Anti-atherogenic properties associated with the antioxidant activity from the hydrophilic extracts of Halimeda incrassata (Chlorophyta, Bryopsidales)

Seaweeds are a source of natural antioxidants having potential application in oxidative stress and associated diseases. In this work, anti-atherogenic properties associated with the antioxidant activity from the hydrophilic extracts of Halimeda incrassata were studied. The phenolic content assessed in the aqueous extract and fraction phenolic acids (FPA) was 0.13 ± 0.05 and 0.47 ± 0.09 mg of gallic acid equivalents (GAE)/g dry seaweed, respectively. In DPPH • , radical scavenging assay fractions exhibited a dependent concentration. The seaweeds extract inhibited the desoxirribose oxidation in the presence or absence of EDTA (IC50 = 1.91± 0.09 mg/mL) (IC50 = 2.95 ± 0.01 mg/mL). In vivo antioxidant properties of FPA-H.incrassata were investigated in rats with a CCl4-induced liver injury. Pre-treatment with H. incrassata led to approximately 50% reductions in liver TBARS levels. The treatment with H. incrassata FPA also increased the activity of the CAT enzyme, which in turn resulted in an enhanced antioxidant defense. The expression of Catalase by PCR-RT technique demonstrated a higher gene expression when compared with that which was observed in the CCl4-treated group. Antiatherogenic properties were studied in the inhibition of lipoprotein oxidation mediated by Cu 2+ or HRP/H2O2, free radical scavenging, and metal ion chelation, and it was dose dependent with a higher concentration needed for the aqueous extract than for the FPA fraction. Antioxidant activity was also improved in macrophages as evaluated in the cell supernatant (by TBARS formation); and by luminol enhanced chemiluminescence after cell activation with zymosan; and a degree of cell lipoperoxidation was decreased by the Halimeda incrassata extract. The results of this work add to the antioxidant potential of the seaweed for its application in oxidative stress associated conditions.


INTRODUCTION
Oxidative stress is involved in a variety of pathologies and indeed it is presently considered a common factor in chronic non transmissible diseases as like atherosclerosis (Sanchez-Recalde and Kaski, 2001).Hence, among the most interesting alternatives for the modulation of oxidative stress as a target to halt the development of atherosclerosis, antioxidant effects have been sought at different stages of the disease: LDL oxidation, macrophage activation, foam cell formation, smooth muscle cell migration and advanced plaque remodeling (Kaliora et al., 2006); and then multiple mechanisms have been proposed for targeting an atherogenesis associated oxidative stress (Stocker and Keaney, 2004).
Seaweeds have combinations of highly developed antioxidant defense systems, allowing them to preserve structural integrity before different kinds of environmental stresses.However, they possess a wide diversity of bioactive compounds from amino acids, such as micosporines, polysaccharides, carotenoids, terpenoids, and an especially high phenolic content (Dutra-Rocha et al., 2007).Their unique composition makes them attractive candidates for their application in the antioxidant field.A correlation has also been found between the consumption of phenolic compounds in general, and seaweeds in particular, and the incidence of cardiovascular diseases (Bocanegra et al., 2009).
In the quest for more potent antioxidants from natural sources, our group has been especially interested in studying the beneficial properties of seaweed from the Halimeda genus for an application in biomedicine in hepato-, neuro-and athero-protection.Several lines of results have documented the ability of a hydrophilic extract from this seaweed to target free-radical mediated processes in vitro cell culture and in vivo experimental models (Rivero et al., 2003;Fallarero et al., 2003;Linares et al., 2004;Mancini-Filho et al., 2009;de Oliveira e Silva et al., 2012).In previous studies, in vitro Halimeda spp seaweeds have been described as having a relationship between antioxidant activity and antiatherogenic properties (Costa-Mugica et al., 2012).Additionally, it has been shown that Halimeda spp.has a high phenolic content (Vidal et al., 2009;Vidal et al., 2011), together with low amounts of other antioxidants, such as ascorbate, β-carotene, chlorophylls, and selenium, and that these compounds may be able to explain antioxidant properties.
Thus, in view of these previous considerations, the aim of this study was to evaluate the antioxidant properties of the hydrophilic fractions obtained from the green seaweed Halimeda incrassata, in free radical scavenging, and in animal models, and a possible relation to lipoprotein oxidation, and macrophage oxidative stress, as pathological stages of atherosclerosis.

Seaw eed collection and hydrophilic extract preparation
The seaw eed Halimeda incrassata (Ellis) Lamouroux (Chlorophyta, Bryopsidales) w as collected during December 2011 in the Bajo de Santa Ana, La Habana, Cuba.Voucher specimens w ere authenticated by Dr. A. M. Suárez from the Seaw eeds Laboratory at the Marine Research Center of the University of Havana.Freshly collected specimens w ere w ashed w ith distilled w ater and dried at room temperature for 3-5 days.After milling and sieving, the dry pow der w as used to obtain the hydrophilic extracts.The dry seaw eed pow der w as extracted w ith distilled w ater (1:5 w /v) at room temperature and centrifuged at 800 g and at 4ºC for 20 min.The supernatant w as recovered, lyophilized, and kept at -20ºC until use.The w eight yield of the final extract in terms of dry seaw eed w as 5%.The lyophilized material w as dissolved in distilled w ater at know n concentrations for the different studies.The lyophilized material w as denominated as being an aqueous extract.Polyphenolic rich fractions w ere obtained according to Krygier et al. (1982).Tetrahydrofurane extraction was performed on the dry seaw eed in order to obtain a fraction rich in free phenolic acids (FPA).The w eight yield of the final dry fraction in terms of dry seaw eed w as 0.8%.

Total phenolic concentration
The total phenolic content w as determined by the Folin-Ciocalteau assay as previously described by Vidal et al. (2009) and expressed as µg of Gallic Acid Equivalents (GAE) /g of sample.The calibration curve w as obtained in the range of 100-1000 µg gallic acid/ml.

DPPH • radical scavenging assay
The free radical scavenging activity of the Halimeda incrassata extract w as done similar to Goupy et al. (1999).Briefly, 0.6 ml of the extract (10 to 40 μg GAE) w as mixed w ith 0.6 mL of a methanolic solution of DPPH • (60 μM).Absorbance was measured in time.Radical scavenging activi ty w as calculated relative to the reference absorption as a Percentage Inhibition (PI) (%) = (1-Asample/Areference) × 100.IC50 w as the antioxidant quantity needed (mg aqueous extract or fraction dry) to scavenge 50% of DPPH • .
Author(s) agree that this article remain permanently open access under the terms of the Creativ e Commons Attribution License 4.0 International License 10 mM, pH 7.4, w ith or w ithout 50 µL EDTA 100 µM and 50 µl FeCl3 25 µM.After 1 min, 50 µl 2-desoxi-D-ribose 2.8 mM and H2O2 2.8 mM w ere added.Reaction w as initiated w ith 50 µL 100 µM ascorbic acid and stopped after 1 h at 37 o C. The degree of oxidation w as assessed by a TBARS formation.Samples w ere incubated w ith 1 ml TBA 1%, 1mL TCA 2.8%, at 80°C for 20 min.Absorbance w as monitored at 532 nm.Without EDTA, the assay indicated the Fe chelating capacity of the extract, w hereas w ith EDTA, it measured the hydroxil radical scavenging activity.

Anim als and treatm ent schedule
Male Wistar rats from the University of São Paulo, Brazil, w eighing 120 g-150 g, w ere maintained under a controlled diet, w ith cycles of 12 h of light/dark, at 25 °C and 60% humidity.The rats had free access to w ater and to a standard food diet according to the care guidelines for laboratory animals used in research.The animal studies w ere approved by the Institutional Ethical Committee for Animal Experimentation from the Faculty of Pharmaceutical Sciences (USP), Brazil.
Hepatic injury w as induced in the rats by an intraperitoneal administration of a single dose of 3 mL CCl4 (mixed 1:1 w ith olive oil) on day 21.Gallic Acid (GA) w as used as a reference.The animals w ere grouped as follow s: Group I: Control, treated daily w ith vehicle (1 ml, p.o.) for 21 days.Group II: Treated daily w ith vehicle (1.0 mL, p.o.) for 21 days, follow ed by treatment w ith CCl4.Group III: Treated daily w ith an aqueous extract of Halimeda incrassata (300 mg/kg, p.o.) for 21 days, follow ed by treatment w ith CCl4.Group IV: Treated daily w ith ferulic acid (20 mg/kg, p.o.) for 21 days, follow ed by treatment w ith CCl4.
At the end of the treatment (day 22), a blood sample and the liver of each animal w as collected.ASAT and ALAT w ere determined by commercial laboratory kits (LABTEST).

TBARS assay
As a marker of lipid peroxidation, the TBARS contents w ere measured in the liver homogenates and serum of the animals using the method of Ohkaw a et al. (1979).The results w ere expressed as nmol/mg protein.

Glutathione (GSH) analysis
The hepatic total of GSH content w as measured using the method of Ellman (1959) as the change in absorbance w as monitored at 410 nm for 5 min, and the GSH level w as calculated by using pure GSH as standard.

Determ ination of superoxide dism utase (SOD) activity
The cytoplasmatic SOD activity w as evaluated according to McCord and Fridovich (1969) by using 100 mM cytochrome C, 500 mM xanthine, 1 mM EDTA, and 200 mM KCN in 0.05 M potassium phosphate, pH 7.8.The xanthine oxidase (same volume in the blank) w as placed in a glass tube along w ith 15 μl of the cytosolic fraction from each liver tissue.The results w ere expressed as U/mg protein.One unit (U) w as the enzyme activity that induced 50% of inhibition of the xanthine reaction at 25 °C, pH 7.8.

Determ ination of catalase (CAT) activity
The activity w as evaluated by the decomposition of hydrogen peroxide caused by the cytoplasmatic enzyme CAT according to Beutler (1975), through the decrement of the optic density at 230 nm (coefficient of the molar extinction 0.0071 mM -1 cm -1 ) at 37 °C.One U of CAT corresponded to the enzyme activity that hydrolyzed 1 molecular w eight of H2O2 per minute at 37 °C, pH 8.0.The activity w as expressed as U/mg of protein.

Expression of hepatic enzym es in rats by RT/PCR
RNA Extraction: CAT and SOD gene evaluation RNA w as extracted from the rat liver utilizing a 100 mg sample and 1 mL of trizol reagent (Invitrogen).The extract w as kept at room temperature for 5 min w ith the addition of 200 μl chloroform (Merck).The samples w ere mixed by vortexing for 15 s and kept at room temperature for 5 min.After this, they w ere centrifuged at 12,000 × g for 15 min at 4 °C.400 μl of the supernatant w as removed, avoiding the interphase, and mixed w ith 500 μl of isopropanol by vortexing for 5 s.These samples w ere then centrifuged at 12,000 × g for 5 min at 4 °C, discarding the supernatant.To the resulting pellet, 1000 μl of ethanol (75%) w as added and gently mixed, follow ed by centrifugation at 7500 × g for 10 min at 4°C, discarding the supernatant.Finally, 20 μl of distilled w ater, RNAse-free, w as added and incubated at 50 °C for 10 min.This material w as then stored at -70°C.

Reverse transcription
2 μg of RNA w ere added to 1.0 μl of primers (SOD or CAT), 1.0 μl of 10 mM dNTP, and 4.0 μL of sterile distilled w ater.The reaction w as started by heating at 65°C for 5 min; then it w as quickly chilled on ice.Next, 4.0 μl of 5× first strand buffer (Invitrogen), 2.0 μl of 0.1 M DTT (Invitrogen), and 1.0 μl of RNAseOut Ribonuclease inhibitor (Invitrogen) w ere added and incubated at 37°C for 2 min.After that, 1.0 μl (200 U) of reverse transcriptase (M-MLV RT-Invitrogen) w as added and incubated at 37°C for 50 min.The reaction w as stopped by heating at 70 °C for 15 min.The PCR product (cDNA) w as stored at -70°C.

Effect of hydrophilic fractions on the inhibition of LDL oxidation m ediated by Cu 2+ and HRP/H2O2
Oxidation experiments w ere conducted w ith heparin precipitated LDL (hep-LDL), a model of LDL that has interacted w ith extracellular matrix, and is, therefore, more prone to oxidation (Upritchard and Sutherland, 1999).Lipoproteins w ere isolated from normolipemic human serum by the method of Wieland and Seidel (Wieland and Seidel, 1983).5 ml sodium citrate buffer (64 mmol/L, pH 5.12) containing heparin (50 000 UI/L) w as added to 0.5 mL serum.After incubating for 10 min at room temperature the sample w as centrifuged at 3000 rpm for 15 min.The Hep-LDL precipitate w as w ashed 3 times w ith a Hepes buffer (5 mM Hepes, 20 mM NaCl, 4 mM CaCl2 and 2 mM MgCl2, pH 7.2), then by centrifuging at 3000 rpm for 15 min; the hep-LDL w as w as next dissolved in a 0.5 mL phosphate buffer, pH 7.4, w ith NaCl 4%.The Hep-LDL fraction w as next divided into aliquots and kept at 4ºC.The cholesterol content w as determined by an enzymatic assay (Boehringer Mannhein Diagnostics) and the protein content by the Low ry method.In brief, for the oxidation, LDL (0.2 μmol cholesterol) w as diluted in a phosphate buffer and incubated in the presence or absence, of hydrophilic fractions (aqueous extract and FPA fraction) for 6 h at 37 o C, w ith 10 μM Cu 2+ , or HRP (119 U)/H2O2 (12.9 µM).The maximum degree of oxidation w as determined by TBARS as described in Frostegard et al. (1990) and expressed as nmoles MDA equivalents/ mg protein, using TMP as standard.

Antioxidant activity of hydrophilic extracts in m acrophages
Cell experiments w ere done w ith the macrophage RAW 264.7 cell line.Cells w ere cultured in DMEM containing fetal bovine serum (FBS), 2 mM L-glutamine and streptomycin/penicillin in 5% CO2.

TBARS form ation by cells
For the assessment of antioxidant activity, cells w ere pre-incubated for 24 h w ith an aqueous extract of Halimeda Incrassata.Lipoperoxidation levels w ere evaluated in the supernatant by a TBARS assay as in Frostegard et al. (2003).

ROS production
ROS production by cells w as determined in conditions similar to Kopprasch et al. (2008).Luminol 4 µM w as added to cells in a 50 mM Hepes buffered DMEM.After adding seaw eed, aqueous extracts cells w ere stimulated w ith opsonized zymosan (OZ) 1 mg/mL.Chimioluminiscent response w as measured in time, and amplitude of the curve w as taken as maximum ROS production.Experiments w ere done w ith a Lumi-Aggregometer from Chrono-Log Corporation w ith AGGRO/LINK softw are version 5.2.3.

Statistical analysis
Values are given as mean + standard deviation (s.d.) of experiments that w ere done in triplicate, and performed at least tw o independent times.In studies of the antioxidant activity in the cell systems, statistical significance w as determined by ANOVA w ith a Tukey posttest.Significant differences w ere concluded for p < 0.05.Data w ere processed using Microcal Origin and GraphPad Prism softw are.

RESULTS AND DISCUSSION
Over the last few years, seaweeds have been widely  investigated as a source of bioactive compounds, with different attributes, and in this context, the genus Halimeda has been studied for different pharmacological properties, including antioxidant activity (Moo-Puc et al., 2008;Nor et al., 2010).

Phenolic content
The phenolic content, as assessed in the Aqueous Extract and by the Fraction Phenolic Acids (FPA), was 0.13 ± 0.05 and 0.47 ± 0.09 mg Gallic Acid Equivalents (GAE)/ g dry seaweed, respectively.Both fractions had a high phenolic content.
The phenolic content found is in the range of the one for seaweeds of Halimeda spp.worked by our research group (12-13) and higher than for other seaweeds informed in the literature like Fucus vesiculosus (Phaeophyceae) and Caulerpa racemosa (Chlorophyta) (Jimenez-Escrig et al., 2001).
The phenolic contribution to antioxidant activity was evaluated in the hydrophilic fractions.When comparing different fractions from the seaweed Eisenia bicyclis (Phaeophyceae), Kim et al. (2011) found that the highest phenolic content was associated with the antioxidant activity and the hepatoprotective effect, against tert-butyl hyperoxide damage (t-BHP) in hydrophilic fractions from the seaweed.
In our previous work, Vidal et al. (2009) identified 8 phenolic acids in Halimeda opuntia and H. monile (Chlrophyta) respectively.They reported that salicylic, cinnamic, gallic, pirogalic and cafeic acids were the principal polyphenolic compounds in both seaweeds.In Halimeda incrassata, it was identified that there were major polyphenolicqq compounds of salicylic and ferulic acids, and they suggested that their levels were related to the antioxidant activity of the seaweed (Vidal et al., 2011).Likewise, the antiatherogenic activity of phenolic compounds has been studied when considering their antioxidant properties as being the main mechanism of action (Bocanegra et al., 2009;Jimenez-Escrig et al., 2001)

DPPH
• radical scavenging has been widely used to study the activity of antioxidant molecules and plant extracts.It is considered, that presently, more than 90% of antioxidant studies use this method in combination with other assays (Goupy et al., 1999).In this work, free radical scavenging by DPPH • assay indicated a dosedependent effect, and in comparison with Halimeda incrassata, a six-fold lower activity for Caulerpa racemosa, a more than 30-fold decrement in Ulva lactuca, and a similar activity in Sargasum spp.( Yangthong et al., 2009).Antioxidant activity by DPPH suggests phenolic compounds are relevant to the effect.Different authors (Dutra-Rocha et al., 2007) have indicated an association between the phenolic content of seaweeds and DPPH • scavenging.Previous results from our group, regarding Halimeda genus, have indicated an association of antioxidant activity in DPPH scavenging with phenolic content (Vidal et al., 2009;2011).Indeed, Katsube et al. (2004) found a tendency of higher antioxidant activity in the inhibition of LDL oxidation, and of DPPH • scavenging in plants with an increasing phenolic content.Hydroxyl radicals are a main initiator of lipid peroxidation.Thereafter, hydroxyl radical scavenging is a relevant indicator of the antioxidant activity of a natural compound, and in this area, plant polyphenols are compounds of interest, as they can react with these radicals to avoid oxidative damage (Stocker and Keaney, 2004).Aqueous seaweed extract inhibited desoxiribose oxidation with a dose-dependent effect both in the presence of EDTA (Figure 2A) (IC 50 = 1.91± 0.09 mg/mL), or in absence of EDTA (Figure 2B) (IC 50 = 2.95 ± 0.01 mg/mL Fe chelating).Additionally, aqueous extracts had a concentration-dependent antioxidant effect, both with and without, EDTA, which is stronger than the one referred to for various antioxidant extracts (Vidal et al., 2006).
Antioxidants in Halimeda incrassata seaweed might offer protection from damage, by avoiding an attack by OH • radicals, generated by a Fenton reaction in the presence of EDTA.On the other hand, when Fe 3+ is added to the reaction milieu (in the absence of EDTA), some ions might join desoxiribose sugar and take part in the Fenton reaction.Antioxidant activity in this assay would then show the Fe 3+ chelating capacity of the extracts.Thus, our results also indicated that the extracts have the capacity to interfere with the site specific generation of OH  confers them with a heavy metal chelating capacity, which is also related to OH .radical scavenging (Vidal et al., 2006;Yuan and Walsh, 2006).

Antioxidant activity protecting against liver damage in CCl 4 -induced Wistar rats
Antioxidant and hepatoprotective properties of the aqueous extract from H. incrassata were investigated in Wistar rats with a CCl 4 -induced liver injury.Through the ASAT and ALAT activities (Table 1), it verified partial liver injuries caused by CCl 4 .We observed that the rats treated with an aqueous extract from H. incrassate, or Ferulic acid, proved to be capable of attenuating the toxic effect produced by CCl 4 .As will be appreciated, it was observed that a partial recuperation occurred with an aqueous extract from H. incrassata.In previous work, Mancini et al. (2009) reported similar results when investigating polyphenol fractions from Halimeda monile.These results are also in accordance with de Oliveira et al. ( 2012) whom investigated the hepatoprotective properties of polyphenolic fractions from H. opuntia under an experimental model CCl 4 injury.Tanigushi et al. (Tanigushi et al., 2004) reported that hepatic damage by CCl 4 may occur in a range of between 6 to 12 h after CCl 4 administration, and that restoration starts after 48 h.It is possible then to suppose that FPA-H.incrassatatreated rats had a faster recovery from liver injury at 24 h than expected, criterion that is concordant with our results.
In Table 1, it may be appreciated that TBARS levels in the liver tissues of CCl 4 -treated rats increased, confirming the successful induction of oxidative damage, while pretreatment with H. incrassata (300 mg/kg) led to an approximate 50% reduction in liver TBARS levels.A previous study from our laboratory showed that an aqueous extract from H. incrassata was effective in significantly reducing serum and brain TBARS levels and other parameters in rats with oxidative stress induced by methyl-mercury (2004).According to de Oliveira e Silva et al. ( 2012), a pre-treatment with polyphenol rich fractions from Halimeda opuntia led to reductions in serum and liver TBARS.Kim et al. (2011) also observed a comparable reduction in hydroperoxide levels in the liver, relative to the CCl 4 -treated group, in a study of rats fed on Saengshik, a non-cooked food containing vegetables and seaweeds.
Animals with liver injuries caused by CCl 4 had GSH levels increased statistically in respect of all groups, while in an aqueous extract from the H. incrassata group was observed with only minor values.According to Chan et al.  (Chan et al., 2001), these increased levels may be explained as an adaptive response of the rats reacting against the oxidative stress introduced by CCl 4 .
The CAT and SOD enzymes are considered to be as a fundamental antioxidant defense system in mammals, and it was demonstrated that CCl 4 treatment significantly reduced the activities of these enzymes.In this study, we observed the ability of CCl 4 to diminish the antioxidant enzyme activities.
As may be appreciated in Figure 3, treatment with the seaweed led to a significant increase in the activity of the CAT enzyme, which in turn resulted in an enhanced antioxidant defense.These results suggest antioxidant and hepatoprotective activities of the phenolic fraction of H. incrassata.Ozturk et al. (2003) observed in the CCl 4treated group significant increases in kidney CAT activity.These results are in agreement with Mancini-Filho et al. (2009) that reported a considerable increase in the activity of CAT in rats treated with a polyphenol-rich fraction similar to that from Halimeda monile.High antioxidant enzyme activity has been reported through repeated administration of Sargassum spp.(Phaeophyceae) extracts (Raghavendran et al., 2005).Treatment with Caulerpa prolifera (Chlorophyta) and Laurencia obtusata (Rhodophyta) extracts also led to a rise in enzyme activity (Abdel-Wahhab et al., 2006).

Expression of CAT hepatic enzymes by PCR-RT
When considering results from antioxidant enzyme activities, only the expression of Catalase by PCR-RT technique was studied.As can be seen in Figure 4, the levels of CAT in liver tissues partially increased with a treatment of seaweed and a posterior CCl 4 administration, which shows alterations in the expression of catalase genes.
Treatment with H. incrassata aqueous extract (band 3) resulted in a higher catalase gene expression when compared with that observed in the CCl 4 -treated group (band 2).A review by Stevenson and Hurst (2007) discusses recent evidence that polyphenols also have an indirect antioxidant effect through the induction of endogenous protective enzymes, and that these inductive or signaling effects may occur at concentrations much lower than those required for effective radical scavenging.Vidal et al. (2011) reported that a total phenolic contents of the hydrophilic fractions from H. incrassata were 255 μg of gallic acid equivalents/g of fresh seaweed, which more than half (63%) corresponds to free phenolic acids, and in this fraction, about 32% was identified as salicylic acid, while a small fraction was associated to ferulic acid.Yeh and Yen (2006) suggested that these three phenolic acids, including ferulic acid, modulate the phase II antioxidant enzymes and the phase II sulphate conjugative enzymes; and they seem to selectively induce hepatic mRNA transcripts for CAT, probably through the up-regulation of gene transcription, as well as the Nrf2 transcription factor.In previous results from our group, Mancini-Filho et al. (2009) reported an over-expression of CAT genes by treatment with FPA from Halimeda monile; while de Oliveira e Silva et al. (2012) showed that by using (RT/PCR) analysis increased the catalase (CAT) gene expression in the group treated with free phenolic acid (FPA) fractions from Halimeda opuntia, suggesting inducing effects on the enzyme.

2+ and HRP/H 2 O 2
The atheroprotective potential of Halimeda incrassata was determined through its effect on LDL oxidation.The inhibition of LDL oxidation by Cu 2+ , or HRP/H 2 O 2 , was dose dependent, with a higher concentration needed for the aqueous extract than for the FPA fraction (Table 2).It was compared with the two oxidation systems relevant to LDL oxidation in the artery wall: by Cu 2+ , or HRP/H 2 O 2 ; and by studying the antioxidant effect in the mediated transition metal and independent LDL oxidation (Stocker and Keaney, 2004).As shown, seaweed hydrophilic fractions had an inhibitory effect on both models of LDL oxidation, indicating its potential in atheroprotection.
The inhibition of LDL oxidation is considered a key target in atherosclerosis management, since oxidized LDL levels are presently one of the main emerging factors for cardiovascular risk (Kang et al., 2003;Levitan et al., 2010).In vivo is a complex process, and there is no certainty as to the precise mechanism of the initiation of lipoperoxidation in the vascular wall ( 2004).In the quest for the atheroprotective potential of natural compounds, different researchers have determined the antioxidant properties of seaweed extracts in the inhibition of LDL oxidation (Bocanegra et al., 2009;Jimenez-Escrig et al., 2001;Yang et al., 2011).
The activity of the inhibition of LDL-oxidation found in this work is promissory when compared to other natural extracts.Hseu et al. (2008) found 37% inhibition of TBARS formation in oxidation mediated by AAPH and 74% in peroxidation mediated by Cu 2+ ions.In this study, the Toona sinensis extracts had 6.5 μg GAE, a phenolic content in the range of the one needed in study for the 50% inhibition of TBARS formation by Halimeda incrassata extracts.Table 2. Inhibitory effect of H. incrassata hydrophilic fractions on LDL oxidation induced by Cu 2+ ions or peroxidase (HRP/H2O2), w ith or w ithout increasing seaw eed extract concentration.TBARS formation w as determined according to Frostegard et al. (1990).

Fractions 50% inhibitory concentration (IC50) of H. incrassata Cu
2+ HRP Aqueous extract (mg) 10.42 ± 0.295 1.09 ± 0.005 FPA (mg) 0.33 ± 0.04 0.66 ± 0.02 LDL oxidation studies were done with LDL that had interacted with heparin as a model of glycosaminoglycan since it has been reported that lipoproteins that have interacted with glycosaminoglycans are more susceptible to oxidation (Upritchard and Sutherland, 1999).In the case of peroxidase, this is associated with a change in the lipoprotein structure, giving an increased access of peroxidase to apo B, and forming free radicals from apo B and vitamin E that would mediate the oxidation of lipids by mieloperoxidase and HRP.The mechanism is different from Cu 2+ mediated oxidation, where free radical formation and peroxidation takes place directly in the lipid phase.
Other authors have evaluated the effect of natural extracts in the inhibition of LDL oxidation by peroxidases.Wang et al. (2003) found a significant decrement in TBARS formation in extracts from C. muk ul on LDL oxidation mediated by lypoxigenases.The extracts also had an antiatherogenic action in the inhibition of cholesterol uptake by macrophages and LDL oxidation mediated by Cu 2+ ions.Few studies approach the effect of antioxidants in LDL oxidation mediated by HRP.It has been shown that in this area, vitamin E acts by transferring radicals, from the aqueous to the lipid phase, and does not protect it from oxidation.However, in the presence of vitamin C, oxidation mediated by peroxidases is inhibited, as in this case, vitamin C acts as a co-antioxidant avoiding the formation of α-tocoferoxil (Upritchard and Sutherland, 1999).Other antioxidants, like some phenolic hydrophilic compounds, act synergistically in the scavenging of free radicals protecting vitamin E, licopene and β-carotene, contained in LDL from oxidation (Kaliora et al., 2006).The antilipoperoxidative activity of the hydrophilic fractions of Halimeda incrassate, in this model, indicates that the extracts are efficient in the inhibition of oxidation mediated by protein radicals, and add evidence to the antiatherogenic and antioxidant potential.
Other authors have obtained an excellent activity of antilipoperoxidation for Halimeda incrassata in β-carotene linoleate systems, with an inhibition of bleaching of 75% for Halimeda incrassate, while for Bryothamnion triquetrum seaweed , the activity was 20% at a dose of 2 mg (Rivero et al., 2003;Vidal et al., 2011).
Our results are also comparable to the work of Yuan and Walsh (2006) with a significant inhibition of conjugated dienes and TBARS formation, during the oxidation of linoleic acid for an aqueous extract from the Palmaria palmate seaweed, suggesting that the antioxidant activity was due to the complex mixture of antioxidants present in the extract, with the presence of chlorophyll, polyphenols, carotene, and ascorbate.

2+
The association of antioxidant activity and the inhibition of LDL-oxidation, evaluated by different methodologies in this paper, were performed to explain the antioxidant mechanisms of Halimeda incrassata.In this work, a positive correlation was found between the scavenging of OH • radicals in the inhibition of the desoxiribose assay in the presence of EDTA (r 2 = 0.997), and the inhibition of LDL oxidation mediated by Cu

2+
. A similar behavior was obtained for the chelating effect of metal ions in the inhibition of desoxiribose oxidation assay in the absence of EDTA (r 2 = 0.943) and the inhibition of LDL oxidation mediated by Cu 2+ .The results of the correlation of antioxidant activity in Fe ion chelation and the inhibition of oxidation of LDL mediated by Cu 2+ suggest that the mechanism of action in the inhibition of LDL oxidation by hydrophilic fractions could be associated with the antioxidant properties of the seaweed in metal ion chelating and free radical scavenging.

Antioxidant activity of hydrophilic extracts of Halimeda incrassata in macrophages
As shown in Figure 6A, the degree of cell lipoperoxidation was decreased by about two-fold in the presence of Halimeda incrassata seaweed extract.The FPA fraction also inhibited TBARS production, indicating the contribution of phenolic compounds to the effect.
The improvement of antioxidant activity was also shown by the 1.2 fold decrement in ROS levels by the treatment with Halimeda incrassata aqueous extract, when aqueous extracts were added immediately before stimulation (Figure 6A), or by a nearly 50% decrement in ROS levels, when aqueous extracts were added 24 h before stimulation of the cells (Figure 6B).
The macrophages associated with oxidative stress are directly involved in the atherosclerosis progression, so when evaluating compounds of interest for atheroprotection antioxidant activity, it is frequently sought at the macrophage level (Kaliora et al., 2006).
A decreased peroxide basal content was found in the cell supernatant as a result of the preincubation with seaweed hydrophilic extracts.About a 3.3 fold higher concentration was required from the FPA fraction for it to reach a similar inhibition of MDA formation as to that of1.5 µg GAE/mL (0.5 mg/mL) for the aqueous extract.To evoke ROS production in macrophages, opsonized zymosan was added to the cells after the addition of aqueous seaweed extracts.About a 20% decrement in ROS production was found with the highest concentrations of seaweed used in the assay.Seaweed preincubation with the cells improved the antioxidant capacity with a 50% decrement in ROS production with the highest concentration tested (1 mg/mL).These results are in agreement with previous studies by our group for the aqueous extracts of Halimeda incrassata (Chlorophyta) and Bryothamnion triquetrum (Rhodophyta) seaweeds, and where basal and H 2 O 2 elicited peroxides were decreased in the GT1-7 hypothalamic cells in the presence of seaweeds (Fallarero et al., 2003).
Indeed, the antioxidant activity of natural extracts in macrophages has also been assessed by other authors that have found a decreased macrophage peroxidation and ROS production by incubation with phenolic rich extracts.Yang et al. (2011) found a 1.5% decrement in the peroxide levels in oxLDL stimulated macrophages, after preincubation with 1mg/mL mulberry leaf extracts (where quercetin, gallocatechin gallate and naringenin were the main polyphenolic constituents identified).The atheroprotective effect was related to an elevation of antioxidant enzymes GPx and SOD by the extracts.
Likewise, a significant inhibition of peroxidation was found in cells stimulated with oxLDL after preincubation with anthocyanin rich purple sweet potato extracts (0.5-0.6 mg/mL); that also inhibited the LDL uptake by macrophages and had antioxidant activity in DPPH • scavenging (Park et al., 2010).
The antioxidant activity in macrophages has been studied by other groups that have found improved in vitro and in vivo antioxidant capacity, as a result of different phenolic rich extract supplementation (Aviram et al., 2008).Targeting oxidative stress as a cause of atherosclerosis progression, they have correlated phenolic content, in vitro antioxidant activity in DPPH • radical scavenging, and the atheroprotective effect in macrophages (evaluated as the inhibition of LDL uptake, decreased macrophage peroxidation, and increased antioxidant enzyme activity) to be in vivo atheroprotection in apo E-/-mice.

Conclusion
In this work, the antioxidant activity in in vitro cell free systems and cell systems for the hydrophilic fractions from Halimeda incrassata was evaluated.Previously, it identified phenolic acids in hydrophilic fractions as being the main active components (salicylic and ferulic acid).It also tested the phenolic contribution to the antioxidant activity for a FPA fraction.A significant antioxidant activity was found in both cell free and cell systems.However, a higher concentration of FPA fraction was required to have a similar effect as for the one with the aqueous extracts.It is then possible to suppose that phenolic acids in the extract to be of relevance for the activity, since both ferulic and salicylic acids have been found to have antioxidant and antinflammatory properties that decrease the macrophage activation.Additionally, different authors have shown a synergic effect in the antioxidant and antiatherogenic properties from seaweed crude extract, and this improved antioxidant activity could also be related to the synergism between the polyphenolic compounds and/or with other antioxidants.Halimeda spp.contains antioxidants like ascorbate, β-carotene, chlorophylls, among other compounds that might contribute to the effect.In summary, this study adds further evidence about the beneficial properties of Halimeda incrassata as an antioxidant and as an antiatherogenic for a future phytotherapeutic application.
was determined for the Aqueous Extract and FPA.Both fractions exhibited a concentration dependent on free radical scavenging activity (Figure1).The FPA fraction had an IC 50 value of 0.46 mg of dry residue (27.1 µg of polyphenol); while the Aqueous Extract had an IC 50 value of 5.75 mg of lyophilized substance (14.8 µg of polyphenol).

Figure 1 .
Figure 1.Scavenging activity of the DPPH • radical by hydrophilic fractions of H. incrassate.(A): Scavenging of DPPH • radicals by aqueous extracts in time.(B): Scavenging of DPPH • radicals by a free phenolic acid fraction (FPA) in time.The assay w as performed according to Goupy et al. (1999).Values are given as a mean  S.D. (n = 3).After incubating, increasing extract concentration (0.4-1 mg for aqueous extract or 10-40 µg total polyphenols for FPA fraction) w ith DPPH • solution, absorbance w as measured at λ = 517 nm in time.

Figure 2 .
Figure 2. Inhibition of the oxidative degradation of 2-Desoxi-D-ribose by the aqueous extract of Halimeda incrassata (A) w ith EDTA, (B) w ithout EDTA.The assay w as performed as described by Aruoma (1994).Oxidation of desoxiribose w as done in the presence, or absence of, increasing concentrations of Halimeda incrassata.Values are given as a mean  S.D. (n=3).

Figure 5 .
Figure 5. Correlation of antioxidant activity by different methodologies. A. Association of antioxidant activity in OH .radical scavenging and the inhibition of LDL oxidation mediated by Cu 2+ ions (r 2 = 0.997).B. Association of antioxidant activity in Fe i on chelation and the inhibition of oxidation of LDL mediated by Cu 2+ (r2= 0.943).
Figure 5A and B indicate the positive correlation between the inhibition of desoxiribose oxidation, with or without EDTA, and the inhibition of LDL oxidation mediated by

Figure 6 .
Figure6.Antioxida nt ac t ivity of hydrophilic extracts of Halimeda incrassata in macrophages.(A).TBARS levels in cell supernatant w ere determined as inFrostegard et al., 1990.(B)  and (C) ROS production by cells after stimulation w ith zymosan w as determined by luminol chemiluminiscence as inKopprasch et al., 2003.Experiments w ere performed by adding aqueous extract immediately before cell stimulation (B) or by preincubating cells w ith extracts for 24 hours before stimulation (C).Significant differences w ere determined by ANOVA w ith a Tukey post test, ** p < 0.001.

Table 1 .
Values of parameters and enzymes in serum and liver tissue relative to oxidative stress.