Brown Algae Padina sanctae-crucis Børgesen: A Potential Nutraceutical

Padina sanctae-crucis Børgesen is distributed worldwide in tropical and subtropical seas; belongs to the Dictyotaceae family, and has proven to be an exceptional source of biologically active compounds. Four compounds were isolated and identified, namely: dolastane diterpene new for the genus Padina; phaeophytin and hidroxy-phaeophytin new for the family Dictyotaceae, and; mannitol first described in this species. Saturated fatty acids as compared to the percentages of unsaturated fatty acids were shown to be present in greater abundance. Palmitic and linolenic acid were the main saturated and unsaturated acids, respectively. Cytotoxic and antioxidant activities were evaluated using human erythrocytes. In vivo evaluations of acute toxicity and genotoxicity were performed in mice. Methanolic extract of P. sanctae-crucis presented antioxidant activity and did not induce cytotoxicity, genotoxicity or acute toxicity. Since Padina sanctae-crucis is already used as food, has essential fatty acids for the nutrition of mammals, does not present toxicity and has antioxidant activity, it can be considered as a potential nutraceutical.


Introduction
The use of seaweed as food has strong roots in Asian countries such as China, Japan and the Republic of Korea, but demand for seaweed as food has now also spread to North America, South America, and Europe, as fresh or dried seaweed, or as an ingredient in prepared foods. It is also an ingredient for the global food and cosmetics industries, and is used as fertilizer and as an animal feed additive. The nutrient composition of seaweed varies, and is affected by species, geographic area, season, and water temperature [1].
Using similar methods to those described above, compounds 2-4 were identified as phaeophytin (2) [21,22], and 13 2 -hydroxy-(13 2 -S)-phaeophytin a (3) [23], and mannitol (4) [24][25][26], with compounds 2 and 3 new for the family Dictyotaceae and compound 4 first described in this species (Figure 1).    The analysis of the 1/8 trans-esterified fraction from the hexane phase of the P. sanctae-crucis ethanol extract [27] under GC-MS allowed identification of 89.82% of unsaponifiable lipids, which were identified as: myristic acid (6.02%); palmitic acid (68.84%); linoleic acid (0.73%); oleic acid (1.61%); linolenic acid (9.75%), and; stearic acid (2.87%). Saturated fatty acids were shown to be present in greater abundance (77.73%) as compared to the unsaturated fatty acids percentage (12.09%) ( Table 2). To evaluate the in vitro toxicity of the methanolic extract (ME), hemolytic assays with human erythrocyte blood groups ABO were performed. The hemolysis percentage increased in a concentration-dependent manner. However, after treatment with ME at a concentration of 1000 µg/mL none of the three blood groups reached 100%. The hemolytic effect to human blood groups A, B and O, at a concentration of 1000 µg/mL, was respectively 45.4%, 24.3% and 30.6% (Figure 2). μg/mL none of the three blood groups reached 100%. The hemolytic effect to human blood groups A, B and O, at a concentration of 1000 μg/mL, was respectively 45.4%, 24.3% and 30.6% ( Figure 2). The oxidant potential was evaluated considering the amount of methemoglobin formed after exposure to ME. The antioxidant potential was measured by effectiveness of ME in inhibiting methemoglobin formation after phenylhydrazine (PH) exposure. As depicted in Figure 3, the amount of methemoglobin formed in the absence and presence of various concentrations of ME were the same, indicating no oxidizing effect. In contrast, the methemoglobin formation was significantly reduced in erythrocytes pretreated with ME in a concentration-dependent manner. It is interesting to note that the erythrocyte antioxidant protection conferred by ME at a concentration of 1 μg/mL (47.6%) and 10 μg/mL (57.9%) was similar the protective effects afforded by Vitamin C (52.7%) [28] (Figure 3). To investigate possible in vivo toxic effects of the ME an acute toxicological study was conducted. With administration of the ME, the animals showed no behavioral changes. All male and female mice remained alive after 14 days of observation. The oxidant potential was evaluated considering the amount of methemoglobin formed after exposure to ME. The antioxidant potential was measured by effectiveness of ME in inhibiting methemoglobin formation after phenylhydrazine (PH) exposure. As depicted in Figure 3, the amount of methemoglobin formed in the absence and presence of various concentrations of ME were the same, indicating no oxidizing effect. In contrast, the methemoglobin formation was significantly reduced in erythrocytes pretreated with ME in a concentration-dependent manner. It is interesting to note that the erythrocyte antioxidant protection conferred by ME at a concentration of 1 µg/mL (47.6%) and 10 µg/mL (57.9%) was similar the protective effects afforded by Vitamin C (52.7%) [28] (Figure 3). μg/mL none of the three blood groups reached 100%. The hemolytic effect to human blood groups A, B and O, at a concentration of 1000 μg/mL, was respectively 45.4%, 24.3% and 30.6% ( Figure 2). The oxidant potential was evaluated considering the amount of methemoglobin formed after exposure to ME. The antioxidant potential was measured by effectiveness of ME in inhibiting methemoglobin formation after phenylhydrazine (PH) exposure. As depicted in Figure 3, the amount of methemoglobin formed in the absence and presence of various concentrations of ME were the same, indicating no oxidizing effect. In contrast, the methemoglobin formation was significantly reduced in erythrocytes pretreated with ME in a concentration-dependent manner. It is interesting to note that the erythrocyte antioxidant protection conferred by ME at a concentration of 1 μg/mL (47.6%) and 10 μg/mL (57.9%) was similar the protective effects afforded by Vitamin C (52.7%) [28] (Figure 3). To investigate possible in vivo toxic effects of the ME an acute toxicological study was conducted. With administration of the ME, the animals showed no behavioral changes. All male and female mice remained alive after 14 days of observation. To investigate possible in vivo toxic effects of the ME an acute toxicological study was conducted. With administration of the ME, the animals showed no behavioral changes. All male and female mice remained alive after 14 days of observation. Table 3 contains values relating to water, feed consumption and weight gain as evaluated during the 14 days of observation. According to the results obtained, after administration of a single dose of ME (2000 mg/kg), no significant differences were observed in water and feed consumption for either male and female groups, as compared to the control group. The body weight of the animals treated with the extract also did not present significant alteration. Data are presented as means ± standard error of mean. Table 4 contains values of the organ indices evaluated after treatment with ME. No significant changes were observed between mice treated with ME or controls. Toxicological analyses of the effects of ME also included assessment of biochemical and hematological parameters. Table 5 contains values for the biochemical parameters evaluated after treatment with ME. No significant changes were observed between ME treated mice and the control. As for hematological evaluations, a significant increase in the female erythrocyte counts when treated with ME as compared to the control group was observed (Table 6). However, despite the observed change in the total number of erythrocytes, it was within the normal range observed for mice [29].
The parameters mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) did not change after the treatment with ME. As for the differential leukocyte count, leukopenia and lymphocytosis were observed in the females treated with ME when compared to the control group (Table 6). To evaluate the possible in vivo genotoxic effects of the ME, we performed micronucleus assaying; the results are presented in Table 7. Treating the animals with the extract did not induce an increase in the micronucleated erythrocytes frequency in the peripheral blood as compared to the control group (5% Tween 80), suggesting no evidence of genotoxicity. Data presented as mean ± s.e.m. of six animals analyzed by ANOVA followed by Dunnett. a p < 0.05 compared to control (5% Tween 80); b p < 0.05 compared to cyclophosphamide.
Palmitic acid increases stability of fixed oils against peroxidation, and contributes to the production of various types of margarines when mixed into the oil in the proportion of 15-25% [30]. Unsaturated fatty acids are of great interest in the cosmetics and biotechnology industries [31]. They have effects on health in various processes such as: regulation of plasma lipid levels; cardiovascular and immune functions; neuronal development; eye function; composition and function of membranes; eicosanoid synthesis; cellular signaling; regulation of gene expression [32]; and even depression [33]. In mammalian nutrition, linoleic and linolenic acids are essential fatty acids [34].
In cytotoxicity assays, the mechanical stability of the erythrocyte membrane is a good indicator of in vitro damage, since drugs can alter this delicate structure [35]. Damage to red cells also provides a preliminary model to study either protective or toxic effects of substances or conditions associated with oxidative stress [36][37][38][39].
The negligible hemolytic effect induced by the ME at all concentrations tested indicates that no damage occurred in the cell membrane of most of the cells and, thus, these have low cytotoxicity. The ABO blood groups were determined by adding different sugars to a protein on the surface of the erythrocytes [40], which contributes to explaining the different hemolytic effect of ME in each blood type.
The ME presented antioxidant effect similar to that of vitamin C, a proven antioxidant agent. Due to concerns about the toxic and carcinogenic effects of synthetic antioxidants [41,42], there is an increasing interest in antioxidants from natural sources [43]. Many epidemiological reports suggest protective effects against atherosclerosis, neuronal degeneration, cancer, rheumatoid arthritis, diabetes mellitus, inflammation and vascular disease [44][45][46]. Reactive oxygen species are toxic since they can oxidize biomolecules, leading to cell death and tissue injury [47].
Human erythrocytes can be used through in vitro experimental models to investigate the antioxidant potential of vegetable extracts. It must be kept in mind, however, that such antioxidant activities found in erythrocytes do not necessarily reflect the antioxidant defenses of the whole organism. In addition, there are genetic variations between individuals that result in altered gene expression, leading to varied and potentially undesired effects in the antioxidant defenses [28].
Throughout the acute toxicological study, the ME did not induce significant differences or changes in behavior, water and feed consumption, body weight, the organ indexes (heart, liver, kidneys, thymus, spleen), biochemical parameters (aspartate aminotransferase, alanine aminotransferase, urea, creatinine), or death. As to the feminine hematological parameters, a significant increase in erythrocyte and lymphocytes counts, and a decrease in total leucocytes, was observed. However, all these changes are within the normal range for female mice [29]. In male mice, we noted leukocytosis, characteristic of a defense response of the immune system.
Treatment of the animals with the ME did not induce micronucleated erythrocyte frequency increases in peripheral blood, thus suggesting no evidence of genotoxicity.
A nutraceutical is any substance that is a food or a part of food and provides medical or health benefits, this includes use for both prevention and treatment of disease. Such products may range from isolated nutrients, dietary supplements and specific diets, to genetically engineered designer foods and herbal products [54]. To develop nutraceuticals and pharmaceutical compounds from marine source, both price and sufficient supply are critical. Among the marine algae, brown algae is a promising candidate for the development of functional foods, since mass production is relatively easy through aquaculture [55]. Brown algae account for approximately 59% of the total macroalgae cultivated in the world and can be cultivated on seashores in large scales. Their growth rate is relatively rapid, and their bioactive compounds such as proteins, polyphenols and pigments can be controlled during production by manipulating the culture conditions [56].
Since Padina sanctae-crucis is used as food, has essential fatty acids for the nutrition of mammals, does not present toxicity, and has antioxidant activity, it can be considered as a potential nutraceutical.

Collection, Extraction and Iolation
The brown alga Two experiments were then carried out: In the first, the dried material (1 kg) was extracted with 95% EtOH (4.0 L) for 72 h at room temperature. The EtOH extract was concentrated under reduced pressure to give 185.75 g of the crude ethanol extract (CEE), which was solubilized in EtOH:H 2 O (7:3), yielding the hydroalcoholic solution. This was then partitioned with hexane, dichloromethane, ethyl acetate and n-butanol, providing 6.21 g of hexane phase, 12.98 g of dichloromethane phase, 114.2 mg of EtOAc phase and 9.03 g n-BuOH phase. The solvents were used alone and in increasing order of polarity.
In the second, the alga was subjected to cold extraction with hexane, dichloromethane, ethyl acetate and methanol; yielding the following extracts: hexane (1.27 g), dichloromethane (3.04 g), ethyl acetate (2.21 g) and methanol (113.34 g). A precipitate was separated from the hexane extract resulting in the isolation of compound 1 in the form of yellowish-white crystals (17.0 mg). The ethyl acetate extract (2.21 g) was subjected to column chromatography with silica gel 60 and the eluents hexane, ethyl acetate and methanol alone or in mixtures, affording 61 fractions of 50 mL each which were concentrated under reduced pressure, analyzed through analytical thin layer chromatography (TLC), and combined according to their retention factors (Rf's). The subfraction 27/51 (97.4 mg) was subjected to preparative TLC, using a mixture of hexane and ethyl acetate in the ratio of 7:3 as eluent; resulting in the isolation and purification of two bluish green amorphous solids, compounds 2 (15.0 mg) and 3 (25.6 mg).
Three (3 g) of the methanolic extract (ME) was subjected to column chromatography using Sephadex LH-20 and the methanol eluent gave 40 fractions of 50 mL each which were concentrated under reduced pressure, and analyzed through analytical TLC. Subfraction 5/22 was again subjected to chromatography following the same methodology, providing 23 subfractions of 50 mL which were concentrated, analyzed and combined, following the previously adopted methods. Subfraction 5/13 yielded a supernatant and 21.2 mg of a white solid precipitate, compound 4.

Fatty Acids Transesterification
The methodology developed by Maia et al. (1993) [27] and Saastamoinen et al. (1989) [30] was adopted to perform trans-esterification of fatty acids from the 1/8 fraction of the hexane phase of the P. sanctae-crucis ethanol extract. The fatty acid methyl esters were prepared as follows: a test tube held the saponification process using 30 mg of the sample together with hydroxide solution sodium (0.5 N) in methanol (4 mL), followed by esterification with 5 mL of ammonium chloride, sulfuric acid and methanol at a ratio of 1:1 and 5:3, respectively. Shortly thereafter, 4 mL of saturated sodium chloride solution was added to the test tube under stirring for 30 s, and then 5 mL of hexane (brand Synth, lot 65741) was also added to the test tube for 30 s and stirring. The solution was then allowed to stand to complete separation of the respective phase. After separation, the upper phase (hexane phase) was transferred using a Pasteur pipette into a capped vial and kept under refrigeration until completion of the un-saponifiable lipids analyses, which were performed in a gas chromatograph coupled to a mass spectrometer (GC-MS, Shimadzu QP-5000) operating with an electron impact of 70 eV).

Experimental Animals
Swiss mice of both sexes, 6-8 weeks of age with an average weight 28-32 g, were obtained from the Federal University of Paraíba (Paraíba, Brazil) and were used throughout the experiments. They were housed in single-sex cage conditions with a 12-h light/dark cycle, at constant temperature (21 ± 1 • C), with free access to water and pellet food. Six hours before each experiment, the animals received only water, to avoid test substance absorption interference with food. The experiments were performed after protocol approval by the Animal Studies Committee of Federal University of Paraíba (0509/109).

Cytotoxicity Assay
Cytotoxic activity is directly related to the hemolytic effect induced by ME [57]. Briefly, human erythrocytes samples were obtained from blood to be discarded by the University Hospital Lauro Wanderley/UFPB Transfusion Unit. To obtain a suspension of erythrocytes, 1.5 mL of whole blood was then made up to 10 mL in NaCl 0.9%, and centrifuged at 3000 rpm for 5 min. The supernatant was then removed by gentle aspiration, and the above process was repeated two more times. Erythrocytes were finally re-suspended in NaCl 0.9% to make 0.5% suspension for the hemolysis assay. ME (at 1, 10, 100 and 1000 µg/mL), dissolved in DMSO (Vetec) (5%), was added to the suspension of erythrocytes and incubated at 100 rpm at 22 ± 2 • C under slow and constant agitation (100 rpm, for 60 min) and then centrifuged at 3000 rpm for 5 min. The absorbance of the supernatants was determined at 540 nm using a UV-Vis Spectrophotometer (UV-1650PC Shimadzu ® ) to measure the extent of erythrocyte lysis. A suspension of erythrocytes was used as a negative control (0% hemolysis), and the erythrocyte suspension plus 1% Triton X-100 (Vetec) (for 100% hemolysis). All the experiments were performed in triplicate and after approval of the protocol by the Ethics Committee in Research of Federal University of Paraíba (0306/11).

Oxidant and Antioxidant Evaluation
The erythrocytes were washed twice as described above, and re-suspended in PBS (NaH 2 PO 4 ·2H 2 O 123 mmol/L, Na 2 HPO 4 27 mmol/L, NaCl 123 mmol/L) supplemented with glucose (200 mg/dL) pH 7.6 to a final hematocrit of 35%. Human erythrocytes in suspension were treated with extracts (1, 10, 100 and 1000 µg) for 60 min at 22 ± 2 • C under slow (100 rpm) and constant agitation. The erythrocyte suspension was the negative control and the erythrocyte suspension plus phenylhydrazine 1 mmol/L (Sigma) was the positive control. The methemoglobin concentration was measured spectrophotometrically (630 nm) as the percentage of total hemoglobin (540 nm) to evaluate the oxidant potential. Afterwards the samples were aerated and exposed to phenylhydrazine (1 mmol/L) for a further 20 min under the same conditions. The methemoglobin concentration as a percentage of the total hemoglobin was measured to evaluate the antioxidant effect [20]. All the experiments were performed in triplicate.

Acute Toxicity Studies
For acute toxicity studies, 12 male and 12 female mice were divided into two groups (6 males or 6 females per group). The extract was administered by gavage to the mice at a dose of 2000 mg/kg, while the control group received vehicle alone. The general behavior of the mice and signs of toxicity were observed continuously for 1 h after the administration of the ME, then intermittently for 4 h, and thereafter over a period of 24 h [58]. The mice were further observed once a day, to 14 days, following the treatment for behavioral changes and signs of toxicity and/or death, and for latency to death. Body weights were measured at the beginning and end of the treatment. On day 14, peripheral blood samples from the controls and treated mice were collected from the retro-orbital plexus under light sodium thiopental anesthesia (40 mg/kg-i.p.). For biochemical analysis, the blood samples from the controls and treated mice were centrifuged, and the levels of urea, creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. For the hematological analysis we used heparinized whole blood; for the hematological parameters: (hemoglobin level, erythrocyte count, hematocrit, red cell indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and total and differential leukocyte count) were performed. The animals were also sacrificed by cervical dislocation; the heart, kidneys, liver, spleen and thymus were excised and weighed for determination of organ indices (organ weight/body weight).

Micronucleus Test
For the micronucleus assay, a group of six Swiss mice males were orally treated with the dose of 2000 mg/kg. A positive control group (cyclophosphamide 50 mg/kg-i.p.) and a negative control group (saline and tween 80 at 5%) were included. After 24 h the animals were anesthetized with sodium thiopental (40 mg/kg-i.p.) and peripheral blood samples were collected by orbital plexus for preparation of the blood extensions. For each animal three blood extensions were prepared and a minimum of 2000 erythrocytes counted to determine the frequency of micronucleated erythrocytes [59].

Statistical Analysis
The results obtained were analyzed with the software GraphPad Prism 5.0 ® (GraphPad Prism Software, San Diego, CA, USA)and expressed in mean ± s.e.m. using unpaired t-test for two-column analysis, and one-way analysis of variance (ANOVA) for comparing more than two columns, followed by the Tukey's test (parametric variables), or Dunnett (non-parametric) and the results were considered significant when p < 0.05.

Conclusions
The chemical study of Padina sanctae-crucis led to the isolation and identification of four secondary metabolites. Substances 1-3 are the first reported for the Padina genus and substance 4 is the first described in the species P. sanctae-crucis. The presence of saturated fatty acids whose major component was palmitic acid, and of unsaturated fatty acids, particularly linolenic acid, suggests that P. sanctae-crucis has great nutritional potential. The methanolic extract was able to prevent oxidative stress and did not present significant cytotoxicity, acute toxicity or genotoxicity in the experimental models evaluated. Since Padina sanctae-crucis possesses essential molecules for nutrition, is able to prevent oxidative stress and does not present significant toxicity, we consider it a potential nutraceutical.