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

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.

Key words

desmarestiaceae - desmarestia antarctica - leishmania amazonensis - neospora caninum - plasmodium falciparum - antiparasitic - molecular targets and activities - marine macroalgae

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, MS^{1 a} : Nist11.lib, MS^{1 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]).

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 (IC_50-PRO), and ^b mammalian cell toxicities are expressed as half-maximal cytotoxic concentrations (IC_50-BALB/c). ^c The selectivity index was calculated as the IC_50-BALB/c/IC_50-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]).

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 (CC_50-VERO). ^c The selectivity index was calculated as the IC_50-VERO/IC_50-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]).

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 (IC_50-TRO), and ^b mammalian cell toxicities are expressed as half-maximal cytotoxic concentrations (IC_50-BALB/c). ^c The selectivity index was calculated as the IC_50-BALB/c/IC_50-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.

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.

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.

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).

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).

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).

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.

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% CO_{2 .} 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.

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].

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%.

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).

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.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Publication History

Received: 06 May 2020
Received: 20 June 2020

Accepted: 13 July 2020

Article published online:
21 August 2020