In vitro susceptibility of Trypanosoma brucei brucei to ...

Experimental Parasitology 190 (2018) 34–40

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In vitro susceptibility of Trypanosoma brucei brucei to selected essential oils and their major components

T

Sonya Costaa,b,c, Cláudia Cavadasc,d, Carlos Cavaleiroc,d, Lígia Salgueiroc,d, Maria do Céu Sousac,d,∗ a

Programme in Experimental Biology and Biomedicine, Centre for Neurosciences and Cell Biology, University of Coimbra, Portugal Institute for Interdisciplinary Research, University of Coimbra, Portugal c CNC-Center for Neurosciences and Cell Biology, University of Coimbra, Portugal d Faculty of Pharmacy, University of Coimbra, Portugal b

A R T I C LE I N FO

A B S T R A C T

Keywords: Human African trypanosomiasis Treatment Juniperus oxycedrus Lavandula luisieri Cymbopogon citratus Plant extracts α-pinene Citral

Aiming for discovering effective and harmless antitrypanosomal agents, 17 essential oils and nine major components were screened for their effects on T. b. brucei. The essential oils were obtained by hydrodistillation from fresh plant material and analyzed by GC and GC-MS. The trypanocidal activity was assessed using blood stream trypomastigotes cultures of T. b. brucei and the colorimetric resazurin method. The MTT test was used to assess the cytotoxicity of essential oils on macrophage cells and Selectivity Indexes were calculated. Of the 17 essential oils screened three showed high trypanocidal activity (IC50 < 10 μg/mL): Juniperus oxycedrus (IC50 of 0.9 μg/ mL), Cymbopogon citratus (IC50 of 3.2 μg/mL) and Lavandula luisieri (IC50 of 5.7 μg/mL). These oils had no cytotoxic effects on macrophage cells showing the highest values of Selectivity Index (63.4, 9.0 and 11.8, respectively). The oils of Distichoselinum tenuifolium, Lavandula viridis, Origanum virens, Seseli tortuosom, Syzygium aromaticum, and Thymbra capitata also exhibited activity (IC50 of 10–25 μg/mL) but showed cytotoxicity on macrophages. Of the nine compounds tested, α-pinene (IC50 of 2.9 μg/mL) and citral (IC50 of 18.9 μg/mL) exhibited the highest anti-trypanosomal activities. Citral is likely the active component of C. citratus and α-pinene is responsible for the antitrypanosomal effects of J. oxycedrus. The present work leads us to propose the J. oxycedrus, C. citratus and L. luisieri oils as valuable sources of new molecules for African Sleeping Sickness treatment.

1. Introduction Human African Trypanosomiasis (HAT), most popularly known as African sleeping sickness, is a vector borne disease that is transmitted to the human host through the bite of a tsetse fly (Glossina species). These tsetse flies are infected with Trypanosoma brucei gambiense or Trypanosoma brucei rhodensiense depending on geographical location. The West African form of the disease (chronic infections) is caused by T. b. gambiense and is responsible for 98% of cases where East African form (acute infections) is caused by T. b. rhodesiense (Franco et al., 2014a; b). More than 10,000 new cases are reported annually, though this number may be underestimated due to underreporting and/or misdiagnosis. Though the WHO reports that HAT is underway to being controlled, over 70 million people are at risk for infection. After inoculation of the parasite by the vector, HAT progresses through two distinct stages: a hemolymphatic stage (early stage) and

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meningoencephalitic stage (late stage). The trypanosomes are found in the blood during the hemolymphatic stage, whereas in the meningoencephalitic stage, the trypanosomes cross the blood brain barrier (BBB) and invade the central nervous system (CNS) (Kennedy PGE, 2004). The blood stream form develops after a short period of initial replication. Then, about one to three weeks after the bite of the vector, a nodule could appear at the bite site along with fever, lymph-nose swelling, skin eruption, headaches and splenomegaly. Symptoms of the meningoencephalitic stage include behavioral changes, confusion, sensory disturbances, poor coordination and the characteristic “sleep disturbances” that lead to death if untreated or inadequately treated. The treatment is dependent on the etiologic agent of disease and the stage. Only four drugs are registered for the treatment of human African trypanosomiasis: pentamidine, suramin, melarsoprol and eflornithine. Sickness caused by T. b. gambiense and T. b. rhodensiense can be treated with pentamidine and suramin during the hemolymphatic stage.

Corresponding author. Faculty of Pharmacy, University of Coimbra, R. do Norte, 3000-295 Coimbra, Portugal. E-mail addresses: [email protected] (S. Costa), ccavadas@ff.uc.pt (C. Cavadas), cavaleir@ff.uc.pt (C. Cavaleiro), ligia@ff.uc.pt (L. Salgueiro), [email protected] (M. do Céu Sousa). https://doi.org/10.1016/j.exppara.2018.05.002 Received 3 April 2017; Received in revised form 16 February 2018; Accepted 16 May 2018 Available online 18 May 2018 0014-4894/ © 2018 Elsevier Inc. All rights reserved.


S. Costa et al.

However, the meningoencephalitic stage of the disease relies on fairly toxic and difficult to administer drugs. Eflornithine and eflornithine/ nifurtimox combination (NECT) can be used only in the second stage of T.b.gambiense infection since it has been found not to be effective against the disease due to T.b rhodesiense (WHO, 2017). Melarsoprol, an arsenic derivative, is the only treatment available for late stage of T. b. rhodesiense, being also used as second line drug for the second or advanced stage of T. b. gambiense infections. However, it can be toxically fatal to patients and increased drug resistance has been observed. Thus, there is a need for new, inexpensive, easily to administer drugs that can treat the chronic and acute sleeping sickness diseases and both stages of HAT. Natural products, particularly those derived from plants, have been valuable sources of active compounds for discovery and development of new drugs. Among them, essential oils (EOs) received special attention. Since ancient times, medicine has relied on EOs to treat several diseases, particularly infectious diseases caused by a broad range of microorganisms (Bakkali et al., 2008; Habila et al., 2010; Nibret and Wink, 2010). EOs are complex mixtures, isolated by distillation of aromatic plants and composed of a diversity of small (up to 300 Da) hydrophobic compounds (typically monoterpenes, sesquiterpenes or phenylpropenoids) that easily cross biological barriers and membranes. The broad biocidal effects and the plausibility that some of the EOs components can cross the blood-brain-barrier (BBB) are credible reasons for the rising interest given to essential oils as potential sources of new anti-trypanosomal agents. Though the majority of the assessments of the activity of essential oils have been tested on Trypanosoma cruzi, only few oils have been evaluated on trypomastigote forms of Trypanosoma brucei brucei (Chenopodium ambrosioides, Cymbopogon citratus, C. giganteus, C. nardus, C. schoenantus, Hagenia abyssinica, Kadsura longipedunculata, Keetia leucantha, Leonotis ocymiolia, Morina stenopetala and Strychnos spinose). Despite the relatively unexplored trypanocidal activities of essential oils, their constituents have been more broadly investigated (Hoet et al., 2006; Monzote et al., 2009; Mulyaningsih et al., 2010; Nibret and Wink, 2010; Bero et al., 2013; Muhd Haffiz et al., 2013; Kpoviessi et al., 2014). Therefore, this study aims to evaluate the in vitro anti-trypanosomal activity of seventeen essential oils and some of their major constituents.

Table 1 Source of essential oils: plant species and family, geographic origin and the part of plant used on the extraction of EOs. Plant Species

Family

Geographic Origin

Material/ Voucher specimens

Cymbopogon citratus L. Distichoselinum tenuifolium (Lag.) García Martiín & Silvestre Juniperus oxycedrus

Poaceae Apiaceae

Angola Beira Litoral, PT

Leaves Aerial Parts (COI) 00005906

Cupressaceae

Trás-os-Montes, PT

Lavandula luisieri L.

Lamiaceae

Beira Alta, PT

Lavandula viridis L’Hér

Lamiaceae

Algarve, PT

Lippia graveolens H.B.K. Mentha cervina L.

Verbenaceae Lamiaceae

Guatemala Minho, PT

Mentha x piperita L. Origanum virens Hoffmans & Link Rosmarinus officinalis L.

Lamiaceae Lamiaceae

Beira Litoral, PT Beira Litoral, PT

Lamiaceae

Ribatejo, PT

Seseli tortuosum L.

Apiaceae

Beira Litoral, PT

Syzygium aromaticum (L.) Merr. & Perry

Myrtaceae

Thymbra capitata (L.) Cav

Lamiaceae

Commercially bought (Segredo da Planta) Algarve, PT

Leaves Berries (COI) CC 045 Inflorescences (FFUC) Zuzarte 02010 Aerial parts (FFUC) Zuzarte 0206 Aerial Parts Leaves (COI) LS 320 Leaves Leaves (COI) LS s.n Leaves (FFUC) CC832 Aerial Parts (COI) ACTavares 108 Floral Buttons

Thymus capitellatus Hoffmanns. & Link. Thymus mastichina L.

Lamiaceae

Ribatejo, PT

Lamiaceae

Algarve, PT

Thymus zygis Loefl. Ex L. subsp. sylvestris (Hoffmanns & Link) Brot. ex Coutinho (geraniol chemotype)

Lamiaceae

Beira Litoral, PT

Aerial Parts (COI) LS430. Aerial Parts (COI) LS220. Leaves (COI) LS120. Leaves (COI) LS64.

2. Material and methods Supelco (Supelco, Bellefonte, PA, USA) fused silica capillary columns with different stationary phases, polymethylsiloxane (SPB-1) and polyethyleneglycol (SupelcoWax-10). GC-MS was carried out in a Hewlett Packard 6890 gas chromatograph fitted with a polymethylsiloxane fused silica column, interfaced with a Hewlett-Packard mass selective detector 5973 (Agilent Technologies) operated by HP Enhanced ChemStation software, version A.03.00. Components of each essential oil were identified by their retention indices, on both columns, calculated by linear interpolation relative to the retention of C8-C23 of n-alkanes and from their mass spectra. Retention indices were compared with those of authentic samples included in laboratory database. Acquired mass spectra were compared with references from the laboratory spectra database, Wiley/NIST database (McLafferty, 2009) and literature data (Adams, 2004). Relative amounts of individual components were calculated based on GC peak areas without FID response factor correction.

2.1. Essential oil isolation Most of the EOs was prepared in the laboratory from fresh plant material collected in different regions of Portugal. Table 1 summarizes the source of the oils, specifying plant species and family, geographic origin and the part used. The oils were isolated by water distillation, for 3 h, using a Clevenger-type apparatus, following the procedure described in the European Pharmacopeia (Council of Europe, 2010). Voucher specimens were deposited at the Herbarium of the Department of Life Sciences of the University of Coimbra, ex. Herbarium of the Department of Botany of the University of Coimbra (COI) and/or at the herbarium of Faculty of Pharmacy of the University of Coimbra (Table 1). 2.2. Essential oil analysis Essential oils analysis was carried out as previously described using gas chromatography (GC) and by gas chromatography-mass spectroscopy (GC/MS) (Cavaleiro et al., 2004). Analytical GC was carried out in a Hewlett-Packard 6890 (Agilent Technologies, Palo Alto, CA, USA) gas chromatograph with a HP GC ChemStation Rev. A.05.04 data handling system, equipped with a single injector and two flame ionization detection (FID) Systems. A graphpak divider (Agilent Technologies, part No. 5021–7148) was used for simultaneous sampling to two

2.3. Compounds and standard drug Borneol, camphor, carvacrol, 1,8-cineole, citral, eugenol, linalool, α-pinene, and thymol were purchased from Sigma–Aldrich Co. The standard drug for HAT treatment, pentamidine was purchased from Sigma–Aldrich Co.

35

36

64.3

84.3

0.8 12.5

18.2

14.8

1.8

Rosmarinus officinalis

Seseli tortuosum

Syzygium aromaticum Thymbra capitata

Thymus capitellatus

Thymus mastichina L.

Thymus zygis sylvestris

28.9

Lippia graveolens

22.5

17.3

Lavandula viridis

Origanum virens

4.7

Lavandula luisieri

8.5 5.6

4.4

84.5

Mentha cervina Mentha x piperita

1.6

77.6

91

80.8

78.8

1.6 79.7

1.4

33.7

74.5

89.7 90.5

41

58.3

75.7

85.6 0.5

12.3 91.2

Cymbopogon citratus Distichoselinum tenuifolium Juniperus oxycedrus (b) Juniperus oxycedrus (l)

0.9

1.0

0.5

9.5 4.7

2.4

1.2

1.3

0.3 1.9

10.3

18.6

2.4

2.4

17

0.1 0.5

Sesquiterpene Hydrocarbons

Monoteerpene Hydrocarbons

Oxygen containing monoterpenes

Sesquiterpenes

Monoterpenes

Table 2 Composition of essential oils.

1.5

0.5

0.5

0.4 0.8

2.3

0.2

0.5

– 0.3

90.3

97.3 98.1

–

– 0.3 – –

1.2

–

85.3 – – –

–

96.3

97.1

97.9

99.8

99.3 0.4

99.5 98.6

90.3

–

–

–

10.8

96.1

0.5

1.9

–

–

86.9

–

1.2

–

3

94.5

2.0 0.3

1.6

–

0.5

97.6

98.7 92.7

% identified

– –

0.8

–

0.6

7 –

– 0.1

– 0.5

Other compounds

Phenylpropanoids

Oxygen containing sesquiterpenes

α-Pinene (54.7%), Germacrene D (10.4%), β-myrcene (17.8%) α-Pinene (65.5%), Δ-3-Carene (5.7%), β-phellandrene (3.2%), β-myrcene (2.7%) 1,1,2,3-Tetramethyl-4-hidroxymethyl-2-cyclopentene (2.4%), 2,3,4,4-Tetramethyl-5-methylene-cyclopent-2-enone (5.2%), trans-α-necrodyl acetate (16.0%) and lyratyl acetate (3.5%), 1,8-cineole (18.9%), Lavandulyl acetate (7.2%), linalool (3.1%), α-pinene (2.3%) 1,8-cineole (29.7%), camphor (10.0%), α-pinene (9.2%) linalool (9.0%), selina-3,7 (11)-diene (6.6%), Z-α-bisabolene (6.3%), borneol (2.7%), camphene (2.7%) Thymol (19.8%), ρ-cymene (16.9%), 1,8-cineole (6.6%), caryophyllene oxide (5.7%), linalool (5.4%), Δ-3-carene (4.3%), α-terpineol (3.6%), myrcene (3.4%), E-caryophyllene (2.4%), trans-sabinene hydrate (2.3%) Pulegone (74.8%), Isomenthone (10.6%), limonene (5.4%) Menthol (44.0%), menthofuran (10.9%), menthone (9.8%), menthyl acetate (7.8%), 1,8-cineole (5.8%), neo-menthol (4.0%) neo-isomenthol (2.9%), pulgone (2.4%) Carvacrol (68.2%), γ-terpinene (7.9%), p-cymene (7.4%), βmyrcene (2.4%), thymol (2.1%) β-myrcene (32.0%), 1,8-cineole (13.7%), camphor (11.9%), α-pinene (11.1%), limonene (6.6%), ρ-cymene (3.8%), camphene (3.4%), linalool (2.1%) α-pinene (27.4%), β-pinene (16.0%), limonene (10.0%), γterpinene (9.3%), Z-β-ocimene (8.0%), β-myrcene (3.0%), camphene (2.1%), sabinene (2.0%) Eugenol (85.3%), α-humulene (6.8%) Carvacrol (74.6%), p-cymene (5.5%), E-caryophyllene (3.9%), γ-terpinene (3.6%), linalool (2.8%) 1,8-cineole (58.6%), borneol (10.0%), camphene (6.5%), αpinene (4.5%), sabinene (3.0%), β-pinene (2.0%) α-pinene (3.0%), sabinene (2.4%), β-pinene (4.0%), 1,8-cineole (67.4%), linalool (4.3%), α-terpineol (3.5%) Geranyl acetate (44.5%), geraniol (33.1%), camphor (3.9%)

Geranial (45.7%), Neral (32.5%), β-myrcene (11.5%) β-myrcene (84.6%), Limonene (2.2%)

Major Compounds (> 2.0%)

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2.4. Trypanosoma and macrophage cultures

3. Results

Trypanosoma brucei brucei blood stream forms (AnTart 1 strain, wild type) were a gift from the Institute of Tropical Medicine in Antwerp, Belgium. Trypomastigotes were grown and maintained in Hirumi's Modified Iscove's medium-9 (HMI 9) at 37 °C with 5.0% CO2 using 24 well plates (serial dilutions, from 1/10 to 1/106) (Van Reet et al., 2011). Parasite number in growth cultures did not exceed 2.0 × 106 cells/mL. Macrophage cell line (RAW 267.4) was grown in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% inactivated foetal bovine serum, penicillin and streptomycin (100 μg/mL) and D-glucose (3.5 g/L) at 37 °C with 5% CO2.

3.1. Composition of essential oils Abridged compositions of the essential oils are reported in Table 2, emphasizing constituents occurring with relative amount over 2%. Most of the essential oils are predominantly composed of monoterpene hydrocarbons and oxygen containing monoterpenes. S. aromaticum oil is chiefly composed of phenylpropanoids., 43 constituents with concentrations over 2%, representatives of the most important classes of volatile phytochemicals were found in the oils: 11 monoterpene hydrocarbons (α-pinene, β-pinene, camphene, sabinene, β-myrcene, Δ-3carene, p-cymene, limonene, β-phellandrene Z-β-ocimene and γ-terpinene); 7 monoterpenic aldehydes and ketones (neral, geranial, camphor, pulgone, menthone, isomenthone and pulgone); 8 monoterpenic alcohols (linalool, borneol, α-terpineol, menthol, neo-menthol, neoisomenthol, geraniol and E-sabinene hydrate); 3 monoterpenic esters (lavandulyl acetate, menthyl acetate and geranyl acetate); 2 monoterpenic oxides (1,8-cineole and menthofuran); 2 monoterpenic phenols (thymol and carvacrol); 4 oxygenated necrodane derivatives (1,1,2,3tetramethyl-4-hidroxymethyl-2-cyclopentene, 2,3,4,4-tetramethyl-5methylene-cyclopent-2-enone, trans-α-necrodyl acetate and lyratyl acetate); 6 sesquiterpenic compounds (E-caryophyllene, germacrene D, selina-3,7 (11)-diene, Z-α-bisabolene and α-humulene, and caryophyllene oxide); and 1 phenylpropanoid (eugenol). Details on the compositions of these oils can be found in literature (Salgueiro et al., 2003, 2004; Boti et al., 2006; Gonçalves et al., 2007; Pinto et al., 2009; Tavares et al., 2010; Zuzarte et al., 2011, 2012; Machado et al., 2012; Vale-Silva et al., 2012; Videira et al., 2013).

2.5. Drug susceptibility assay In a 96 multiwell plate, trypomastigotes (4.0 × 104 cells/mL) in log growth phase were incubated at 37 °C and 5% CO2 for 48 h in fresh HMI 9 medium with a range of drug/essential oils concentrations (1–100 μg/ mL). The effect on the cells viability was tested by Resazurin based in vitro Toxicology Assay Kit (Sigma Aldrich Co). Resazurin (blue color) is reduced to resorufin (highly fluorescent pink color) by mitochondrial dehydrogenase enzymes that are responsible for transferring electrons from NADPH +H+ to resazurin. Briefly, after incubation, 25 μL (10% of the final volume) of resazurin was added to each well and allowed to incubate for an additional 24 h at 37 °C with 5% CO2. The absorbance were read at 600 nm and 690 nm on an ELISA plate reader (Synergy HT, Bio-TEK) and the viability was calculated as previously described (Rolón et al., 2006) using the following formula:

Viability (%) =

3.2. Anti-trypanosomal activity

A600 − (A690 x Ro) test well x 100 [A600 − (A690 x Ro) control well]

Of the 17 essential oils screened three showed high trypanocidal activity; the oil from fruits of J. oxycedrus (b) (IC50 of 0.9 μg/mL), the oil of C. citratus (IC50 of 3.2 μg/mL) and the oil of L. luisieri (IC50 of 5.7 μg/mL) (Table 3). These active oils had low cytotoxic effects on macrophage cells showing the highest values of Selectivity Indexes (SI),

Where A600: absorbance at 600 nm; A690: absorbance at 690 nm; Ro: A600/A690; correction factor for resazurin in the medium. The concentration that inhibited viability by 50% (IC50) was determined through dose-response regression analysis using GraphPad Prism 6.0.

Table 3 Anti-trypanosomal activity and cytotoxic effects of essential oils.

2.6. Mammalian cell cytotoxicity In order to evaluate the cytotoxicity on mammalian cells, a series of increasing concentrations of essential oils and compounds were tested on macrophage cells (RAW 264.7). Log phase of macrophages (4.0 × 104/ml) were incubated at 37○C in 96-well tissue culture plates in growth medium under microaerophilic condition. When the monolayers reached confluence, the medium was removed and the cells were incubated at 37○C for 72 h with fresh medium plus a range of drug concentrations (10–100 μg/mL). The viability was determined using an MTT (3-[4, 5-methylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) colorimetric method, where 25 μL of MTT solution (5 mg/mL) was added to each well and allowed to incubate at 37 °C with 5% CO2 for 1 h. The medium was removed, the cells were washed with PBS and 250 μL of dimethylsulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was read at 530 nm on an ELISA plate reader (Synergy HT, Bio-TEK) and the viability was calculated using the following formula: [(L2/L1) x 100], where L1 is the absorbance of control cells and L2 is the absorbance of treated cells. The concentration that induced macrophage death by 50% (CC50) was determined through dose-response regression analysis using GraphPad Prism 6.0. The selectivity index (SI) was determined as the ratio of CC50 macrophage/ IC50 parasite.

37

Essential Oil

IC50 * (μg/mL) (95%Confidence Intervals) T. b. brucei

CC50 (μg/mL) (95%Confidence Intervals) Macrophages

Selectivity Index (SI)

Cymbopogon citratus Distichoselinum tenuifolium Juniperus oxycedrus (b) Juniperus oxycedrus (l) Lavandula luisieri Lavandula viridis Lippia graveolens Mentha cervina Mentha x piperita Origanum virens Rosmarinus officinalis Seseli tortuosum Syzygium aromaticum Thymbra capitata Thymus capitellatus Thymus mastichina Thymus zygis sylvestris

3.2 (2.7–3.7)

28.7 (15.5–41.4)

9.0

20.0 (15.9–25.1)

72.7 (64.0–82.7)

3.6

0.9 (0.8–1.1)

57.1 (50.5–64.5)

63.4

36.2 (31.0–42.4)

–

–

5.7 (4.3–7.4) 20.6 (14.9–28.4) > 100.0 > 100.0 63.9 (56.4–72.4) 24.0 (19.8–29.0) > 100.0

67.1 (59.3–75.9) 19.8 (17.1–22.9) – – – 48.4 (36.4–64.3) –

11.9 1.0 – – – 2.0 –

8.2 (5.8–11.6) 10.4 (7.4–14.3)

19.6 (15.3–25.1) 22.4 (18.6–27.0)

2.4 2.2

14.2 (11.9–17.2) 44.1 (40.5–48.1)

86.4 (82.1–91.0) –

6.1 –

> 100.0 > 100.0

– –

– –


S. Costa et al.

however, they have been previously studied on T. cruzi and did not exhibit exceptional activity (Santoro et al., 2007b; Azeredo et al., 2014). In our study R. officinalis behaved similarly, whereas S. aromaticum essential oil and O. virens exhibited lower activity with an IC50 value of 10.4 μg/mL and 24.0 μg/mL. Essential oils belonging to other species of Lippia (L. alba, L. citriodora, L. dulcis, L. origanoides, and L. sidoides) and Thymus (T. vulgaris) have been studied on T. cruzi and the Lippia species showed a great variation in their activity (Santoro et al., 2007a; Escobar et al., 2010). The one representative of the Lippia genus that we tested did not exhibit anti-trypanosomal activity at the concentrations tested. The activity of the oil from J. oxycedrus berries (b) can, in part, be accounted by the presence of α-pinene which makes up 54.7% of the composition (Table 2). This compound exhibited the highest antitrypanosomal activity (IC50 of 2.9 μg/mL) and is likely one of the active components of the oil. Interestingly, the percentage of α-pinene in J. oxycedrus leaf is higher than in the essential oil from the berries (Table 2), however, that essential oil has a lower antitrypanosomal activity. These differences may be due a synergistic interactions of other constituents of the berries oil, such as germacrene D, which is not constituent of the leaves oil. Germacrene D has not been previously tested on Trypanosoma, however, oils rich in this compound, as well as in E-caryophyllene, showed activity on various forms of T. cruzi (da Silva et al., 2013). Another important constituent of both J. oxycedrus oils, β-myrcene, has recently been evaluated on T. b. brucei exhibiting high antitrypanosomal activity (IC50 of 2.24 μg/mL) (Kpoviessi et al., 2014). The antitrypanosomal activity of the C. citratus oil is probably due to its major constituents, the monoterpenic aldehydes, neral (the Zisomer) and geranial (the E-isomer). The mixture of these isomers, usually named citral, showed activity on T. brucei with an IC50 of 18.9 μg/mL (Table 4) according with previous results (Kpoviessi et al., 2014). Moreover, C. citratus essential oil and citral have been previously tested on another trypanosomatid, Leishmania, revealing leishmanicidal effects attributed to programmed cell death (apoptosis) (Cardoso and Soares, 2010; Machado et al., 2012). Citral is thought to permeate the parasitic cell membrane, leading to mitochondrial membrane impairment and depolarization and cell lysis. Literature also reports other activities for the oil of C. citratus, particularly antifungal (Khan and Ahmad, 2012; Sessou et al., 2013) antiparasitic (Santoro et al., 2007c; Machado et al., 2010, 2011; 2012; Kpoviessi et al., 2014) and antibacterial (Korenblum et al., 2013). The essential oil of L. luisieri, the third most active on T. b. brucei, is primarily composed of oxygen containing monoterpene 1,8 cineole, lavandulyl acetate, linalool as well as necrodane oxygenated derivatives, such as necrodyl and lyratyl acetate (Table 2). The 1,8-cineole exhibited weak activity with an IC50 greater than 100 μg/mL and so is not seems responsible for the oil activity. Nibret and Wink (2010) also evaluated this constituent and obtained similar results. Linalool, found in the oil composition at 3.1%, has also a weak activity (IC50 > 100 μg/mL). However, two studies that evaluated this compound on T. b. brucei reported a higher activity for this compound (IC50 values ranging from 2.5 to 39.6 μg/mL) (Hoet et al., 2006; Nibret and Wink, 2010). The α-pinene, aforementioned as active on T. b. brucei, is found in L. luisieri oil in low concentration (2.3%), so it cannot support alone the activity observed for this oil. Thus, the oxygenated necrodanes, which can make up to 37% of oil, may be responsible for the antitrypanosomal activity (Table 2). Further investigation is required to elucidate whether or not the antitrypanosomal activity is caused by these necrodane derivatives. The oils from the aerial parts of S. tortuosum, from the buds of S. aromaticum and from the aerial parts of T. capitata exhibited antiparasitic activity (IC50 < 15 μg/mL) but a poor selectivity indexes (SI ranging from 2.2 to 6.1). The major constituents of S. tortuosum were α-pinene, β-pinene, limonene and γ-terpinene (Table 2). It is possible that the

Table 4 Activity of essential oil constituents on T. b. brucei and on mammalian cells. Compound

T.b. brucei IC50 (μg/mL)

Macrophage cells CC50 (μg/mL)

Selectivity Index SI (CC50/IC50)

1,8-Cineole Borneol Camphor Carvacrol Citral Eugenol Linalool Thymol α-Pinene

> 100 > 100 > 100 31.4 (27.5–36.0) 18.9 (17.2–20.7) > 100 > 100 28.9 (27.5–36.01) 2.9 (1.5–5.8)

> 100 > 100 > 100 > 100

> 3.8 > 5.3 > 3.5 > 34.5

63.4, 9.0 and 11.8, respectively. The oils of D. tenuifolium, L. viridis, O. virens, S. tortuosum, S. aromaticum and T. capitata also exhibited activity with IC50 values ranging between 10 and 25 μg/mL (Table 3). However, these oils showed cytotoxicity on macrophages and a low SI. Essential oils from the leaves of J. oxycedrus (l), M. piperita, and T. capitellatus showed moderate activity with IC50 values ranging between 36 and 64 μg/mL. The oils of L. graveolens, M. cervina, R. officinalis, T. mastichina and T. zygis subspecies sylvestris are weakly active against T. b. brucei with IC50 values higher than 100 μg/mL and so their SI was not determined. The antitrypanosomal activity of the major constituents of essential oils, 1,8-cineole, borneol, camphor, carvacrol, citral (mixture of neral and geranial), eugenol, linalool, thymol and α-pinene were also determined (Table 4). Of the nine compounds tested, four exhibited antitrypanosomal activity at the concentrations tested: α-pinene and citral exhibited the highest activities with IC50 values of 2.9 and 18.9 μg/mL, respectively. 4. Discussion The trypanosomal activity of the essential oils of C. citratus, D. tenuifolium, J. oxcycedrus (leaves or berries), L. luisieri, L. viridis, L. graveolens, M. cervina, M. piperita, O. virens, R. officinalis, S. tortuosum, S. aromaticum, T. capitata, T. capitellatus, T. mastichina and T. sylvestris on T .b. brucei was assessed. The most active oils were those from J. oxycedrus berries (b) (IC50 of 0.9 μg/mL; SI = 63.4), C. citratus leaves (IC50 of 3.2 μg/mL; SI = 9.0) and L. luisieri inflorescences (IC50 of 5.7 μg/mL; SI = 11.9). These oils have a high trypanocidal activity, with IC50 values < 10 μg/mL and with suitable selectivity (SI values > 9.0), allowing to optimistically use them for further research, seeking for active compounds on Trypanosoma. Several essential oils had been evaluated on T.b. brucei (Hoet et al., 2006; Monzote et al., 2009; Mulyaningsih et al., 2010; Nibret and Wink, 2010; Bero et al., 2013; Muhd Haffiz et al., 2013; Kpoviessi et al., 2014). Of these, three have been identified as the most active essential oils on trypomastigotes with Chenopodium ambrosioides as the most active (Monzote et al., 2009) followed by Cymbopogon giganteus and Cymbpogon nardus (whole plant) (Muhd Haffiz et al., 2013; Kpoviessi et al., 2014). To this list we can now add J. oxycedrus berry essential oil which also exhibited an IC50 value below 1 μg/mL. Based on previous literature, Cymbopogon citratus, Cymbopogon nardus (leaf) and Moringa stenopetala also exhibited IC50 values below 10 μg/mL (1.83 μg/mL, 5.03 μg/mL and 5.7 μg/mL, respectively) (Nibret and Wink, 2010; Kpoviessi et al., 2014). C. citratus leaf essential oil, the only oil described in literature that was also tested on this study, had an IC50 that did not waver far from that obtained in our laboratory (IC50 = 3.2). Lavandula luisieri, one of the promising essential oils identified in this study, also fit within this category. Furthermore, essential oils derived from O. vulgare subsp virens, R. officinalis and S. aromaticum, have not been tested before on T. b. brucei, 38


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MTT

antitrypanosomal activity results from the effects of associations of these compounds. Kpoviessi et al. (2014) and Nibret and Wink (2010) reported for β-pinene and limonene IC50 values of 4.2 and 35.6 μg/mL, respectively, whereas Mikus et al. (2000) and Kpoviessi et al. (2014) reported IC50/ED50 values for β-pinene between 47 and 55 μg/mL. Though the strong activity of S. tortuosum on the parasite, the essential oil also revealed cytotoxic activity on macrophage cell lines (SI of 2.8), thus is not an ideal candidate for further evaluation. Likewise, S. aromaticum oil exhibited low specificity for T. b. brucei parasites (SI of 2.2). The oil was mainly composed of eugenol (85.3%) (Table 2), but this constituent was not active on T. b. brucei at the concentrations tested. In other trypanosomatid parasite, T. cruzi, the eugenol also exhibited weakly antiparasitic activity (246 μg/mL) (Santoro et al., 2007b). To our knowledge, α-humulene, the second most abundant constituent in the S. aromaticum oil (Table 2), has not been tested on trypanosomal species, but has exhibited activity on L. donovani (19 μg/mL) (Zheljazkov et al., 2008). T. capitata oil is mainly composed of carvacrol (75%) (Table 2) and the IC50 value of this phenol monoterpene on T. b. brucei was 31.4 μg/ mL (Table 4). The activity of this constituent was previously reported by Nibret and Wink (2010) and seems to contribute to the antiparasitic activity of oil on T. b. brucei. Conversely the p-cymene and linalool, two other constituents of the T. capitata oil, were weakly active (Nibret and Wink, 2010; Kpoviessi et al., 2014). Of all the moderate active oils, the T. capitata oil is the less cytotoxic and the most selective. Machado et al. (2010) suggested that T. capitata acts on Giardia lamblia parasite through impairment of osmoregulation caused by plasma membrane alterations. Antifungal (Salgueiro et al., 2004; Palmeira-de-Oliveira et al., 2012) and antibacterial (Faleiro et al., 2005) activities were also reported. Considering the high IC50 and/or low SI values, the oils of Distichoselinum tenuiolium, Juniperus oxycedrus (l), Lavandula viridis, Lippia graveolens, Mentha cervina, Mentha x piperita, Origanum virens, Rosmarinus officinalis, Seseli tortusoum, Syzygium aromaticum, Thymbra capitata, Thymus capitellatus, and Thymus zygis sylvestris are irrelevant when concerning their antitrypanosomal potential.

NADPH SI

3-[4, 5-methylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide Nicotinamide Adenine Dinucleotide Phosphate Selectivity Index

Funding This work was supported by FCT POCTI (FEDER) and COMPETE [PEst-C/SAU/LA000172013-2014], and FCT [SFRH/BD/51201/2010]. Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declarations Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Ethical approval Not applicable. Authors' contributions Conceived and designed the experiments: CC, MCS. Worked on data acquisition and sample collection: SC, LS, CC, MCS. Performed the experiments: SC. Analyzed the data: SC, CC, MCS. Contributed reagents/materials/analysis tools: CC, CC, MCS. Wrote and revised the paper: SC, CC, CC, MCS. All authors read and approved the final version of the manuscript. References

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Juniperus oxycedrus berries oil, Cymbopogon citratus leaves oil and Lavandula luisieri inflorescences exhibited an excellent antiparasitic activity (IC50 < 10 μg/mL). J. oxycedrus showed exceptional antitrypanosomal activity with low cytotoxicity on macrophage cell lines. α-pinene is likely the active compound of the J. oxycedrus oil and citral are likely the responsible for the activity of C. citratus oil. Acknowledgments Authors are grateful to Philippe Buscher from Institute of Tropical Medicine in Antwerp, Belgium for supplying the tryposmatigotes of T. b. brucei and to Professor Jorge Paiva for help in plant taxonomy. List of abbreviations BBB CNS COI

Blood Brain Barrier Central Nervous System Herbarium of the Department of Botany of the University of Coimbra DMSO – dimethylsulfoxide EOs Essential Oils FID Flame Ionization Detection GC Gas Chromatography GC/MS gas chromatography-mass spectroscopy HAT Human African Trypanosomiasis HMI 9 Hirumi's Modified Iscove's medium-9 IC50 – half maximal inhibitory concentration 39


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