CC BY-NC-ND 4.0 · Planta Medica International Open 2020; 07(03): e122-e132
DOI: 10.1055/a-1219-2207
Original Papers

GC-MS Analysis, Bioactivity-based Molecular Networking and Antiparasitic Potential of the Antarctic Alga Desmarestia antarctica

1  Department of Biomolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
Karen Cristina Rangel
2  Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
1  Department of Biomolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
Lorena Rigo Gaspar
2  Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
Péricles Gama Abreu-Filho
3  Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
Luíz Miguel Pereira
3  Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
Ana Patrícia Yatsuda
3  Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
Marília Elias Gallon
1  Department of Biomolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
Leonardo Gobbo-Neto
1  Department of Biomolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
,
Leandro da Costa Clementino
5  Institute of Chemistry, State University Júlio de Mesquita Filho, Araraquara, SP, Brazil
,
Márcia Aparecida Silva Graminha
4  Department of Clinical Analysis, School of Pharmaceutical Sciences of São Paulo State University Júlio de Mesquita Filho, Araraquara, SP, Brazil
,
Laís Garcia Jordão
6  Technology and Innovation Department, National Institute of Amazon Research, Manaus, AM, Brazil
,
Adrian Martin Pohlit
6  Technology and Innovation Department, National Institute of Amazon Research, Manaus, AM, Brazil
,
Pio Colepicolo-Neto
7  Chemistry Institute, University of São Paulo, São Paulo, SP, Brazil
,
Hosana Maria Debonsi
1  Department of Biomolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil
› Author Affiliations
Funding: This study had financial and logistic support from the Brazilian Antarctic Program (PROANTAR/MCT/CNPq N°64/2013), Brazilian Marine Force, National Institute of Science and Technology (INCT: BioNat), Grant # 465637/2014–0, and the State of São Paulo Research Foundation (FAPESP), Grant # 2014/50926–0 and Grant # 2017/03552–5. The authors are thankful to the University of São Paulo for providing access to necessary resources, the financial and fellowship support from the Brazilian research funding agencies Coordination of Improvement of Higher-Level Personnel (CAPES), and the National Council for Scientific and Technological Development (CNPq) for the scholarship provided, Grant #1408011/2018–4. The department of Biomolecular Sciences and the Núcleo de Pesquisas em Produtos Naturais e Sintéticos – NPPNS are acknowledged.
 

Abstract

Leishmaniasis, malaria, and neosporosis are parasitic diseases that affect humans and animals, causing public health problems and billions in economic losses. Despite the advances in the development of new drugs, the severe side effects of available leishmaniasis treatments, the Plasmodium spp. resistance to antimalarial drugs, and the lack of a specific treatment against neosporosis lead us to the search for new anti-protozoan molecules from underexplored sources such as the Antarctic marine environment. Herein, we describe for the first time the chemical profile of Desmarestia antarctica crude extract and fractions using GC-MS and LC-MS/MS (molecular networking) approaches, and evaluate their antiparasitic activity against Leishmania amazonensis, Neospora caninum, and multi-drug-resistant Plasmodium falciparum. Furthermore, the cytotoxicity in 3T3 BALB/c fibroblasts and Vero cells was evaluated. D. antarctica fraction E ( IC50 of 53.8±4.4 μg mL− 1 and selectivity index of 3.3) exhibited anti-promastigote activity and was fourfold more selective to L. amazonensis rather than to the host cells. D. antarctica fraction D (IC50 of 1.6±1.3 μg mL− 1 and selectivity index of 27.8), D. antarctica fraction F (IC50 of 3.1±2.1 μg mL− 1 and selectivity index of 23.1), and D. antarctica fraction H (IC50 of 3.1±2.0 μg mL− 1 and selectivity index of 12.9) presented the highest antiparasitic effects against N. caninum with no cytotoxic effects. Also, D. antarctica fraction D presented a significant antiplasmodial inhibitory effect (IC50 of 19.1±3.9 μg mL− 1 and selectivity index of 6.0). GC-MS analysis indicated palmitic acid, myristic acid, fucosterol, phthalic acid, di(2-methylbutyl) ester, loliolide, and neophytadiene as the main components in the active fractions. In addition, this is the first report of a biological screening of macroalgae secondary metabolites against N. caninum parasites.


#
Abbreviations

VLC: vacuum liquid chromatography

NPs: natural products

DA-FD: Desmarestia antarctica fraction D

DA-FE:Desmarestia antarctica fraction E

DA-FF: Desmarestia antarctica fraction F

DA-FG: Desmarestia antarctica fraction G

DA-FH: Desmarestia antarctica fraction H

DA-FI: Desmarestia antarctica fraction I

FA: fatty acid

SI: selectivity index

Introduction

Humans and animals are host to a myriad of protozoan parasites that cause severe public health problems and affect millions of people throughout the world, costing billions of dollars for developing countries each year [1].

Leishmaniasis is found in about 89 countries and accounts for 1.5 to 2 million new cases annually and causes approximately 70 000 deaths per year. The treatment consists of chemotherapeutic agents such as the pentavalent antimonials amphotericin B, paromomycin, and miltefosine. However, the effectiveness of these drugs is holdback by its severe side effects [2].

The phylum Apicomplexa includes several important human and animal disease-causing parasites, including the agents of human malaria Plasmodium spp. and the animal agent of neosporosis, Neospora caninum [3] [4] . N. caninum infects mammalian species, including cattle, sheep, goats, horses, and dogs, and despite the effort of many research groups and industry, neosporosis lacks an effective chemotherapy, leading to relevant economic losses mainly in developing and underdeveloped countries since the parasite is related to an abortive syndrome in cattle [5].

Human malaria is caused by four different species of Plasmodium spp., but the severe form is caused by Plasmodium falciparum [3] . There were an estimated 219 million cases and 43 500 deaths related to the disease in 2017 [6]. Malaria chemotherapy is effective and nontoxic but protozoa resistance to antimalarial drugs became a debilitating point towards its control and elimination [7]. The new drug artefenomel, a novel synthetic trioxolane antimalarial drug, is currently in phase 2 clinical tests [8], but with regard to the rapidly acquired Plasmodium spp. resistance to antimalarial drugs, the search for bioactive compounds against this parasite needs to be continued.

NPs have been used by humans through the ages [9] and are part of the backbone of traditional therapies [10]. The diversity of molecular structures from natural origins is responsible for the pronounced biological potential presented by these molecules, which represent a promising source of new drug leads [9].

Currently, many of the available drugs for parasitic disease treatment are NPs or derivatives [10] [11] [12]. Despite the fact that most of these molecules were isolated from terrestrial plants, for the past decades, we have been witnessing the growing advance of NPs obtained from marine sources in the field of drug discovery [13]. Indeed, marine organisms synthesize sophisticated molecules, aiming their survival in an environment with limited resources and predatory pressure [14]. Among the great diversity of marine organisms, macroalgae standout as an interesting source of bioactive molecules and is largely explored in the field of drug discovery for neglected diseases [1].

Extreme marine environments such as the Southern Ocean and the cold waters surrounding the Antarctic Peninsula are geographically and biologically isolated [15]. In Antarctica, the Desmarestiales are the main constituents of the benthic algal flora. Taxonomic studies of Desmarestia antarctica are well established, however, the secondary metabolites produced by this macroalga and its biological potential are poorly explored [16].

Even though progress has already been achieved in the past years in the field of NPs from polar regions, the climate conditions and remoteness of these environments still pose difficulties to the improvement of this area [17]. Moreover, another common issue regarding NPs and therapeutic lead discovery is that despite the potential results obtained in the bioassays in the initial extracts or fractions, the bioactive compounds may not be isolated or they are known molecules with established biological potential [18]. As a strategy, the use of new approaches such as MS/MS-based Global Natural Products Social (GNPS) Molecular Networking (MN) has contributed to avoiding the loss of chemical information and improved bioguided fractionation and isolation of new and bioactive molecules [19].

Herein, we report for the first time the chemical profile of volatile compounds in the macroalga D. antarctica. Furthermore, the antiparasitic activity of this species was evaluated against L. amazonensis, N. caninum, and P. falciparum, and its cytotoxicity on 3T3 BALB/c fibroblasts and Vero cells was assessed.


#

Results and Discussion

The chemical profile of volatile compounds in the crude extract of D. antarctica was accessed using GC-MS, and the identified constituents are shown in [Table 1]. Among the identified compounds, FAs, esters, terpenes, and sterols were the most prevalent. Twenty-five compounds were identified. The major FAs were palmitic acid (27.8%), oleic acid (15.8%), and myristic acid (12.0%). Phytol (2.9%) and neophytadiene (2.2%) were the prevalent terpenes. The sterol composition presented fucosterol (14.5%) as the major constituent of this class of compounds, followed by stigmasterol (7.9%), desmosterol (1.0%), and brassicasterol (0.3%). Our investigation of the D. antarctica chemical profile corroborates with previous investigations that show palmitic acid as the most predominant FA in the Antarctic brown macroalgae Ascoseira mirabilis, Adenocystis utricularis, and Phaeurus antarcticus [20]. The composition of macroalgal FAs varies in quality and quantity according to different environmental conditions such as light, temperature, and salinity [21] [22]. Cold-water macroalgae present a higher lipid content than tropical species [23].

Table 1 Chemical profile of volatile constituents identified in the D. antarctica crude extract obtained by GC-MS analysis (%) of total area.

Compound

RI

ID

(%)

Benzene carboxylic acid/C7H6O2

1180

MS1 a

0.33

Loliolide/C11H16O3

1667

MS1 b

1.11

Myristic acid/C14H28O2

1740

MS1 a

12.00

Phthalic acid, butyl undecyl ester /C23H36O4

1805

MS1 a

1.25

Neophytadiene/C20H38

1811

MS1 b

2.22

2-Pentadecanone, 6,10,14-trimethyl/C18H36O

1842

MS1 b

0.59

Phytol acetate/C22H42O2

1847

MS1 a

1.19

Palmitoleic acid/C16H30O2

1928

MS1 a

1.44

Palmitic acid/C16H32O2

1943

MS1 a

27.81

Oleic acid, methyl ester/C19H36O2

2094

MS1 b

0.30

Phytol/C20H40O

2108

MS1 b

2.19

Linoleic acid/C18H32O2

2115

MS1 a

1.51

Cervonic acid/C22H32O2

2119

MS1 a

1.47

Elaidic acid/C18H34O2

2123

MS1 a

15.77

Stearic acid /C18H36O2

2141

MS1 a

0.96

Arachidonic acid/C20H32O2

2286

MS1 a

1.26

Cis-5,8,11,14,17-Eicosapentaenoic acid, methyl ester/C21H31O2

2299

MS1 a

1.16

Elaidic acid, 2,3-dihydroxypropyl ester /C21H40O4

2315

MS1 b

1.35

2-Hexadecanoyl glycerol /C19H38O4

2448

MS1 a

0.42

Decanedioic acid, bis(2-ethylhexyl) ester/C26H50O4

2792

MS1 a

0.17

α-Tocopherol/C29H50O2

3120

MS1 a

0.11

Stigmasterol/C29H48O

3205

MS1 a

7.90

Brassicasterol/C28H46O

3235

MS1 a

0.33

Fucosterol/C29H48O

3299

MS1 b

14.56

Desmosterol/C27H44O

3468

MS1 b

1.09

RI: relative retention index, ID: identification by GC-MS fragmentation profile, MS1 a : Nist11.lib, MS1 b : Wiley7.lib.

In a previous study, the crude extract of D. antarctica presented antifouling activity against model strains of sympatric diatoms that could potentially foul it in nature [24]. However, no description of the secondary metabolites present in the extract was provided, highlighting the need to chemically characterize this macroalga species. In addition, the molecules identified in D. antarctica crude extract could be used as a fingerprint to future ecological studies and it is an important contribution to literature data.

The VLC fractionation of the crude extract led to nine different fractions. However, fractions DA-FA – DA-FC presented low yields, therefore only fractions DA-FD – DA-FI were used for antiparasitic activity evaluation.

In the antileishmanial assay against L. amazonensis promastigotes, only fraction DA-FE (IC50 of 53.8 μg mL− 1 ) presented anti-promastigote activity ([Fig. 1]). Cytotoxicity on 3T3 BALB/c fibroblasts ([Fig. 1S], Supporting Information) demonstrates that the DA-FE was fourfold more selective to the parasites than the reference drug amphotericin B ([Table 2]).

Zoom Image
Fig. 1 Inhibition growth (%) of L. amazonensis promastigotes after treatment with different concentrations (μg mL− 1) of fractions DA-FD, DA-FE, DA-FF, and DA-FG. The results are expressed as the mean (n=3)±SD of three independent experiments.

Table 2 Anti-L. amazonensis promastigote activity and 3T3 BALB/c fibroblast cytotoxicity of the crude extract and fractions from D. antarctica.

Sample

IC50-PRO a (μg mL−1; mean±SD)

IC50-BALB/c b (μg mL−1; mean±SD)

SI c

Crude extract

>500

815.6±2.7

DA-FD

96.5±5.5

115.2±1.1

1.1

DA-FE

53.8±4.4

179.1±1.4

3.3

DA-FF

102.2±5.7

134.0±1.1

1.3

DA-FG

223.2±9.7

DA-FH

>250

138.7±1.2

2.4

DA-FI

>250

Amphotericin B

5.8±0.8

4.6±1.0

0.8

aAntiparasitic activities are expressed as half-maximal inhibitory concentrations (IC50-PRO), and b mammalian cell toxicities are expressed as half-maximal cytotoxic concentrations (IC50-BALB/c). c The selectivity index was calculated as the IC50-BALB/c/IC50-PRO.

Results indicated that all fractions significantly inhibited the proliferation of N. caninum tachyzoites ([Fig. 2]). Among these fractions, DA-FD, DA-FE, DA-FF, and DA-FH demonstrated IC50 values below 5.5 µg mL− 1 on N. caninum (1.6 µg mL− 1, 4.2 µg mL− 1 3.1 µg mL− 1, and 3.1 µg mL− 1, respectively). On the other hand, DA-FI and the crude extract presented the lowest capacities to inhibit N. caninum with IC50 values of 12.5 µg mL− 1 and 20.6 µg mL− 1, respectively ([Table 3]). All fractions demonstrated higher CC50 values on Vero cells compared to IC50 values on N. caninum tachyzoites. No toxic effects were observed for concentrations below 40 µg mL− 1 ([Table 3] and [Fig. 2S], Supporting Information). The CC50 value was used to calculate the SI, which represents the relation between the cytotoxicity and the anti-parasite concentrations (CC50/IC50), indicating the effectiveness and safety of a compound for further in vivo applications. Among the fractions, DA-FD indicated the highest SI (27.8), followed by DA-FE (> 23.8), DA-FF (23.2), DA-FG (> 18.2), DA-FH (12.9), DA-FI (7.0), and the crude extract (6.6) ([Table 3]).

Zoom Image
Fig. 2 Inhibition growth (%) of N. caninum tachyzoites after treatment with different concentrations (μg mL− 1) of the crude extract and fractions DA-FD, DA-FE, DA-FF, DA-FG, DA-FH, and DA-FI. The results are expressed as the mean (n=3)±SD of three independent experiments.

Table 3 Anti-N. caninum tachyzoites activity and Vero cell cytotoxicity of the crude extract and fractions from D. antarctica.

Sample

IC50-TAC a (μg mL− 1; mean±SD)

CC50-VERO b (μg mL− 1; mean±SD)

SI c

Crude extract

20.6±6.3

75.1±22.6

3.6

DA-FD

1.6±1.3

44.5±21.3

27.8

DA-FE

4.2±2.4

>100

23.8

DA-FF

3.1±2.1

71.9±16.6

23.2

DA-FG

5.5±1.3

>100

18.2

DA-FH

3.1±2.0

40.1±6.9

12.9

DA-FI

12.5±17.2

87.3±57.2

7.0

Pyrimethamine

0.7

aAntiparasitic activities are expressed as half-maximal inhibitory concentrations (IC50-TAC), and b mammalian cell toxicities are expressed as half-maximal cytotoxic concentrations (CC50-VERO). c The selectivity index was calculated as the IC50-VERO/IC50-TAC,

Results presented fraction DA-FD as the most active in the anti-Neospora evaluation. Concerning this bioactivity, DA-FD was screened against P. falciparum and showed promising antiplasmodial activity (IC50 of 19.1 μg mL− 1) ([Table 4] and [Fig. 3]).

Zoom Image
Fig. 3 Inhibition growth (%) of P. falciparum trophozoites after treatment with different concentrations (μg mL− 1) of fraction DA-FD. The results are expressed as the mean (n=3)±SD of two independent experiments.

Table 4 Antiplasmodial activity in P. falciparum trophozoites and cytotoxicity in 3T3 BALB/c fibroblasts of the DA-FD fraction from D. antarctica.

Sample

IC50-TRO a (μg mL− 1; mean±SD)

IC50-BALB/c b (μg mL− 1; mean±SD)

SI c

DA-FD

19.1±3.9

115.2±1.1

6.0

Chloroquine diphosphate

0.1±0.3

aAntiparasitic activities are expressed as half-maximal inhibitory concentrations (IC50-TRO), and b mammalian cell toxicities are expressed as half-maximal cytotoxic concentrations (IC50-BALB/c). c The selectivity index was calculated as the IC50-BALB/c/IC50-TRO.

The volatile constituents of the most active fractions in the antiparasitic bioassays were identified using GC-MS analysis and are shown in [Table 5]. The fractions DA-FD, DA-FE, DA-FF, and DA-FH presented promising antiparasitic effects. The major identified compounds in fraction DA-FD were palmitic acid (29.3%), myristic acid (21.3%), fucosterol (8.8%), oleic acid, methyl ester (3.2%), and desmosterol (2.8%). Myristic acid (14.9%) and oleic acid (12.6%) represent the major FAs in fraction DA-FE. In fraction DA-FF, the major constituents were phthalic acid, di(2-methylbutyl) ester (27.0%), loliolide (15.1%), phytol acetate (4.2%), and neophytadiene (4.0%). Fraction DA-FH presented loliolide (16.7%) as the major compound, followed by neophytadiene (15.3%), tetradecanal (8.0%), phytol acetate (6.5%), phthalic acid, bis(2-ethylhexyl) ester (6.1%), and 2-pentadecanone, 6,10,14-trimethyl (3.2%).

Table 5 Chemical profile of volatile constituents identified in DA-FD, DA-FE, DA-FF, and DA-FH obtained by GC-MS analysis (%) of the total area.

Compounds

RI/ ID

Peak area (%)

DA-FD

DA-FE

DA-FF

DA-FH

Trans-2-Heptenal/C7H12O

780/ b

0.15

0.22

0.57

1,4-Hexadiene, 3-ethyl/C8H14

999/ a

0.18

Trans-6-Tetradecene/C14H28

1053/ a

0.28

6-Undecanone/C11H22O

1085/ b

0.35

Nonanal/C9H18O

1112/ a

0.15

0.35

0.24

2-Ethyl-3-methylmaleimide/C7H9NO2

1192/ b

0.13

0.67

Trans-Decenal/C10H18O

1254/ a

0.57

0.25

1.43

1.52

Heptanoic acid, anhydride/C14H26O3

1277/ a

0.85

0.11

Trans-2,Cis-4-Decadienal/C10H16O

1283/ b

0.13

Trans-2-Decen-1-ol/C10H20O

1334/ a

0.09

0.42

Dodecane, 2,6,10-trimethyl/C15H32

1436/ a

0.11

0.35

Dihydroactinolide/C11H16O2

1490/ a

0.36

2,11-Dioxatetracyclo-undec-4-ene, 3,7,7,10-tetramethyl/C13H18O2

1497/ a

4.48

Tetradecanal/C14H28O

1607/ a

8.00

Loliolide/C11H16O3

1667/ b

0.22

15.08

16.66

Palmictoleic acid, hexadecylester/C32H64O2

1715/ b

0.98

Myristic acid, methylester/C15H30O2

1717/ a

1.41

0.32

Myristic acid/C14H28O2

1740/ a

21.30

14.92

1.68

Palmitic acid/C16H32O2

1720/ b

29.31

Neophytadiene/C20H38

1811/ b

2.31

1.04

3.97

15.30

Elaidic acid /C18H34O2

1832/ a

12.69

2–Pentadecanone, 6,10,14–trimethyl/C18H36O

1842/ a

1.79

2.09

1.31

3.20

Phytol acetate/C22H42O2

1847/ b

4.23

6.52

Phthalic acid, diisobutyl ester/C16H22O4

1848/ a

2.74

Palmitoleic acid, methylester/C17H32O2

1907/ b

0.44

Palmitic acid, methylester/C17H34O2

1918/ b

1.60

0.20

Phthalic acid, butyl isobutyl ester/C16H22O4

1978/ a

0.34

1-Nonadecene/C19H38

1993/ a

0.24

1,2-Oxathiane, 6-dodecyl-, 2,2-dioxide/ C16H32O3S

1995/ a

0.34

1,8-Dioxacyclohexadecane-2,10-dione, 5,6:12,13-diepoxy-8,16-dimethyl/C16H24O6

2037/ a

0.59

Palmitic acid, 3-hydroxy-, methylester/C17H34O3

2052/ a

0.11

Linoleic acid, methylester/C19H34O2

2088/ a

0.24

Oleic acid, methylester/C19H36O2

2095/ a

3.24

14β-Pregnane/C21H36

1906/ a

1.16

3.44

Phthalic acid, di(2-methylbutyl) ester/C18H26O4

1959/ b

27.00

Cis-Phytol/C20H40O

2104/ b

0.76

Phytol/C20H40O

2108/ b

0.66

1.37

1.56

2.61

Palmitaldehyde, diallyl acetal/C22H42O2

2137/a

0.81

1.78

Triacontane/C30H62

2323/ c

1.93

17.64

0.77

Phthalic acid, bis(2-ethylhexyl) ester/C24H38O4

2528/ a

1.17

2.56

2.42

6.06

Cis-13-Docosenamide/C22H43NO

2627/ a

0.57

α-Tocopherol/C29H50O2

3120/ a

0.33

0.76

Desmosterol/C27H44O

3253/ a

2.85

Isofucosterol/C29H48O

3290/ b

0.72

Fucosterol/C29H48O

3299/ b

8.80

RI: relative retention index, ID: identification by GC-MS fragmentation profile, a Nist11.lib, b Wiley7.lib; c FFNSC1.3.lib.

Continuing our efforts to chemically characterize the bioactive fractions in the N. caninum assay, we used the data generated through LC-MS/MS analysis to construct a bioactivity-based MN of D. antarctica. After the data process, the ions that presented r>0.65 and p value < 0.1 (larger nodes) were pointed as the bioactive components and are presented as larger nodes in [Fig. 4]. The GNPS library was used to identify annotated molecules. However, none of the ions were identified. In [Table 6], we summarize the ions that were presented as bioactive and present probable molecular formulas of these molecules.

Zoom Image
Fig. 4 Molecular networking of D. antarctica fractions analyzed by LC-MS in the positive ionization mode. Nodes represent detected compounds and are colored according to the type of sample. Larger node forms represent ions with a high score of bioactivities (r>0.65 and p value < 0.1) in N. caninum assays. Edges between nodes represent molecular structural similarity between compounds. Nodes without GNPS spectral library matching with other nodes are represented as self-loops.

Table 6 Molecular formula of ions indicated as bioactive (r>0.65 and p value < 0.1) against N. caninum.

RT

m/z

Molecular formula

Error (ppm)

18.3

309.2009

C13H28N2O6

5.3

C20H24N2O

13.7

C18H28O4

18.3

23.2

155.1049

C5H10N6

2.5

C9H14O2

14.8

25.5

381.2970

C18H40N2O6

1.5

C23H40O4

9.1

C24H36N4

12.6

C25H36N2O

16.9

26.5

137.1263

C10H16

8.1

27.6

183.1374

C11H18O2

6.0

C7H14N6

8.7

34.2

221.1771

C12H20N4

2.2

C11H24O4

8.3

34.4

435.2974

C23H38N4O4

0.7

C22H42O8

3.7

C28H38N2O2

8.6

C29H38O3

17.2

36.9

691.4970

C37H70O11

3.8

C44H66O6

4.7

C51H62O

13.2

C48H66O3

17.3

RT: retention time in minutes, m/z: mass to charge ratio.

The FAs present antibacterial properties due their ability to kill or inhibit the growth of bacteria, and are used by many organisms as defense against parasitic or pathogenic bacteria [25]. Fucosterol is the characteristic sterol of brown macroalgae [26]. Previous studies report the antileishmanial activity of fucosterol isolated from the brown macroalga Lessonia vadosa towards amastigotes of Leishmania infantum (IC50 of 10 μM, SI>10) and L. amazonensis (IC50 of 8 μM, SI>12), despite the lower activity when compared to amphotericin B (IC50 of 0.2 μM). The higher SI values of fucosterol turn it into a promising lead for the development of leishmanicidal drugs with less toxic effects [27]. Fucosterol isolated from the brown macroalgae Sargassum linearifolium also exerted high inhibitory effects against 3D7 chloroquine sensitive P. falciparum (IC50 of 7.48 μg mL− 1), and morphological changes of P. falciparum were observed [28].

Phthalates have been isolated from Brevibacterium mcbrellneri and showed bactericidal and mosquito larvicidal activities [29]. A study reporting the antileishmanial potential of the Antarctic red alga Irideae cordata against L. amazonensis amastigotes led to the identification of phthalic acid, diisobutyl ester, phthalic acid, di(2-methylbutyl) ester and palmitic acid beta-monoglyceride in the most active fractions of I. cordata through GC-MS analysis [17]. Loliolide is found in many macroalgae and plants and its biological activity has been described as a repellent [30] and inducer of herbivore resistance [31]. The active hexane and dichloromethane fractions of the red macroalgae Centroceras clavulatum against Trypanosoma cruzi forms (epimastigote IC50 of 19.1 μg mL− 1 and trypomastigote IC50 of 76.2 μg mL− 1) were analyzed through GC-MS and compounds such as loliolide, neophytadiene, and phytol were identified [32]. The antiparasitic activity of phytol against Schistosomiasis mansoni has been described. In vitro, phytol reduced the motor activity of worms, caused by their death. In vivo, a single dose of phytol (40 mg/kg) administered orally to mice infected with adult S. mansoni resulted in total and female worm burden reductions of 51.2 and 70.3%, respectively [33]. A previous study has identified neophytadiene in cytotoxic fractions of Senna spp. (Leguminosae, Caesalpinioideae) tested against human colon and human glioblastoma cell lines [34]. Interestingly, the increase of neophytadiene concentration in fraction DA-FH (15.3%) compared to DA-FF (3.9%) led to higher cytotoxic effects in Vero cells. The fractions of D. antarctica demonstrated low toxicity in the tested mammal cells.

Despite the advances for the screening of drug candidates against N. caninum [35], there are few works reporting the bioactivity of NPs against this parasite. Only plant extracts such as Thai Piperaceae, Thalassomya japonica, and Sophora flavescens were screened to date [36] [37]. Comparatively, the D. antarctica extract and fractions demonstrated a lower IC50 in relation to the Thai piperaceae extract (IC50 22.1 μg mL− 1).

To our knowledge, there is no reports concerning the use of macroalgae secondary metabolites against N. caninum. In this way, this work reveals the novelty of macroalgae use to treat N. caninum infections. Thus, the use of macroalgae derivatives may be a promising strategy to develop forms to control coccidian parasites in humans and animals. Moreover, special attention is necessary to develop the use of alternative sources of compounds to the N. caninum control, once there is no commercial strategy for the neosporosis treatment.


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Material and Methods

Algal material

Samples of D. antarctica R. L. Moe & P.C. Silva Desmarestiaceae specimens were collected at King’s George Island, Demay Point, Antarctica (62°12' 60.0" S 58°25' 59.9" W) in January 2016 during the Brazilian Antarctic Expedition OPERANTAR XXXIV. A total of 350 g of macroalga were collected and manually cleaned with local seawater to remove surface contaminants. A voucher specimen was authenticated by Dr. Beatriz Castelar Duque Estrada and MSc. Jônatas Martinez Canuto Souza and deposited in the herbarium Maria Eneyda P. Kauffman Fidalgo, Institute of Botany (São Paulo – Brazil) under number SP 470155.


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Extraction and fractionation

A sample (300 g) was fragmented and extracted (×3) with dichloromethane (CH2Cl2):methanol (MeOH) 2:1 v/v (500 mL) for 30 min under stirring in a thermal blanket with a controlled temperature (30°C). Solvents (CH2Cl2:MeOH) were selected to obtain both polar and nonpolar compounds. The combined resulting solutions were evaporated under reduced pressure at 30°C, resulting in 2 g of crude extract (CE). The CE was fractionated by silica gel 60 (Mesh 70–230) VLC in a 500-mL glass Buchner funnel. Elution using the organic solvents, n-hexane (HX), ethyl acetate (EtOAc), and methanol (MeOH) with 300 mL of a stepwise polarity gradient yielded nine fractions: DA-FA (HX), DA-FB (HX:EtOAc, 9:1), DA-FC (HX:EtOAc, 8:2), DA-FD (HX:EtOAc, 6:4), DA-FE (HX:EtOAc, 4:6), DA-FF (HX:EtOAc, 2:8), DA-FG (EtOAc), DA-FH (EtOAc:MeOH, 7.5:2.5), and DA-FI (MeOH).


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Chemical profile of crude extract and bioactive fractions using a GC-MS approach

The GC-MS analysis was performed using a Gas Chromatograph Mass Spectrometer Mod GCMS-QP2010-Ultra (Shimadzu). Analyses were performed using a nonpolar RTx-5MS (30 m×0.25 mm×0.25 um) column and helium (H2) as the carrier gas at a flow rate of 1 mL min− 1. The temperature was increased at the rate of 3°C min− 1 from 60 to 260°C and was held isothermally for 60 min. The injection and transfer line temperatures were 260°C. The detection was carried out in the full scan mode ranging between 50 and 650 m/z. The ionization mode employed was electron impact (EI) with a collision energy of 70 eV, and the mass spectrometer ion source was maintained at 260°C. The relative retention index (RI) values were calculated by evaluating external standard sets of n-alkanes (C9-C35) under the same conditions and column using the formula obtained by Vandendool and Kratz equation [38]. The confirmation of the compounds was obtained by comparing the calculated RI values with library matches (Wiley 7, Nist 11s, and FFNSC1.3 libraries).


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LC-MS/MS analysis and molecular networking approach

The crude extract and fractions from D. antarctica were analyzed on a Shimadzu UFLC system coupled to a quadrupole time-of-flight tanden mass spectrometer (micrOTOF QII, Bruker Daltonics) using a C18 Supelco column (5 µm, 15 cm×3.0 mm; Ascenti Express C18). The mobile phase was composed of water (A) and MeOH (B), both with 0.1% formic acid at a flow rate of 1.0 mL min− 1. The gradient was 0–35 min, 30–90% B; 35–45 min, 90% B; 45–55 min 90–100% B; 55–60, min 100–30% B; 60–65 min, 30% B. The column oven was set at 35°C and 20 µL of each sample were injected.

Chemical profiles were obtained in the positive ionization mode. The mass spectrometer parameters were as follow: capillary voltage 3.5 kV; end plate offset 500 V; nebulizer 5 bar; dry gas (N2) flow 10 L · min− 1; dry temperature 220°C; scan mode MS/MS (auto) between m/z 100 and 1500; precursor average 4; number of precursors 3; exclusion activation 1 spectra; exclusion release 36 s; charged ions group length 5.

LC-MSn data were converted to the .mzXML format using MSconvert software (Proteowizard Software Foundation) and processed using MzMineTM (BMC Bioinformatics). The following parameters were employed: mass detection using the centroid algorithm, scan MS level 1 (noise level, 1.0E3) and scan MS level 2 (noise level, 1.0E2); ADAP chromatogram builder (min group size in # of scans, 1.0E3; group intensity threshold, 1.0E3; min highest intensity, 3.0E3; m/z tolerance, 0.01 m/z or 20 ppm); chromatogram deconvolution using wavelets (ADAP) algorithm (S/N threshold, 10; S/N estimator, intensity window SN; min feature height, 3.0E3; coefficient/area threshold, 10; peak duration range, 0.02–2.00; RT wavelet range, 0.02–0.20) (m/z center calculation – median; m/z range for MS2 scan pairing, 0.01; RT for MS2 scan pairing, 0.2); isotopic peak grouper (m/z tolerance, 0.01 m/z or 20 ppm; retention time tolerance in minutes, 0.2; maximum charge, 2; representative isotope, most intense) and alignment using the join aligner [m/z tolerance, 0.02 m/z or 20 ppm; weight for m/z, 75; retention time tolerance, 0.2 (abs); weight for retention time, 25]. After data processing, peaks with the MS2 scan were exported for GNPS analysis (.csv quantification spreadsheet and .mgf file).

The output data were uploaded to the GNPS platform and a feature-based MN was created, employing the following parameters: precursor ion mass tolerance, 2.0 Da; fragment ion mass tolerance, 0.5 Da; minutes pairs cos, 0.6; minimum matched fragment ions, 4; maximum shift between precursors, 500 Da; network topK, 10; maximum connected component size, 100; library search minutes matched peaks, 4; score threshold, 0.7; search analogues, don’t search; maximum analog search mass difference, 100 Da; top results to report per query, 1; minimum peak intensity, 0; filter precursor window, filter; filter library, filter library; filter peaks in 500 Da window, filter; normalization per file, no norm; aggregation method for peak abundances per group, sum. The generated MN was visualized and analyzed with Cytoscape version 3.8 (Institute for Systems Biology).

The bioactivity score significance was predicted and mapped onto the MN according to Nothias et al. [18]. Briefly, the .csv spreadsheet generated by MzMine was used for the calculation of the bioactivity score for N. caninum and L. amazonensis, separately, using the selective index and an R-based Jupyter notebook available from GitHub at http://github.com/DorresteinLaboratory/Bioactive_Molecular_Networks. The output table was imported into the Cytoscape software and the nodes with r>0.65 and p value < 0.1 were selected (larger nodes).


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Antileishmanial activity

Promastigote forms of L. amazonensis (MPRO/BR/1972/M1841-LV-79) were cultivated at 27°C in liver infusion tryptose medium supplemented with 10% FBS (Sigma-Aldrich), penicillin, and streptomycin (Sigma-Aldrich). Cultured promastigotes at the end of the exponential growth phase (6–7 days) were seeded at 1×107 parasites mL− 1 in 96-well flat-bottom plates (TPP; Sigma-Aldrich). Samples were dissolved in DMSO (Sigma-Aldrich) (the highest concentration was 1.4%), then they were added to the parasite suspension at final concentrations from 7.8–500 μg mL− 1 to the crude extract and from 3.9–250 μg mL− 1 to the fractions, and incubated at 27°C for 72 h. Amphotericin B (purity>95%; Sigma-Aldrich) was used as a reference drug from 1.6–100 µg mL− 1. The assays were carried out in triplicate. The cell viability was assessed by the MTT method [39]. Briefly, the plates were kept at 28°C for 72 h. Then, an aliquot of 10 μL of 6 mM MTT and 0.7 mM PMS (phenazine methosulfate) was added to each well, and the plates were incubated at 28°C for 75 min. Subsequently, 100 μL of 10% sodium dodecyl sulfate (SDS) were added and maintained at room temperature for 30 min, and, finally, the samples were read at 595 nm. All the incubations were performed in the dark. The 50% of promastigote parasite growth inhibition is expressed as the inhibitory concentration (IC50-PRO) in μg.


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Anti-neospora activity

A proliferation assay was performed as previously described [5] using β-galactosidase-expressing tachyzoites (NcLacZ). Briefly, purified LacZ N. caninum tachyzoites were distributed (1×103/well) on Vero cell cultures in a 96-well plate and incubated for 2 h at 37°C and 5% CO2 to allow the invasion after the invasion process. Seven serial dilutions (starting from 100 μg mL− 1) of the crude extract and fractions of D. antarctica were added to the cultures and incubated for 72 h at 37°C and 5% CO2 . Following the treatment step, the wells were washed with PBS and lysed with the lysis buffer [100 mM 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 8.0; 1 mM CaCl2; 1% Triton X-100, 0.5% SDS; 5 mM dithiothreitol] for 1 h at 50°C. The lysed cultures were incubated with chlorophenol red-β-D-galactopyranoside (CPRG) buffer (5 mM CPRG, 5 mM 2-mercaptoethanol in PBS) for 2 hat 37°C and the plates were read with an ELISA reader (Synergy H1, Biotek) at 570 nm. Pyrimethamine (purity>95%; Sigma-Aldrich) was used as a control drug. The percentage of parasite inhibition and cell toxicity was calculated from the mean absorbance of samples in relation to the non-treated controls. Three independent assays were performed.


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Cytotoxicity on Vero cells

The MTT assay [40] was applied for toxicity evaluation on Vero cells. The cells were cultivated in Roswell Park Memorial Institute (RPMI) supplemented with 5% FBS (RPMI-FBS) in 75 cm2 flasks. For the MTT assay, the cultures were distributed in 96-well plates (5×103/well in RPMI-FBS) and cultivated at 37°C and 5% CO2. After cell confluence, the plates were incubated with serial dilutions of D. antarctica crude extract and fractions (starting from 100 μg mL− 1 in phenol-free RPMI) for 72 h, 37°C, and 5% CO2. The media was removed, and the treated cultures were incubated with 100 μL of MTT (purity>98%; Sigma-Aldrich) solution (500 μg mL− 1) for 4 h, 37°C, and 5% CO2, followed by formazan crystal dilution with DMSO. The plates were read at 570 nm in an ELISA reader (Synergy H1; Biotek), and the percentage of cytotoxicity (in relation to non-treated controls) was calculated from three independent assays. The positive control was composed of 5% DMSO (purity>98%; Sigma-Aldrich), which led to > 95% cytotoxicity compared to the non-treated group. For all groups treated with D. antarctica fractions, the DMSO concentration was < 1%. From the percentages of tachyzoite/Vero cell inhibition, the IC50 and CC50 values were calculated using Compusyn software (http://www.combosyn.com/) [41].


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Antiplasmodial activity

In vitro cultures of chloroquine-resistant P. falciparum K1 strains (MRA-159, MR4, ATCC) were established using A+type blood cells and RPMI-1640 culture medium enriched with 10% plasma. The in vitro inhibition of the growth of P. falciparum K1 from these cultures by DA-FD was evaluated as described previously [42]. Briefly, as the initial condition for the susceptibility assay, the synchronization of cultures with 5% D-sorbitol provided young trophozoites (ring stage). The sample was diluted in DMSO (10 mg mL− 1) to aid stock solutions. Stock solutions underwent sequential dilution in culture medium (RPMI-1640), resulting in seven diluted samples with concentrations in the range of 0.13–100 μg mL− 1 (well concentrations) and final (well) DMSO concentrations of 1%. The test solution was transferred to 96-well test plates containing parasitized red blood cells with initial 2% hematocrit and 1% parasitemia. The sample was evaluated in duplicate and the test plate was incubated for 48 h at 37°C. After the incubation period, analysis of thin blood smears of the contents of each well using an optical microscope provided the parasitemia of each well. Chloroquine diphosphate (purity > 98%; Sigma-Aldrich) was used as a control drug (0.003–2.5 μg mL− 1). Interpolation of the nonlinear curve using GraphPad Prism software permitted the calculation of estimates of the sample concentrations able to inhibit 50% of parasite growth (IC50) compared to drug-free controls. The IC50 values represent the results from two independent experiments with a confidence interval of 95%.


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Cytotoxicity on 3T3 BALB/c fibroblasts

Cytotoxicity was evaluated by the neutral red uptake method [43]. The 3T3 BALB/c fibroblasts (Banco de células do Rio de Janeiro – BCRJ, Brazil) were cultivated in DMEM (Gibco) supplemented with 10% FBS, 4 mM glutamine, penicillin (100 IU mL− 1) and streptomycin (100 µg mL -1). The cell suspension (1×105 cells mL− 1) was seeded in 96-well plates and incubated for 24 h at 37°C in an atmosphere of 5% CO2. The samples and the positive control were firstly dissolved in DMSO (the highest final concentration was 1%). Next, they were diluted into eight concentrations in DMEM (5% FBS), with the final concentration ranging from 6–100 μg mL− 1 for DA-FD, from 23–100 μg mL− 1 for DA-FH, and from 0.33–5 μg mL− 1 for amphotericin B. The positive control used was sodium lauryl sulfate (Invitrogen). After incubation, the cells were treated with the diluted samples for 24 h at 37°C in an atmosphere of 5% CO2, then the plates were washed with PBS (pH=7.2). The cell viability was evaluated using the neutral red uptake method, and the plates were read at 540 nm. The assays were carried in triplicate. The concentration producing 50% of cytotoxicity is expressed as the IC50-BALB/c value in μg mL− 1. The IC50 and CC50 values were calculated by nonlinear regression analysis using the software GraphPad Prism version 5.0, and are presented as the mean (n=3)±standard deviation (SD).


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Supporting information

Dose-response curves that support the calculation of CC50 values of D. antarctica fractions on 3T3 BALB/c and D. antarctica crude extract and fractions on Vero cells are available as Supporting Information.


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Conflict of Interest

The authors declare that they have no conflict of interest.

Supporting Information


Correspondence

Prof. Dr. Hosana Maria Debonsi
Departamento de Ciências Biomoleculares, Faculdade de
Ciências Farmacêutica de
Ribeirão Preto, Universidade de
São Paulo
Avenida do Café s/n
Ribeirão Preto
São Paulo
14040-903
Brasil   
Phone: +55 16 3315 4713   
Fax: + 55 16 3315 4243   

Publication History

Received: 06 May 2020
Received: 20 June 2020

Accepted: 13 July 2020

Publication Date:
21 August 2020 (online)

© 2020. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).

© Georg Thieme Verlag KG
Stuttgart · New York


  
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Fig. 1 Inhibition growth (%) of L. amazonensis promastigotes after treatment with different concentrations (μg mL− 1) of fractions DA-FD, DA-FE, DA-FF, and DA-FG. The results are expressed as the mean (n=3)±SD of three independent experiments.
Zoom Image
Fig. 2 Inhibition growth (%) of N. caninum tachyzoites after treatment with different concentrations (μg mL− 1) of the crude extract and fractions DA-FD, DA-FE, DA-FF, DA-FG, DA-FH, and DA-FI. The results are expressed as the mean (n=3)±SD of three independent experiments.
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Fig. 3 Inhibition growth (%) of P. falciparum trophozoites after treatment with different concentrations (μg mL− 1) of fraction DA-FD. The results are expressed as the mean (n=3)±SD of two independent experiments.
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Fig. 4 Molecular networking of D. antarctica fractions analyzed by LC-MS in the positive ionization mode. Nodes represent detected compounds and are colored according to the type of sample. Larger node forms represent ions with a high score of bioactivities (r>0.65 and p value < 0.1) in N. caninum assays. Edges between nodes represent molecular structural similarity between compounds. Nodes without GNPS spectral library matching with other nodes are represented as self-loops.