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Botanica Marina 52 (2009): 268–277 2009 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BOT.2009.037

Antioxidant and cytotoxic activities of some red (Rhodophyta) from Brittany coasts (France)

Mayalen Zubia*, Marie-Sophie Fabre, over, the interest in employing antioxidants from natural Ve´ ronique Kerjean and Eric Deslandes sources has been considerably enhanced by consumers’ preferences for natural products and concerns about the Plateforme BIODIMAR/UBO, Universite´ de Bretagne toxic effects of synthetic antioxidants (Ito et al. 1986). Occidentale, 305 rue Claude Shannon, 29280 Plouzane´, Algae, as photosynthetic organisms, are exposed to a France, e-mail: [email protected] combination of light and high oxygen concentrations, which induces the formation of free radicals and other * Corresponding author oxidative reagents. The absence of structural damage in the algae implies that these organisms are able to gen- erate antioxidants to protect themselves against oxida- Abstract tion. In this regard, macroalgae are considered to be rich in natural antioxidants (e.g., phlorotannins, ascorbic acid, We assessed the antioxidant activity of crude extracts tocopherols, carotenoids) (Plaza et al. 2008). from 24 rhodophyte species from Brittany coasts using Another area that has been the focus of much attention three complementary methods (2,2-diphenyl-1-picryhy- is the search for anticancer drugs, as marine molecules drazyl, reducing activity, and b-carotene-linoleic acid have shown promising results for different stages of can- system). We also examined phenolic contents. Cytotoxic cer development (Mayer and Gustafson 2006). Among activities were determined with three different cancer cell marine organisms, numerous macroalgae have potent lines. Four species ( pseudobyssoides, cytotoxic activities (see reviews in Gu¨ ven et al. 1990, Furcellaria fastigiata, lanosa, and Heterosi- Smit 2004, Mayer and Gustafson 2006) and algal con- phonia plumosa) had high antioxidant activity and high sumption has been suggested as a chemo-preventive phenolic content. The extract from bys- agent against several cancers (Funahashi et al. 2001, soides had the highest antioxidant potential, which was Yuan and Walsh 2006). Halogenated terpenoids from red also found to be equivalent to the antioxidant activities algae are considered to be promising secondary metab- of some commercial antioxidants. In the b-carotene sys- olites in anticancer research. Indeed, dehydrothyrsiferol tem, extracts from Porphyra leucosticta and Porphyra and halomon, extracted from viridis (Pec et al. purpurea had some specific antioxidant activity. Further- 2003) and Portieria hornemanii (Egorin et al. 1997), more, Asparagopsis armata, B. byssoides and H. plumosa respectively, have been developed to the preclinical extracts had strong cytotoxic activities against Daudi and phase. Jurkat cells. The coasts of Brittany (France) are characterized by great macroalgal species richness (around 700 species; Keywords: antioxidant activity; cytotoxic activity; Dizerbo and Herpe´ 2003). Among them, only few, mainly macroalgae. brown algae, have been studied for their antioxidant capacities (Cerantola et al. 2006, Connan et al. 2006, 2007), their anti-fouling (Hellio et al. 2000, 2001, 2004), Introduction and antitumoral (Moreau et al. 2006) activities. This study aimed to assess the antioxidative and cyto- In Asian countries, macroalgae are traditionally traded as toxic capabilities of 24 growing along the food items, e.g., sushi wrappings, seasonings, condi- shorelines of Brittany. To gain more insight into antioxi- ments, and vegetables, and for the phycocolloid industry. dant processes, the antioxidative activities of the crude In recent years, the market for macroalgae has consid- extracts were characterized by three biochemical meth- erably expanded into the pharmaceutical and para-phar- ods: 2,2-diphenyl-1-picryhydrazyl (DPPH) radical-scav- maceutical sectors owing of their exceptional richness in enging activity, reducing activity, and b-carotene-linoleic bioactive compounds (e.g., antimicrobial, anti-inflamma- acid system. The total phenolic contents of these tory, and antitumoral activities) (Smit 2004, Kornprobst extracts were also examined, and their cytotoxic activi- 2005). There is a huge potential for macroalgae in the ties were determined with three different cancer cell lines food, medical, and cosmetic industries, especially red (Daudi, Jurkat, and K562). macroalgae that form a very heterogeneous group with regard to their chemical composition (Kornprobst 2005). Among the most relevant compounds found in algae, Materials and methods antioxidants are probably those that have attracted most interest. Antioxidants are considered key compounds in Collection the fight against various diseases (e.g., cancer, chronic inflammation, atherosclerosis, cardiovascular disorders) Samples of 24 species of red macroalgae were collected and the aging process (Kohen and Nyska 2002). More- along the coasts of Brittany between 2006 and 2007

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M. Zubia et al.: Biological activities of some red algae 269

(Table 1). Once harvested, they were stored in plastic Antioxidant assays bags for transport to the laboratory. Voucher specimens of all species were pressed and stored in 4% formalin DPPH radical-scavenging activity 2,2-Diphenyl-1-picry- for identification (Cabioc’h et al. 2006). All of the samples hydrazyl radical (DPPH) radical-scavenging activity was were washed thoroughly with freshwater to remove salts, determined using the method developed by Brand-Wil- sand, and epiphytes, and then stored at -208C. Each liams et al. (1995) and modified by Fukumoto and Mazza macroalgal sample was lyophilized and pulverized into (2000). The fundamental principle of the DPPH method is the reduction of the DPPH radical in alcoholic solution powder before extraction. by H-donor antioxidant (AH) to form the non-radical form DPPH-H. In a 96-well microplate, 22 ml of sample were Preparation of crude algal extracts mixed with 200 ml of the DPPH solution (25 mg l-1)pre- pared daily. Samples were prepared in triplicate and at Extraction was carried out with an Accelerated Solvent least seven different concentrations (0–40 mg ml-1) were Extraction system (ASE 300) equipped with a solvent used for each extract. The DPPH solution and the sam- controller unit (Dionex, Yvelines, France). A preliminary ples were prepared with 80% methanol. Due to the color study was carried out to improve the extraction yields by of the extracts, blanks had to be prepared by mixing optimizing parameters (unpublished data). The red algal 22 ml of sample with 200 ml of 80% methanol. The reac- extractions performed in this study were carried out with tion was incubated for 2 h in the dark at room temper- the optimized parameters as follows: 10 g of lyophilized ature, and then the absorbance was read at 515 nm with samples were mixed with 10 g of Fontainebleau sand as a multi-well spectrophotometer (Sunrise, TECAN, Lyon, a dispersing agent, and placed in a 66-ml stainless steel France). The DPPH concentration in the reaction medium extraction cell equipped at its outlet with a cellulose filter. was calculated from a calibration curve (ns8, rs0.99) All extractions were performed with dichloromethane determined by the following linear regression: methanol (1:1, v:v) at 758C and 1500 psi (103.42 bar) wDPPHxs(Abs–0.0398)/0.0137 to further deduce the per- during two static 7-min cycles. Each sample was extract- centage of remaining DPPH (% DPPH). A curve of extract ed twice, and then the cell was rinsed with solvent and concentration against % DPPH was generated to esti- purged with a flow of nitrogen. The extracts were then mate the concentration of extract needed to cause a filtered through a grade-4 Whatman filter and concen- 50% reduction of the initial DPPH concentration. This trated to 10 ml under reduced pressure prior to storage value is known as EC50 (efficient concentration when at -208C. 50% oxidation is achieved, also called oxidation index)

Table 1 Information on the red macroalgae studied.

Number Species Order Family Place of Date of collection collection

1 Aglaothamnion pseudobyssoides (P.L. Crouan et Ceramiaceae Callot June 2007 H.M. Crouan) Halos 2 Asparagopsis armata Harvey Bonnemaisoniales Bonnemaisoniaceae Anse de Melon March 2007 3 (Goodenough et Ceramiales Callot June 2007 Woodward) F. Schmitz 4 Calliblepharis jubata (Goodenough et Woodward) Cystocloniaceae Anse de Melon March 2007 Ku¨ tzing 5 Callithamnion tetragonum (Withering) S.F. Gray Ceramiales Ceramiaceae Anse de Melon May 2007 6 Callophyllis laciniata (Hudson) Ku¨ tzing Gigartinales Kallymeniaceae Anse de Melon May 2007 7 Caulacanthus ustulatus (Mertens ex Turner) Gigartinales Caulacanthaceae Callot June 2007 Ku¨ tzing 8 ciliatum (J. Ellis) Ducluzeau Ceramiales Ceramiaceae Anse de Melon May 2007 9 Cystoclonium purpureum (Hudson) Batters Gigartinales Cystocloniaceae Anse de Melon May 2007 10 carnosa (Schmidel) Kuntze Gigartinales Bertheaume July 2007 11 contorta (S.G. Gmelin) Ruprecht Gigartinales Dumontiaceae Callot March 2006 12 Furcellaria fastigiata (Turner) J.V. Lamouroux Gigartinales Furcellariaceae Anse de Melon March 2007 13 Gastroclonium ovatum (Hudson) Papenfuss Rhodymeniales Champiaceae Anse de Melon April 2007 14 Gracilaria gracilis (Stackhouse) M. Steentoft, Gracilariales Gracilariaceae Callot June 2007 L.M. Irvine et W.F. Farnham 15 Grateloupia filicina (J.V. Lamouroux) C. Agardh Halymeniales Halymeniaceae Callot June 2007 16 Heterosiphonia plumosa (J. Ellis) Batters Ceramiales Dasyaceae Anse de Melon May 2007 17 Kallymenia reniformis (Turner) J. Agardh Gigartinales Kallymeniaceae Porz Liogan June 2007 18 Lomentaria articulata (Hudson) Lyngbye Rhodymeniales Lomentariaceae Anse de Melon March 2007 19 Lomentaria clavellosa (Turner) Gaillon Rhodymeniales Lomentariaceae Anse de Melon May 2007 20 Plocamium cartilagineum (Linnaeus) P.S. Dixon Plocamiales Plocamiaceae Anse de Melon May 2007 21 Polyneura bonnemaisonii (C. Agardh) Ceramiales Anse de Melon March 2007 Maggs et Hommersand 22 (Linnaeus) Tandy Ceramiales Rhodomelaceae Anse de Melon May 2007 23 Porphyra leucosticta Thuret Bangiales Bangiaceae Anse de Melon April 2007 24 Porphyra purpurea (Roth) C. Agardh Bangiales Bangiaceae Iˆle de Bec July 2007 Article in press - uncorrected proof

270 M. Zubia et al.: Biological activities of some red algae

and was expressed in units of mg ml-1. This assay was Total phenolic content carried out in triplicate for each sample, and the mean The total phenolic content of algal extracts was deter- values were used to calculate the EC50. Ascorbic acid, a- tocopherol, butylated hydroxyanisole (BHA) and butylat- mined by spectrophotometry using the Folin-Ciocalteu ed hydroxytoluene (BHT) were used as positive controls. method from Connan (2004). The samples (100 ml) were mixed with 50 ml Folin-Ciocalteu reagent, 200 ml of 20% sodium carbonate solution, and 650 ml of distilled water. Reducing activity The method of Kuda et al. (2005) The mixture was incubated at 708C in the dark for 10 min. was used, after slight modifications (incubation time and After production of a blue color, absorbance was read at wavelength), to assess the reducing activity of each 700 nm. The total content of phenolic compounds was extract. This method relies on the evaluation of total anti- expressed as % of dry weight (% dw) based on a stan- oxidant capacity of a given extract from the redox poten- dard curve of phloroglucinol. This analysis was carried tials of the compounds. Aliquots of extracts (0.2 ml) at out in triplicate for each extract. four different concentrations (50, 100, 200, and 500 mg l-1) were mixed with phosphate buffer (0.2 ml, 0.2 M, Cell culture w x pH 7.2) and potassium ferricyanide K3Fe(CN)6 (0.2 ml, 1%). After incubation at 508C for 30 min, the mixture was Daudi (Human Burkitt’s lymphoma), Jurkat (Human leu- cooled down prior to the addition of 0.2 ml of trichloro- kemic T-cell lymphoblast), and K562 (Human chronic acetic acid (10%). Then, 125-ml aliquots of this mixture myelogenous leukemia) cells were obtained from ECACC were transferred to a 96-well microplate before addition (European Collection of Cell Cultures, Salisbury, UK) and = grown in RPMI 1640 medium with L-glutamine (Lonza, of 20 ml of 0.1% FeCl3 6H2O to each well. Increased absorbance of the reaction mixture at 620 nm indicated Basel, Switzerland) supplemented with 10% of heat inac- increased reducing activity. Absorbance was read with a tivated fetal bovine serum (FBS; Cambrex Bioscience, multi-well spectrophotometer (Sunrise, TECAN) and Saint-Beauzire, France), and 20% for Daudi cells. Cells = 4 then transformed into a percentage of inhibition in com- were plated at 5 10 cells in 200 ml per well onto 96- parison to a blank (ethanol). This assay was carried out flat-bottomed well ELISA plates and incubated at 378C in triplicate for each sample and positive controls (ascor- in 5% CO2 for 24 h. Blanks (medium without cells) were bic acid, a-tocopherol, BHA and BHT). prepared under the same conditions. Seaweed extracts were added to the cells at a concentration of 100 mgml-1. Dimethyl sulfoxide (DMSO) was used as control and was b-Carotene-linoleic acid system The antioxidant added to the cells at a concentration of 100 mgml-1. activities of samples assayed by the b-carotene-linoleic Seaweed extracts, blank, and control were prepared in acid system were measured according to the method triplicate. Subsequently, the plates were incubated for developed by Koleva et al. (2002) (emulsion preparation) 24 h. and by Zhang et al. (2007). This method is based on the loss of the yellow color from b-carotene due to its reac- Cytotoxic assay tion with the radicals formed by linoleic acid oxidation; b-carotene bleaching is, thus, slowed down in the pres- The cytotoxic activities of these crude extracts were ence of antioxidants. After dissolution of b-carotene investigated by a cytotoxic assay adapted from the (1 mg) in 5 ml of chloroform, 1 ml of this solution was method by Ishiyama et al. (1995). To assess mitochon- mixed with linoleic acid (25 ml) and Tween-40 (200 mg). drial function, mitochondrial dehydrogenase (succinate- After removal of chloroform by evaporation at 408C under tetrazolium-reductase) activity was determined with the w vacuum, 50 ml of distilled water oxygenated by air-bub- WST-1 (4- 3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetra- x bling were added slowly to the semi-solid residue under zolio -1,3-benzene disulfonate) colorimetric assay (Roche vigorous stirring to form an emulsion. A 96-well micro- Diagnostics, Meylan, France); WST-1 is a tetrazolium dye plate was loaded with 50 ml per well of the samples or containing an electron coupling reagent which is cleaved positive controls (a-tocopherol, BHA and BHT prepared by the mitochondrial dehydrogenase enzyme to a water- in ethanol) and 200 ml of the emulsion. Four final con- soluble formazan dye, and there is a direct correlation between this reaction and the number of metabolically centrations were tested (50, 100, 200, and 500 mg l-1) active cells. After 24 h of incubation, 10 ml of the for- and ethanol was used as blank. All determinations were mazan dye were added to each well prior to a 3-h incu- carried out in triplicate. The absorbance value at 450 nm bation at 378C. Absorbance was measured in triplicate at was read with a multi-well spectrophotometer (Sunrise, 450 nm with a multi-well spectrophotometer (Infinite, TECAN) with addition of the emulsion considered as the TECAN) in blank, control, and tested cells (with sea- starting time of the reaction (ts0 min). After covering the weeds extracts). plate with a film (Seal Plate, VWR, Fontenay sur Bois, Cells were plated a second time and the experiment France) and incubation at 508C for 3 h, the absorbance was repeated. The results were then expressed in per- was measured every 30 min. The antioxidant activity (AA) centage of viability in comparison to the control. of the extracts was evaluated as the percentage inhibi- tion of bleaching of b-carotene using the following for- Statistical analysis sw x= mula: AA 1–(A0–At)/(A90–A9t) 100, where A0 is the s absorbance of the sample at t 0 min and A90 is the All statistical analyses were performed with Statistica 6 s absorbance of the control at t 0 min, A t and A9t are software (StatSoft Inc., Maison-Alfort, France). The data absorbances of the sample and control at ts3h. were tested for normality (Shapiro-Wilk’s test) and sub- Article in press - uncorrected proof

M. Zubia et al.: Biological activities of some red algae 271

jected to the Bartlett test to verify the homogeneity of Results variance. One-way analysis of variance was carried out to compare antioxidant activities between extracts, after Antioxidant activity transformation of data (arcsin or log) to obtain homoge- neity of variance when required. A post-hoc test (Tukey Figure 1, Tables 2 and 3 report main results of the HSD) was carried out when data showed significant dif- assessment by DPPH radical-scavenging-activity, reduc- ferences (p-0.05) to classify antioxidant activity means ing activity, and the b-carotene-linoleic acid system, measured for extracts and positive controls. Student’s respectively, of the crude extract antioxidative activities. t-test was used to compare cytotoxic activity of the All the species collected for this study had DPPH rad- extracts with the control (p-0.01). ical-scavenging activities (Figure 1). The crude extract

The Pearson correlation coefficient (r) was also calcu- from Brongniartella byssoides had the lowest EC50 value lated (p-0.05) to evaluate the relationship between the (0.14"0.01 mg ml-1), which was similar to the DPPH rad- different methods used to measure antioxidant activities ical-scavenging activity of a-tocopherol (0.14"0.01 mg of the extracts and to determine whether phenolic com- ml-1). Furcellaria fastigiata (1.39"0.04 mg ml-1), Polysi- pounds were implicated in the antioxidant capacity and phonia lanosa (2.71"0.01 mg ml-1), Aglaothamnion pseu- in the cytotoxic activities. dobyssoides (3.01"0.05 mg ml-1), and Heterosiphonia

-1 " s Figure 1 DPPH radical-scavenging activities expressed as the oxidation index EC50 (mg ml , mean SD, n 3) of red algal crude extracts and controls (ascorbic acid, a-tocopherol, BHA and BHT). Significant differences between radical-scavenging activities of the crude extracts and the controls were determined by the Tukey HSD test (p-0.05) and are indicated by different letters (a–u). Article in press - uncorrected proof

272 M. Zubia et al.: Biological activities of some red algae

Table 2 Reducing activity of red algal crude extracts at different concentrations (50, 100, 200, and 500 mg l-1) and expressed as % inhibition.

50 mg l-1 100 mg l-1 200 mg l-1 500 mg l-1

Aglaothamnion pseudobyssoides 33.85"2.64 (c, d) 52.53"0.26 (d) 68.41"0.63 (c) 79.42"0.15 (b) Asparagopsis armata 28.56"1.13 (e, f) 42.77"1.50 (e, f) 44.41"1.79 (g, h) 65.73"0.40 (h, i) Brongniartella byssoides 74.86"2.34 (b) 86.21"0.11 (c) 91.65"0.17 (b) 94.80"0.08 (a) Calliblepharis jubata 10.51"1.57 (h) 21.50"2.12 (l) 43.73"1.28 (h) 68.10"1.00 (f–h) Callithamnion tetragonum 24.71"1.80 (f) 37.27"0.83 (f, g) 45.42"0.36 (f–h) 73.17"0.46 (c, d) Callophyllis laciniata 19.26"1.26 (g) 31.72"0.52 (h, i) 47.63"0.31 (f, g) 68.93"0.39 (e–g) Caulacanthus ustulatus 17.40"1.23 (g) 29.35"1.05 (i–k) 48.12"0.75 (f) 66.73"0.30 (g–i) 18.75"2.04 (g) 30.95"1.95 (i, j) 46.08"2.37 (f–h) 66.15"1.75 (h, i) Cystoclonium purpureum 15.74"1.53 (g) 26.06"0.68 (k) 42.79"0.71 (h) 62.23"0.18 (j, k) 27.00"0.64 (e, f) 34.82"0.51 (g, h) 51.81"0.48 (e) 70.68"0.30 (d–f) 19.28"1.42 (g) 29.95"0.62 (i, j) 48.11"0.90 (f) 65.78"1.78 (h, i) Furcellaria fastigiata 24.23"1.71 (f) 43.52"1.42 (e, f) 60.53"1.55 (d) 75.19"0.79 (c) Gastroclonium ovatum 11.11"0.00 (h) 17.39"0.77 (m) 32.73"1.36 (j) 52.05"0.52 (l) Gracilaria gracilis 18.32"1.36 (g) 28.29"0.60 (i–k) 44.69"1.89 (f–h) 62.78"0.58 (j, k) Grateloupia filicina 2.40"1.01 (i) 14.27"1.36 (m) 32.74"2.10 (j) 51.47"1.28 (l) Heterosiphonia plumosa 28.12"0.56 (e, f) 46.15"0.31 (e) 61.11"0.28 (d) 75.15"0.31 (c) Kallymenia reniformis 1.69"0.00 (i) 9.33"2.42 (n) 20.18"0.64 (l) 42.75"0.86 (m) Lomentaria articulate 9.98"1.80 (h) 17.68"0.87 (m) 35.09"0.54 (j) 50.89"1.10 (l) Lomentaria clavellosa 16.08"1.45 (g) 27.72"0.62 (j, k) 38.65"0.44 (i) 64.21"0.46 (i, j) Plocamium cartilagineum 30.07"1.92 (d, e) 40.09"0.48 (f, g) 52.20"0.31 (e) 70.85"0.41 (d, e) Polyneura bonnemaisonii 8.72"1.07 (h) 10.52"1.03 (n) 23.56"1.95 (k) 38.97"2.05 (n) Polysiphonia lanosa 38.16"1.17 (c) 55.65"0.40 (d) 66.21"0.60 (c) 80.88"0.72 (b) Porphyra leucosticta 16.18"2.09 (g) 25.44"2.27 (k) 44.23"0.92 (g, h) 61.00"1.82 (k) Porphyra purpurea 25.37"0.00 (f) 28.56"1.02 (i–k) 44.03"0.36 (h) 62.59"0.16 (j, k) BHA 87.42"0.07 (a) 94.02"0.08 (a) 94.17"0.08 (a) 95.64"0.02 (a) BHT 84.63"0.37 (a) 90.18"0.17 (b) 93.65"0.17 (a) 94.93"0.11 (a) Ascorbic acid 90.04"0.32 (a) 92.91"0.14 (a, b) 93.53"0.09 (a) 94.87"0.01 (a) a-Tocopherol 77.02"0.39 (b) 85.08"0.16 (c) 90.30"0.13 (b) 94.61"0.04 (a) Values are means"SD (ns3). Significant differences determined by the Tukey HSD test (p-0.05) are indicated by different letters (a–n).

Table 3 Antioxidant activities as % inhibition in red algal crude extracts at different concentrations (50, 100, 200, and 500 mg l-1) assayed by the b-carotene-linoleic acid system.

50 mg l-1 100 mg l-1 200 mg l-1 500 mg l-1 Aglaothamnion pseudobyssoides 18.04"1.12 (d) 31.53"1.00 (f) 39.90"0.83 (e) 41.89"0.58 (j) Asparagopsis armata 8.02"1.19 (h–j) 7.95"1.33 (l, m) 5.49"0.74 (p) 4.92"0.83 (o) Brongniartella byssoides 75.90"0.74 (c) 83.86"1.42 (b, c) 89.33"1.03 (b) 91.40"0.51 (b) Calliblepharis jubata 15.01"0.61 (d–f) 28.11"1.57 (f–h) 39.73"1.21 (e) 47.45"1.11 (f–h) Callithamnion tetragonum 16.83"1.03 (d) 30.35"1.32 (f, g) 38.24"1.08 (e, f) 47.40"1.99 (f–h) Callophyllis laciniata 7.84"0.70 (h–j) 16.00"0.49 (j) 22.48"0.88 (j, k) 28.48"0.80 (m) Caulacanthus ustulatus 8.80"0.74 (h, i) 26.40"0.79 (g, h) 38.94"0.97 (e, f) 51.18"0.78 (e, f) Ceramium ciliatum 12.18"0.78 (f, g) 25.82"1.76 (h) 36.02"0.41 (f, g) 46.22"1.44 (g–i) Cystoclonium purpureum 8.15"1.01 (h–j) 16.00"0.58 (j) 18.49"0.56 (l, m) 20.45"0.77 (n) Dilsea carnosa 9.43"0.27 (g–i) 20.98"1.51 (i) 31.77"1.19 (h, i) 42.99"0.37 (i, j) Dumontia contorta 8.61"0.49 (h–j) 15.87"0.33 (j) 31.81"0.34 (h, i) 49.76"0.61 (f, g) Furcellaria fastigiata 6.00"1.01 (j, k) 6.91"0.32 (l, m) 15.39"0.40 (n, o) 33.33"0.57 (k) Gastroclonium ovatum 7.26"1.25 (i, j) 18.81"0.97 (i, j) 33.96"2.08 (g, h) 45.09"1.26 (h–j) Gracilaria gracilis 4.37"0.12 (k) 7.31"0.14 (l, m) 18.31"0.45 (l–n) 33.16"1.41 (k, l) Grateloupia filicina 7.08"0.02 (i, j) 12.36"1.15 (k) 24.44"0.30 (j) 33.96"2.69 (k) Heterosiphonia plumosa 16.28"0.18 (d, e) 26.48"0.98 (g, h) 34.23"0.49 (g, h) 42.13"1.07 (i, j) Kallymenia reniformis 1.93"0.88 (l) 5.61"0.77 (m) 15.91"0.59 (m–o) 20.60"0.83 (n) Lomentaria articulata 10.33"1.80 (g, h) 17.34"2.68 (i, j) 20.25"1.80 (k, l) 27.26"1.58 (m) Lomentaria clavellosa 12.66"1.15 (e–g) 20.13"1.26 (i) 24.80"1.31 (j) 34.05"2.82 (k) Plocamium cartilagineum 5.97"0.63 (j, k) 16.26"0.25 (j) 22.08"0.90 (j, k) 29.05"0.67 (l, m) Polyneura bonnemaisonii 6.13"0.42 (i–k) 9.44"0.45 (k, l) 14.58"0.18 (o) 20.70"1.71 (n) Polysiphonia lanosa 24.08"0.90 (c) 47.92"1.34 (d) 61.16"0.49 (c) 66.75"0.72 (c) Porphyra leucosticta 18.60"0.25 (d) 37.48"1.38 (e) 51.09"0.27 (d) 61.45"0.30 (d) Porphyra purpurea 0.59"0.15 (m) 6.46"0.37 (m) 30.52"1.08 (i) 54.16"1.12 (e) BHA 82.40"1.92 (b) 87.18"1.19 (a, b) 87.45"1.20 (b) 90.45"1.55 (b) BHT 80.44"1.41 (b) 80.70"1.76 (c) 87.41"0.88 (b) 91.06"1.88 (b) a-Tocopherol 89.01"1.39 (a) 90.16"0.18 (a) 93.42"0.59 (a) 97.20"0.54 (a) Values are means"SD (ns3). Significant differences determined by the Tukey HSD test (p-0.05) are indicated by different letters (a–p). Article in press - uncorrected proof

M. Zubia et al.: Biological activities of some red algae 273

plumosa (4.03"0.06 mg ml-1) provided the next most Table 4 Total phenolic content of red algal crude extracts " active extracts (Figure 1). (means SD; ns3) expressed in % of dry weight (% dw). The reducing activity assay confirmed the results obtained by the DPPH assay. Table 2 shows that the Species % dw Brongniartella byssoides extract had the highest reducing Aglaothamnion pseudobyssoides 5.73"0.04 activity (94.80"0.08% at 500 mg l-1) among the species, Asparagopsis armata 1.13"0.05 " and its activity elicited a typical dose response. More- Brongniartella byssoides 3.45 0.01 " over, statistical analysis showed that at 500 mg ml-1, Calliblepharis jubata 1.74 0.03 Callithamnion tetragonum 1.85"0.03 reducing activity of this extract was equivalent to the Callophyllis laciniata 2.77"0.10 reducing activities of any commercial antioxidants tested Caulacanthus ustulatus 2.77"0.09 and to that of a-tocopherol at lower concentrations. The Ceramium ciliatum 1.82"0.03 crude extracts from Polysiphonia lanosa, Aglaothamnion Cystoclonium purpureum 0.89"0.03 pseudobyssoides, Furcellaria fastigiata, and Heterosipho- Dilsea carnosa 1.89"0.07 " nia plumosa also had high reducing activities (80.88" Dumontia contorta 0.56 0.01 " 0.72%, 79.42"0.15%, 75.19"0.79%, and 75.15"0.31% Furcellaria fastigiata 3.25 0.02 Gastroclonium ovatum 0.73"0.03 -1 at 500 mg l , respectively), and this finding is in agree- Gracilaria gracilis 2.11"0.06 ment with the DPPH assay results. The correlation Grateloupia filicina 2.51"0.11 between DPPH radical-scavenging activities and the Heterosiphonia plumosa 3.29"0.03 reducing activities measured in red algae extracts was Kallymenia reniformis 1.65"0.01 found to be significant (rs-0.843). Lomentaria articulata 1.07"0.01 " Table 3 shows that the strong antioxidant activity of Lomentaria clavellosa 1.85 0.03 Plocamium cartilagineum 3.30"0.09 Brongniartella byssoides extract obtained in the previous Polyneura bonnemaisonii 0.49"0.01 assays was confirmed by the b-carotene-linoleic acid Polysiphonia lanosa 3.64"0.06 system assay, with this species having the highest per- Porphyra leucosticta 2.05"0.01 cent inhibition of b-carotene bleaching (91.40"0.51% at Porphyra purpurea 1.32"0.06 500 mg l-1). The antioxidant activity of B. byssoides extract was not significantly different from those of BHA and BHT at concentrations of 500, 200, and 100 mg l-1. Cytotoxic activity In the case of Polysiphonia lanosa, our finding of a high The cytotoxic activities of the crude extracts were tested antioxidant activity (66.75"0.72% at 500 mg l-1) is con- against three cancer cell lines (Daudi, Jurkat, and K562). sistent with the results of the DPPH and reducing activity Figure 2 illustrates the results of the tests and highlights assays. On the other hand, for Aglaothamnion pseudo- five crude extracts with significant cytotoxic activities byssoides, Furcellaria fastigiata, and Heterosiphonia plu- against Daudi cell lines (p-0.01): Asparagopsis armata mosa extracts, the results of the b-carotene-linoleic acid (32.52"7.33%), Brongniartella byssoides (57.92" system assay were not in full agreement with those from 12.65%), Heterosiphonia plumosa (60.89"10.89%), Plo- the two other assays; the correlations between DPPH camium cartilagineum (63.49"14.64%), and Ceramium radical-scavenging activities, or reducing activities, and ciliatum (83.91"4.92%). Jurkat cell viability was signifi- the antioxidant activities measured with the b-carotene- cantly decreased in treatments with extracts from A. linoleic acid system (rs-0.518 and rs0.596, respectively) armata (30.07"7.24%, ps0.0000), B. byssoides were rather low, although they were significant. However, (38.31"8.35%, ps0.0000), H. plumosa (47.60"18.24%, with this method, the antioxidant activities in the crude ps0.0009), and Calliblepharis jubata (76.74"12.74%, extracts (500 mg l-1)fromPorphyra species were quite ps0.0066). For K562 cell lines, only the B. byssoides high (61.45"0.30% and 54.16"1.12%). extract had significant cytotoxic activity (77.35"9.08%, ps0.0016). Total phenolic content No significant correlation was found between phenolic Table 4 shows that the total phenolic contents of the content and cytotoxic activities in the red algae under seaweed extracts varied from 0.49% dw (Polyneura study for all cancer cell lines (rs0.130, rs0.128, and bonnemaisonii) to 5.73% dw (Aglaothamnion pseudobys- rs0.079 for Daudi, Jurkat, and K562 cell lines, soides). It is worth noting that the extracts with the high- respectively). est antioxidant activities measured with the DPPH and reducing activity assays, i.e., A. pseudobyssoides, Poly- siphonia lanosa, Brongniartella byssoides, Heterosipho- Discussion nia plumosa, and Furcellaria fastigiata, were also the richest in phenolic compounds (5.73"0.04% dw, The antioxidative activities of red algal crude extracts 3.64"0.06% dw, 3.45"0.01% dw, 3.29"0.03% dw, were characterized by three complementary biochemical 3.25"0.02% dw, respectively). Indeed, significant corre- methods to overcome the inability of a simple one- lations were found between the total phenolic contents dimensional test of antioxidant capacity to accurately and the DPPH radical-scavenging activities (rs-0.645) mirror the complex in vivo interactions between antioxi- and the reducing activities (rs0.665) measured in the dants (Frankel and Meyer 2000, Huang et al. 2005). extracts. In contrast, there was no significant correlation DPPH, reducing activity, and b-carotene-linoleic acid between phenolic contents and antioxidant activities system assays are known to be simple, fast, and reliable, measured by the b-carotene assay (rs0.333). and they proved their relevance here in assessment of Article in press - uncorrected proof

274 M. Zubia et al.: Biological activities of some red algae

Figure 2 Percentage cell viability following treatment and in a control (means"SD, ns6). Three different cancer cell lines (Daudi, Jurkat, and K562) were exposed for 24 h to the red algal crude extracts (a key to the red algal extract numbers is given in Table 1) and to the control (DMSO, 100 mgml-1). * Indicates significant differences (p-0.01) compared to the control. the total antioxidant activity of the extracts. The finding of transition metal ion catalysts, decomposition of per- of a significant correlation between the DPPH radical- oxides) in addition to free radical trapping (Frankel and scavenging activities and the reducing activities dis- Meyer 2000). played by the red algal extracts under study is explained Among the species under study, Brongniartella bys- by the fact that both assays rely on electron/hydrogen soides had the highest antioxidant activity in all assays, donation. On the other hand, the emulsified lipid used in and its antioxidant activity was equivalent to commercial the b-carotene assay introduced additional variables that antioxidant compounds tested in this experiment: ascor- affect the oxidation process in comparison to the other bic acid, a-tocopherol, BHA and BHT. To the best of our methods. In the more complex b-carotene system, anti- knowledge, this study is the first report to provide evi- oxidant molecules may inhibit lipid oxidation by several dence of high antioxidant activity in B. byssoides. mechanisms (e.g., prevention of chain initiation, binding Although this species belongs to the order Ceramiales, Article in press - uncorrected proof

M. Zubia et al.: Biological activities of some red algae 275

which is the subject of extensive studies due to its huge extract (3.25% dw) suggests that phenolic compounds variety of bioactive compounds (mainly halogenated, may be responsible for the antioxidant activity. Indeed, e.g., terpenoids, bromophenols, acetogenins; Korn- some bioactive halogenated phloroglucinols and nitro- probst 2005), literature data are scarce. In this study, genous derivative compounds have been previously extracts of four species in the order Ceramiales, i.e., identified in carrageenophyte species, such as Furcellaria Aglaothamnion pseudobyssoides, B. byssoides, Hetero- (Kornprobst 2005). There is a lack of available literature siphonia plumosa, and Polysiphonia lanosa, were among on its bioactivity, but our preliminary result would encour- the five most active. This finding constitutes additional age additional investigations on this species because it evidence for the bioactive potential of this order. More- is easily cultivated. Sufficient amounts of cultured algae over, it is worth noting that both B. byssoides and P. lanosa with similar biochemical and chemical characteristics belong to the family Rhodomelaceae. Numerous chemi- could be provided for purification and isolation of the cal investigations have led to the isolation of an impres- bioactive molecules, and the traceability could be sive array of bioactive compounds in this family, ensured. especially halogenated terpenoids in the genus Laurencia With the b-carotene system assay, Porphyra leucostic- (Kornprobst 2005). Few studies have evidenced a ta and Porphyra purpurea extracts showed some specific marked antioxidant activity in Polysiphonia extracts (Fuji- antioxidant activity. As Porphyra, commonly known as moto 1990, Yan et al. 1998, Dummermuth et al. 2003), nori or laver, is widely used in Asia as part of the human and some bromophenols isolated from Polysiphonia diet, it has been the subject of numerous studies per- urceolata have been identified as the antioxidant mole- formed in order to identify its functional properties. Sev- cules (Fujimoto 1990, Duan et al. 2006, Li et al. 2007, eral antioxidant molecules have been identified in the 2008). Bromophenols from P. lanosa have also demon- genus Porphyra: histidine-related compounds (Tamura et strated antimicrobial (Glombitza et al. 1985, Fariq 1991) al. 1998), chlorophyll analogs and mycosporine-like ami- and cytotoxic (Shoeib et al. 2004) activities. These no acids (Nakayama et al. 1999), sulfated polysaccha- results, as well as the finding here of a high phenolic rides (Zhang et al. 2003, 2004) and oligosaccharides (Wu content in B. byssoides and P. lanosa extracts, suggest and Pan 2004). It has been suggested that the accu- that bromophenols might be responsible for the antioxi- mulation of the UV-absorbing mycosporine-like amino dant activity of these extracts. acids, such as porphyra-344, provides a photoprotection Aglaothamnion pseudobyssoides and Heterosiphonia to P. leucosticta, and thus these compounds may func- plumosa also proved to be active, but there is a lack of tion as biological antioxidants (Dunlap and Yamamoto published information about their antioxidant capacities. 1995, Korbee et al. 2005). Our study suggests that the antioxidant mechanisms at This study highlighted the strong cytotoxicity of crude play in these extracts rely mainly on the trapping of a free extracts from Asparagopsis armata, Brongniartella bys- soides, and Heterosiphonia plumosa against the human radical, because the strong antioxidant activity exhibited cancer cell lines Daudi and Jurkat. This is the first report by these extracts was not confirmed by the b-carotene of cytotoxic activities for these red macroalgae. The assay. This result might also be explained by the inter- literature contains reports of antimicrobial activity of actions (synergistic, additive or antagonistic effects) halogenated metabolites produced by A. armata (Korn- between the emulsified medium used in the b-carotene probst 2005), but until now there has been no evidence assay and the complex composition of the crude of its cytotoxic activity. This screening has also confirmed extracts. The high phenolic contents measured in A. the great bioactivity of species in the Ceramiales, espe- pseudobyssoides (5.73% dw) and H. plumosa (3.29% cially B. byssoides, which had cytotoxic activities against dw) extracts could partly explain their high radical-scav- all human cancer cell lines tested. Numerous cytotoxic enging activity. This finding is in agreement with a pre- compounds have been isolated previously from species vious study (Zubia et al. 2007), which showed that of Ceramiales, especially from the genus Laurencia (e.g., marked DPPH radical-scavenging activity correlated with Pec et al. 2003, Mohammed et al. 2004, Sun et al. 2005), high phenolic content in a Heterosiphonia extract. In fact, and a promising anticancer drug (dehydrothyrsiferol) phenolic compounds are thought to protect the algal extracted from Laurencia viridis has been developed to thallus against photodestruction by UV radiation and to the preclinical phase (Pec et al. 2003). Phenolic com- have radical-scavenging properties (Connan et al. 2006). pounds, such as bromophenols, have also been identi- Numerous studies have demonstrated a highly significant fied as the cytotoxic molecules in Rhodomela confer- correlation between phenolic content and antioxidant voides (Hudson) P.C. Silva (Xu et al. 2004), but no signif- activity in seaweed extracts (e.g., Kim et al. 2005, Zhang icant correlation was found in this study between cyto- s et al. 2007), as confirmed in this study (r -0.645 and toxic activities and phenolic content. The ability of 0.665). However, in this study, the highest antioxidant phenols to protect cells from oxidative stress has been activity found in Brongniartella byssoides extract did not demonstrated, but these compounds have a contradic- correspond to the highest phenolic content (found in A. tory behavior characterized by anti- and protumoral pseudobyssoides extract). A possible explanation may be activities according to their chemical structure, the sys- that there are other types of antioxidant molecules struc- tem and conditions used in the study (Gomes et al. turally different from phenolic compounds in red algae 2003). Further purifications are required to isolate and (e.g., carotenoids, terpenoids, ascorbic acid). identify the cytotoxic molecules in A. armata, B. bys- This study also highlighted marked antioxidant activity soides, and H. plumosa extracts. in Furcellaria fastigiata, which belongs to the order Gigar- The finding of a positive correlation between cytotoxic tinales. The high phenolic content in the F. fastigiata effects and the strong DPPH radical-scavenging activity Article in press - uncorrected proof

276 M. Zubia et al.: Biological activities of some red algae

in Brongniartella byssoides extract makes this species a Ce´ rantola, S., F. Breton, E. Ar Gall and E. Deslandes. 2006. Co- promising candidate for further investigations. Oxidative occurrence and antioxidant activities of fucol and fucophlor- stress is known to be implicated in the process of car- ethol classes of polymeric phenols in spiralis. Bot. Mar. 49: 347–351. cinogenesis through damage to cellular molecules, such Connan, S. 2004. Etude de la diversite´ spe´ cifique des as proteins, lipids and nucleic acids. Hence, the preva- macroalgues de la Pointe de Bretagne et analyse des lence of both antioxidant and cytotoxic properties in a compose´ s phe´ noliques des Phe´ ophyce´ es dominantes. PhD single compound could be beneficial in terms of rational, thesis, University of Western Brittany, Brest, France. pp. 280. preventive or therapeutic purposes. However, additional Connan, S., F. Delisle, E. Deslandes and E. Ar Gall. 2006. Intra- studies are needed to demonstrate that this extract thallus phlorotannin content and antioxidant activity in exhibits no cytotoxicity towards normal cells. Detailed Phaeophyceae of temperate waters. Bot. Mar. 49: 39–46. Connan, S., E. Deslandes and E. Ar Gall. 2007. Influence of day- examination of the mechanism of action of the isolated night and tidal cycles on phenol content and antioxidant and purified compounds of B. byssoides extract should capacity in three temperate intertidal brown seaweeds. J. be investigated, e.g., their impact on cell cycle, their abil- Exp. Mar. Biol. Ecol. 349: 359–369. ity to activate caspases or induce mitochondrial and DNA Dizerbo, A.H. and H. Herpe´ . 2003. Liste et re´ partition des algues damages. marines des coˆ tes francaises¸ de la Manche et de l’Atlantique, ıˆles Anglo-Normandes incluses. Anaximandre, Landerneau. pp. 315. Duan, X.J., W.W. Zhang, X.M. Li and B.G. Wang. 2006. Evalua- Conclusion tion of antioxidant property of extract and fractions obtained from a red alga Polysiphonia urceolata. Food Chem. 95: This study constitutes the largest screening of antioxi- 37–43. dant and cytotoxic activities in red macroalgae from the Dummermuth, A.L., U. Karsten, K.M. Fisch, G.M. Ko¨ nig and C. Brittany coasts to date. The results clearly indicate that Wiencke. 2003. Responses of marine macroalgae to hydro- extracts of the 24 species of red algae tested possess gen-peroxide stress. J. Exp. Mar. Biol. Ecol. 289: 103–121. Dunlap, W.C. and Y. Yamamoto. 1995. Small-molecule antioxi- antioxidant activity to varying degrees. This screening dants in marine organisms: antioxidant activity of mycospor- emphasized the great antioxidant potential of Brongniar- ine-glycine. Comp. Biochem. Physiol. 112B(1): 105–114. tella byssoides, which was found to be equivalent to the Egorin, M.J., D.M. Rosen, S.E. Benjamin, P.S. Callery, D.L. Sentz antioxidant activity of commercial antioxidant molecules and J.L. Eiseman. 1997. In vitro metabolism by mouse and assessed in the same study. Moreover, three other Cera- human liver preparations of halomon, an antitumor haloge- miales species (Aglaothamnion pseudobyssoides, Hete- nated monoterpene. Cancer Chemother. Pharmacol. 41: rosiphonia plumosa, and Polysiphonia lanosa) had high 9–14. Fariq, V.N. 1991. Antifungal activity in crude extracts of marine antioxidant activities, as did one member of the Gigarti- red algae. Mycol. Res. 95: 1433–1440. nales (Furcellaria fastigiata) and two species of Bangiales Frankel, E.N. and A.S. Meyer. 2000. The problems of using one- (Porphyra leucosticta and Porphyra purpurea). The cor- dimensional methods to evaluate multifunctional food and relation usually found between marked radical-scaveng- biological antioxidants. J. Sci. Food Agric. 80: 1925–1941. ing activities and a high total phenolic content supports Fujimoto, K. 1990. 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