Chemical Profile and Biological Potential of Non-Polar Fractions from Centroceras clavulatum (C. Agardh) Montagne (Ceramiales, Rhodophyta)

The present study reports the Gas Chromatography-Mass Spectrometry (GC-MS) evaluation of the hexanes and dichloromethane fractions from extracts of the red alga Centroceras clavulatum (C. Agardh) Montagne. Twenty three compounds were identified, totaling ca. 42% of both fractions (0.18 g mass extract). The main constituents of the fractions were hexadecanoic acid (17.6%) and pentadecanoic acid (15.9%). Several secondary metabolites with interesting biological activity, such as (-)-loliolide, neophytadiene, phytol were identified. In addition, several classes of secondary metabolites, including phenolic compounds (e.g., phenylacetic acid), terpene derivatives, fatty acids, halogenated compound (e.g., 2-chlorocyclohexenol), lignoids, steroids, esters, amides (e.g., hexadecanamide), ketones, carboxylic acids, aldehydes and alcohols were observed. The occurrence of several of these structural classes is described for the first time in this species. The same fractions analyzed by GC-MS, and a separate set of polar fractions, were evaluated against two life cycle stages (epimastigote and trypomastigote forms) of the protozoan Trypanosoma cruzi and against phytopatogenic fungi Cladosporium cladosporiodes and C. sphaerospermum. The dichloromethane fraction was active against both T. cruzi forms (epimastigote IC50 = 19.1 μg.mL−1 and trypomastigote IC50 = 76.2 μg.mL−1). The hexanes and ethyl acetate fractions also displayed activity against both fungi species (200 μg) by TLC-bioautography.


Introduction
The family Ceramiaceae (Ceramiales, Rhodophyta) consists of more than one hundred genera, and 28 of them are found along the Brazilian coast [1,2], including the type genus Ceramium Roth, and Centroceras Kützing. Centroceras clavulatum (C. Agardh) Montagne has a wide distribution and common occurrence around the World, and it is the only species of the genus reported in Brazil [3].
Recent reports have shown biological activity in red algae. An interesting antifouling property, for example, was demonstrated by Ceramium botryocarpum A.W.Griffiths ex Harvey, which has been used in France in aquaculture tanks to limit the growth of invasive green algae (Cladophora, Bryopsis and Ulva genus) [4].
Hayee-Memenon and co-workers identified secondary metabolites of Centroceras clavulatum from Karachi (Pakistan) by GC-MS analysis; fatty acids and sterols were detected [5]. The present study reports the identification of volatile compounds of C. clavulatum collected from the coast of São Paulo, in southeastern Brazil, in order to investigate chemical profile of Brazilian C. clavulatum. In addition, this research was encouraging since essential fatty acids are precursors of prostaglandins, which are becoming increasingly important to the pharmaceutical industry [6].
The biological potential demonstrated by marine red algae [7][8][9], suggests that additional analysis to evaluate the trypanocidal activity would be worthwhile. Activity in this study was assessed against the flagellated protozoan Trypanosoma cruzi [10], the causative agent of Chagas' disease, which affects 16-18 million people around the World and more than 100 million live in endemic areas, making it one of the major neglected tropical diseases [11][12][13]. Currently, between 12 and 14 million people are infected by T. cruzi. Also, one of the routes of transmission of infection by T. cruzi is by the placenta, resulting in the emigration of large numbers of infected women from rural areas to cities where there are no vectors of transmission [14]. Organ transplantation is another source of transmission. This mechanism of transmission may trigger the acute phase of the disease, since the individuals who receive transplanted organs take immunosuppressive drugs, and consequently are less resistant to infection [15]. There is no vaccine available, and few effective treatments. Benznidazole (Roche) is the most commonly drug used in Brazil. The identification of new chemotherapeutic methods and/or vaccines is the greatest challenge for controlling Chagas' disease. The increase of the use of natural products for medicinal purposes has caused renewed interest in understanding the characteristics of substances with trypanocidal activity.
Antiparasitic activity against Leishmania and Trypanosoma genera has been described in extracts and compounds isolated from marine organisms and red algae [16,17]. Some results using extracts of seaweed such as Fucus evanescense and Pelvetia babingtonii showed a potential inhibition on DHOD (dihydroorotate dehydrogenase), an essential enzyme in T. cruzi [10]. Trypanocidal and antifungal activities of some non-polar fractions were also reported in the marine red alga Bostrychia tenella (J.V. Lamouroux) J. Agardh [9] showing high trypanocidal potential for this species.
In this way, the present work reports the chemical constituents of non-polar fractions from Centroceras clavulatum, as well as its biological activity against two life cycle stages (epimastigote and trypomastigote forms) of Trypanosoma cruzi. Also, considering the availability of red algae species in Brazil, our biological studies are directed towards the discovery of new antifungal agents based on a simple bioautographic assay. Thus, the fractions of differing polarity from C. clavulatum were selected aiming to discovery any antifungal potential against the phytopatogenic fungi Cladosporium sphaerospermum and C. cladosporioides [18].

Trypanocidal and Antifungal Activities of CC-H and CC-D Fractions
The dichloromethane fraction was active against the two forms of Trypanosoma cruzi, epimastigote IC 50 = 19.1 μg/mL and trypomastigote IC 50 = 76.2 μg/mL, respectively, and the hexane fraction also showed interesting antifungal activity against Cladosporium cladosporiodes and C. sphaerospermum ( Table 2). Table 1. Identification of compounds from hexanes and dichloromethane fractions from Centroceras clavulatum (Ceramiales, Rhodophyta).

Chemical Composition of CC-H and CC-D Fractions
The highlighted technique, GC-MS, is a useful tool in modern food, medicine and biological research for the separation and identification of components of organic mixtures, and this method has already been applied successfully for the analysis of terpenoids, especially mono-and sesquiterpenes in various essential oils [20]. In addition, the trypanocidal activity of the dichromethane and hexane fractions, IC 50 of 19.1 and 21.7 μg/mL, respectively for epimastigote forms; as well as the methanol extract, IC 50 of 18.2 μg/mL (trypomastigote forms) was more potent than the positive control, gentian violet (IC 50 of 31 μg/mL), so the non-polar fractions were selected for the chromatographic separation in order to obtain pure substances. The GC-MS analysis of the red alga, Centroceras clavulatum led to the identification of 23 major components (Table 1), accounting for ~42% of the total components present.
The major identified components of CC-H and CC-D fractions of C. clavulatum were fatty acids, specifically tetradecanoic acid (1.6%), pentadecanoic acid (15.9%), hexadecanoic acid (17.6%), eicosanoic acid (1.1%), and the unsaturated fatty acid (Z)-9-octadecenoic acid (1.7%) ( Table 1). This result is in accordance with previous reports about fatty acids detected in Rhodophyta [6,10,21]. The total fatty acid composition of nine species of Rhodophyta showed a characteristic distribution, with high relative levels of the acids 16:0 (hexadecanoic acid), 18:1(n-7), 18:1(n-9), 20:5(n-3) and 20:4(n-6) [21]. The GC-MS results obtained in this study however were significantly different from the one previously reported by Hayee-Memenon and coworkers [6], mainly due to their methodology. In their experiments, the crude extract of C. clavulatum was chemically treated with alkali and acid solutions, and the organic fractions were submitted to methylation in order to promote esterification of free fatty acids. Based on this methodology, the major compounds of fatty acids from C. clavulatum were methyl tetradecanoate, methyl pentadecanoate, methyl hexadecanoate and methyl heptadecanoate. In addition, this previous study identified some unsaturated fatty acids and different sterols. On the other hand, we identified other classes of chemical compounds, showing the great metabolic diversity this species is able to synthesize. Also, this study highlights the need for future chemical investigation of this species, since many compounds, like amides, are known to possess a large range of biological activities that was not evaluated in this study. The differences in the GC-MS profiles of C. clavulatum collected from different locations could be explained by the fact that plants often produce different amounts of phycochemicals when are growing in different geographical regions [22].

Biological Activities of CC-H and CC-D Fractions
The CC-H fraction of Centroceras clavulatum showed activity against the epimastigote form and it was inactive against the trypomastigote form of Trypanosoma cruzi. However, the CC-D fraction was active against both T. cruzi forms. In both fractions, fatty acids were present, but not necessarily in equal proportion. The antifungal activities of fractions against phytopatogenic fungi Cladosporium cladosporioides and Cladosporium sphaerospermum were also evaluated in this work, and the hexanes fraction showed activity in different levels of lethality (Table 2). This result suggests the selectivity of some compounds or the synergism of them against the pathological agent. Our results can be related to some antibacterial and antifungal properties of algal extracts which contain saturated and unsaturated fatty acids [25,26]. Furthermore, mixtures well-defined among fatty acids of up to 22 carbons could improve the penetration of herbicides, contributing indirectly as antifungal agents [27].

Extraction and Chromatographic Separation
The dried and cleaned algae were powdered (120 g) and extracted by maceration three times using CH 2 Cl 2 -MeOH (2:1 v/v) at room temperature. The solvent was evaporated under reduced pressure, and the obtained crude extract (3.24 g) was partitioned with organic solvents, yielding the hexanes [CC-H] (0.29 g), dichloromethane [CC-D] (0.14 g), ethyl acetate [CC-A] (0.10 g) and methanol [CC-M] (0.73 g) fractions, besides the resulting salt residue. In this study, the CC-H and CC-D fractions were analyzed by GC-MS in order to identify non-polar metabolites and evaluate their biological activities. In addition, all the fractions were evaluated by various bioassays to determine the potential activity of this algal species.

Trypanocidal Activity (Epimastigote Form)
Epimastigote forms of Trypanosoma cruzi (Y strain) were maintained in liver-infusion tryptose medium (LIT), supplemented with 10% fetal calf serum [28]. For the in vitro assay, the parasites (log phase) were diluted to 10 5 parasite/mL and 100 μL of the suspension was distributed in 96-wells plate. The fractions CC-H and CC-D were dissolved in DMSO, added in different concentrations to the wells and the plate was incubated in humid chamber for 72h at 28 °C. In the negative control was added the same amount of DMSO. All assays were performed in triplicate. The trypanosomes were counted using the Neubauer chamber and the percentage of dead parasites was calculated based on the positive control. The activities of the compounds were expressed as IC 50 values, corresponding to the concentration that causes death of 50% of the parasites. The IC 50 of benznidazole was determined by the same methodology.

Trypanocidal Activity (Trypomastigote Form)
The CC-H and CC-D fractions were evaluated in vitro against trypomastigote blood forms of Trypanosoma cruzi (Y strain), which were obtained from parasitic culture in LLCMK2 cells [29]. The cells were cultivated in RPMI 1640 medium, supplemented with 2 nM L-glutamine, 10 nM NaHCO 3 , 100 U/mL of penicillin, 100 μg/mL of streptomycin and 5% inactivated fetal bovine serum, in culture bottles at 37 °C in an atmosphere with 5% CO 2 , and 95% humidity. Infected blood with trypomastigote forms was added to the culture, and the mixture was incubated for 24 hours. Following this initial treatment, cells were washed with culture medium, only leaving the parasite-infected cells. After about 5 days, when the process of cellular lysis had started and the trypomastigote forms in the medium were observed, the supernatant containing trypomastigote forms was removed, parasite concentration was determined and measured (10 6 parasites/mL) into 96-well microplates. Samples fractions of C. clavulatum in concentrations of 0.5, 2.0, 8.0 and 32.0 μg/mL were added to the mixtures.
After 24 hours of incubation, the biological activity was determined using a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, ACROS Organics). First MTT (10 μL, 5 mg/mL in each plate) was added and incubated at 37 °C for 4 hours. Subsequently, acidified isopropanol [100 μL of 0.04 N HCl (aq) in isopropanol] was added to the solutions, and the microplate absorbances were measured in a Sunrise -Tecan apparatus with a 570 nm filter (reference at 630 nm). The data were processed by the program Magelan 3 (TECAN). A DMSO solution was used as the negative control with the same concentrations as the evaluated samples. The activity of the positive control (benznidazol) was verified quantitatively using a previously described methodology [30]. The fractions were prepared in triplicate and measured for their trypanocidal activity, as expressed by the percentage of activity (%AE) according to the formula: where AE represents the "absorbance of tested plates," AEB is the "absorbance of plates containing medium and sample," AC is the "absorbance of plates containing negative control," and ACB is the "absorbance of plates containing culture medium." All the IC 50 values were calculated by nonlinear regression equation, using the computer program GraphPad Prism v.4.02.

Antifungal Activity
The phytopathogenic microorganisms used in the antifungal assays Cladosporium sphaerospermum (Penzig) CCIBt 491 and C. cladosporioides (Fresen) de Vries CCIBt 140 were cultured at the Instituto de Botânica, São Paulo, SP, Brazil. Ten microliters of a solution corresponding to 200 µg of fractions were dissolved in polarity-compatible solvents and applied on pre-coated TLC plates. For the hexane fraction, the TLC plates were developed with hexanes-ethyl acetate (9:1) and for the dichloromethane fraction with dichloromethane-methanol (9:1) as the mobile phase. After eluting, the plates were dried for complete removal of the solvents. The chromatograms were sprayed with a conidia suspension of C. sphaerospermum or C. cladosporioides (≥10 6 conidia mL −1 ) in a glucose and salt solution and incubated for 48 h in a dark moistened chamber at 28 °C. A clear inhibition zone appeared against a dark background, indicating favorable sample activity. Nystatin (5 µg) was used as standard control [31].

Conclusions
In conclusion, the present study describes for the first time the occurrence of alkaloids in non-polar fractions of Centroceras clavulatum. Besides, the organic fractions obtained from Centroceras clavulatum showed interesting anti-protozoa results when submitted to Tripanosoma cruzi and antifungal potential against Cladosporium sphaerospermum and C. cladosporioides. Further chemical composition analyses showed that the environment can cause in some variations related to the structural metabolite diversity, such as when the Pakistan algae composition is compared with the Brazil species. So, it is clear that the environment and/or biodiversity can modify the species chemical constitution, showing us that the characteristics about one organism cannot be inferred for the same species from another ambient.