [58]. The L1 larvae, however, also have a thin cuticle organized into

three layers: a basal layer composed of proteins, mainly by collagen; an epicuticular and outer cortical layer comprising other structural

proteins; and an external surface formed from non-structural proteins [59]. The L3 larvae are coated by two cuticles, and between them is a

space filled with a liquid rich in glycoproteins that serves as a lubricant and prevents abrasion between the layers [60]. The compounds from

plant extracts can interact with proteins of the cuticle, oral cavity, oesophagus, cloaca and vulva of nematodes and modify their

physicochemical properties [61].

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Accepted Article

Figure 1. GC analysis of Tagetes patula essential oil. (A) GC-FID chromatogram. (B) Total ion chromatogram (TIC) from the MS detector. (C-D)

Representative GC-MS spectra for piperitone and piperitenone, respectively.

Figure 2. Chemical structures of compounds 1, 2, 3 and 4 isolated from the ethanolic extract of the aerial parts of Tagetes patula.

Table 1. Composition of Tagetes patula essential oil obtained by GC-MS.

Rta RIb Component Area % Rta RIb Component Area %

7.263 1027 limonene 1.1 16.887 1285 dimethoxy-cymene 0.3

7.540 1036 (Z)-β-ocimene 1.1 17.118 1291 thymol 0.1

7.868 1046 (E)-β-ocimene 0.1 17.377 1297 carvacrol 0.3

8.016 1050 dihydro tagetone 0.4 17.992 1313 p-vinylguaiacol 0.5

9.236 1088 terpinolene 6.6 19.175 1342 piperitenone 23.5

9.381 1093 p-cymene 0.1 19.727 1356 eugenol 0.5

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 9.619 1100 linalool 0.6 20.080 1364 piperitenone oxide 1.5

10.039 1111 1,3,8-mentatriene 0.1 20.858 1384 geranyl acetate 0.5

10.687 1128 alloocimene 0.1 22.106 1415 (E)-caryophyllene 2.8

Accepted Article
10.818 1131 (Z)-epoxy-ocimeno 0.3 23.530 1451 α-humulene 0.1

11.184 1141 (E)-epoxy-ocimene 0.1 23.693 1456 (E)-β-farnesene 0.3

11.304 1144 (E)-tagetone 2.3 24.498 1476 germacrene D 0.7

11.626 1152 (Z)-tagetone 4.7 24.754 1483 (E)-β-ionone 0.1

12.044 1163 borneol 0.3 25.188 1494 bicyclogermacrene 0.1

12.509 1176 1,8-mentadien-4-ol 3.7 25.682 1507 (E,E)-α-farnesene 0.1

12.852 1185 p-cymen-8-ol 3.6 26.164 1519 δ-cadiene 0.1

13.030 1189 α-terpineol 0.5 27.752 1562 (E)-nerolidol 0.2

14.693 1231 (Z)-ocimenone 3.5 28.154 1572 spathulenol 1.7

14.806 1234 thymol methyl ether 0.4 28.333 1577 caryophyllene oxide 3.9

15.032 1239 (E)-ocimenone 2.2 30.344 1632 iso-spathulenol 0.2

15.666 1255 piperitone 20.1 37.395 1837 neofitadiene 1.8

c
16.838 1284 bornyl acetate 0.2 - - n.i. 8.4

a
Retention time; bRetention index relative to n-alkanes on the HP-5 MS capillary column; c Not identified

Table 2. Efficacy of the crude ethanolic extract of Tagetes patula against Haemonchus contortus by Egg Hatch Test (EHT) and Larval Development Test (LDT).

EHT LDT
Test Treatment
Concentration (mg.mL-1) Efficacy ± SD Concentration (mg.mL-1) Efficacy ± SD

100.0 100 ± 0a 3.125 100 ± 0a

50.0 91.04 ± 4.07b 1.56 100 ± 0a

25.0 62.78 ± 4.26c 0.78 85.98 ± 10.53b

TpEtOH
12.5 38.06 ± 5.53d 0.39 36.12 ± 12.64c

6.25 28.77 ± 6.65e 0.18 29.10 ± 18.84d

3.125 28.28 ± 6.27e 0.09 19.96 ± 4.09d

Distilled water 9.72 ± 3.13f Distilled water 3.09 ± 1.66e

C-
Tween 80 2% 4.93 ± 1.17f DMSO 0.5% 9.54 ± 0.93e

TpEtOH: ethanolic extract of T. patula; C-: negative controls; Means with the same letter within the same column are not significantly different by Tukey Test (p ≤
0.05).

Table 3. Efficacy of the essential of Tagetes patula against Haemonchus contortus by Egg Hatch Test (EHT) and Larval Development Test (LDT).

EHT LDT
Test Treatment
Concentration (mg.mL-1) Efficacy ± SD Concentration (mg.mL-1) Efficacy ± SD

0.75 100 ± 0a 0.375 100 ± 0a

EO 0.375 95.3 ± 3.35a 0.188 68.18 ± 13.00b

0.188 75.39 ± 9.48b 0.094 58.07 ± 11.38c

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 0.094 35.46 ± 5.36c 0.047 47.08 ± 19.66d

0.047 17.51 ± 3.51d 0.023 25.17 ± 21.30e

0.023 16.44 ± 5.69d 0.012 24.68 ± 6.58e

Accepted Article
0.012 16.24 ± 4.06d 0.006 20.88 ± 6.44e

0.006 15.52 ± 7.36d 0.003 16.71 ± 8.14e,f

Distilled Water 2.58 ± 1.32e Distilled Water 2.54 ± 1.92g

C-
Tween 80 2% 4.93 ± 1.17e DMSO 0.5% 9.54 ± 0.93f,g

EO: essential oil of aerial parts of T. patula; C-: negative controls; Means with the same letter within the same column are not significantly different by Tukey
Test (p ≤ 0.05).

Table 4. LC50 and LC90 values of Tagetes patula extractives on Haemonchus contortus by EHT and LDT tests.

EHT LDT
Sample
LC50 (mg.mL-1) LC90 (mg.mL-1) LC50 (mg.mL-1) LC90 (mg.mL-1)

12.8 67.2 0.34 1.259

TpEtOH
(11.9 - 13.84) (58.93 - 78.17) (0.315 - 0.364) (1.117 - 1.444)

0.078 0.452 0.04 0.59

EO
(0.072 - 0.084) (0.398 - 0.521) (0.035 - 0.044) (0.476 - 0.748)

TpEtOH = ethanolic extract of aerial parts of T. patula; EO = essential oil of aerial parts of T. patula; values in parentheses corresponds to the confidence interval
(CI) of 95%.

Conclusions

In conclusion, both of extractives obtained from the aerial parts of T. patula were effective anthelmintics, eliminating eggs and larvae at

concentrations below others described in the literature, and they could therefore be used in the formulation of an anthelmintic product. The

essential oil and the ethanolic extract have rich phytochemical compositions, and the variety of substances present in these matrices may

explain their biocidal effects since several compounds are synergistically and simultaneously acting against different molecular targets.

Experimental Section

General

Analytical TLC analyses were performed using silica gel 60 PF254 (Merck). Silica gel (Merck 60-230 mesh) was used for column

chromatography (CC). Analytical and preparative HPLC analyses were performed by using an HPLC-DAD system (Shimadzu®, Japan)

equipped with a degasser (DGU-20A5R), two solvent pumps (LC-6DA), and a photodiode array detector (SPD-M20A). Separations were

achieved using a reverse phase in a Phenomenex® octadecylsilane (C18) column; a 250 mm × 4.6 mm column was used for analytical

separations, and a 150 mm × 21.2 mm column was used for preparative separations. One-dimensional (1H and 13C) and two-dimensional (1H-
1
H gCOSY, gHMQC, and gHMBC) NMR experiments were recorded on a Bruker Advance III HD 600 spectrometer (14.1 T) operated at

600 MHz for 1H and at 150 MHz for 13C. Deuterated solvent (CDCl3) was used, and the δ values are reported relative to TMS. All solvents

used in this study were high-purity American Chemical Society (ACS) solvents from Sigma-Aldrich.

Plant material

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 Aerial parts of T. patula (stems, leaves and flowers) were obtained from the Collection of Medicinal and Aromatic Plants (CPMA) of the

Multidisciplinary Center for Chemical, Biological and Agricultural Research (CPQBA), State University of Campinas (UNICAMP). The

cultivation was carried out from seeds from the Top Seed Garden line (Agristar ®). A voucher specimen was deposited in the CPQBA
Accepted Article
Herbarium with a process number of 1421.

Extraction and isolation

Dried and powdered aerial parts of T. patula (1600 g) were extracted at room temperature with ethanol for 72 h. The solution was

concentrated by distillation under reduced pressure to yield a crude extract (204 g). A 50.0 g aliquot of this extract was fractionated eluting in

n-hexane (Hex), Hex/ethyl acetate and methanol (MeOH) to yield 9 fractions (F1–F9). F3 (2.46 g), F5 (0.89 g) and F6 (1.22 g) were analysed

by HPLC-DAD and then submitted to reverse phase preparative HPLC using a Phenomenex C 18 column with an isocratic elution of MeOH

(54%): ACN (13%): H2O (33%) over 20 minutes at a flow rate of 8 mL.min-1. This resulted in the isolation of 1 (59.0 mg) and 2 (20.6 mg)

from F3, 3 (12.8 mg) from F5 and 4 from F6 (20.3 mg). The compounds were identified by comparison with data reported in the literature

(1H-NMR and 13C-NMR) [54, 55], and the assignments were based on two-dimensional NMR experiments.

Obtaining the essential oil

The essential oil was extracted using hydrodistillation with a modified Clevenger. After manual sorting to remove strange organic matter,

approximately 200 g of the distillate was placed in a 6.0 L flask, which was filled to half its volume with distilled water. The extraction was

carried out over 4 h. The oil was collected in a beaker, and water droplets were removed by adding anhydrous sodium sulfate (Na2SO4). The

procedure was repeated until a satisfactory volume of oil was obtained. The oil was stored in 1.5 mL amber vials at 4 °C.

Identification of the chemical compounds of the essential oil by GC-MS

Chemical analysis of the plant material was carried out by gas chromatography coupled to mass spectrometry (GC-MS) in an Agilent 5973 N

system (Agilent Technologies®) equipped with an HP-5MS capillary column (5% diphenyl, 95% dimethyl silicone, 30 m x 0.25 mm; film

thickness 0.25 μm). Helium was used as the carrier gas (1.0 mL.min-1). A total of 1.0 μL of a 1% solution of the oil in dichloromethane was

injected, the injector was heated to 250 °C, and the system was operating in split mode (split ratio 1:100). The oven temperature increased

from 60-240 °C at a heating rate of 3 °C min-1. The mass detector was operated in electronic ionization mode (70 eV), the mass analyser was

maintained at 150 °C, the ionization source was maintained at 220 °C, and the transfer line was maintained at 260 °C. For quantification, the

essential oils were also analysed in an Agilent 7890A (Agilent Technologies ®) chromatograph equipped with a flame ionization detector

(FID) kept at 280 °C and fitted with the same HP-5MS capillary column. The same injection and chromatographic conditions described

above were applied in the GC-FID, but hydrogen was used as the carrier gas at 1.5 mL min -1. The results are expressed as relative area

(percent area). Linear retention indices were calculated by injection of a series of n-alkanes (C7-C26, Sigma-Aldrich®) in the same column

and conditions stated for GC-FID analyses. The essential oil components were identified by comparing their mass spectra and retention

indices with the Wiley 6th Edition Spectral Database and other literature data.

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 In vitro bioassays using Haemonchus contortus

The use of parasites in the tests was conducted under SISBIO research authorization no. 37006-4. All experimental protocols were approved

by the CPPSE Animal Care and Use Committee (CPPSE/Protocol 02/2014).

Accepted Article
Collection of eggs

Faecal matter was collected from the rectum of Santa Inês sheep artificially infected with the isolate of H. contortus Embrapa 2010, which is

resistant to benzimidazoles, macrocyclic lactones and imidazothiazoles [62]. The eggs were recovered according to the methodology

described by Coles et al. [63]. The faeces were homogenized in water and filtered through a set of sieves. The eggs that were retained on a 25

µm sieve were washed with distilled water and centrifuged at 3000 rpm for 5 minutes in 50 mL tubes. The supernatant was discarded, and

saturated saline was added to resuspend the pellet. After further centrifugation under the same conditions, the supernatant was passed through

a 25 µm sieve and the eggs were collected. The eggs were counted into aliquots of 10 µL each.

Egg Hatch Test (EHT)

Approximately 100 eggs per volume were placed in each well of a 24-well cell culture dish. The plant extract was added at 6 different

concentrations, and the essential oil was added at 12 different concentrations. The negative control was composed of distilled water and 2%

(v/v) Tween 80 solution. The test was run with six replicates. The plates were manually homogenized and stored in a BOD chamber (27 °C

and RH > 80%) for 24 h. A drop of lugol was added to each well, and the L1 hatched larvae and eggs were quantitated using an inverted

microscope (x40 magnification). The percentage of egg hatch inhibition was calculated according to the following equation:

(N Eggs)
Egg Hatch inhibition (%) = x 100
(N Eggs + N Larvae)

Larval Development Test (LDT)

Approximately 100 eggs, obtained according to the technique previously described, were added to each well of a 24-well plate along with 80

µL of a nutrient solution containing Escherichia coli and distilled water to a volume of 250 µL [64]. The material was manually

homogenized and incubated in a BOD chamber (27 °C and RH > 80%) for 24 h. After this period, 250 µL aliquots of 8 different dilutions of

the extract and 12 dilutions of the essential oil were added. The negative control consisted of 0.5% (v/v) DMSO solution and distilled water.

The test was run with six replicates. The plates were incubated for six days, and after adding the lugol, counting of L 1, L2 and L3 survived

larvae was performed using an inverted microscope (x40 magnification) in order to calculate the percentage of larval development inhibition.

Statistical analysis

Data were subjected to analysis of variance employing the GLM procedure of SAS (SAS 2010) using the tested concentrations as the

treatments and the percentages of inhibition observed in the experiments as a variable of response. The Tukey test was adopted for multiple

comparisons of the means (p ≤ 0.05). The PROBIT procedure of SAS was also used to estimate the LC50 and LC90 values and their

confidence intervals (CI 95%) for each experiment.

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 Acknowledgements

We thank the São Paulo Research Foundation (FAPESP, process number 2013/03493-8 and CIBFar 2013/07600-3), National Council for

Scientific and Technological Development (CNPq) and Embrapa project n° 03.11.01.023.00.00 for their financial support, and the Collection
Accepted Article
of Medicinal and Aromatic Plants (CPMA) of the Multidisciplinary Center for Chemical, Biological and Agricultural Research

(CPQBA/UNICAMP) for providing the plant material. M.F. is also grateful to the National Council for Scientific and Technological

Development (CNPq) for the research fellowship (307550/2013-3).

Author Contribution Statement

F.A.S. Politi conducted the experiments, analysed the results and wrote the manuscript. A.A. Souza-Júnior performed the NMR experiments

and chromatographic analyses. R.R. Fantatto and M.D. Rabelo assisted in the biological tests. H.R. Bizzo performed the GC analyses. W.

Barioni-Júnior assisted in the statistical analysis. R.C.L.R. Pietro, A.C.S. Chagas and M. Furlan conceived the research and reviewed the

final manuscript.

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