Antioxidant and Cytotoxic Activities of Some Red Algae (Rhodophyta) from Brittany Coasts (France)
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Article in press - uncorrected proof Botanica Marina 52 (2009): 268–277 ᮊ 2009 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BOT.2009.037 Antioxidant and cytotoxic activities of some red algae (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 (Aglaothamnion pseudobyssoides, cytotoxic activities (see reviews in Gu¨ ven et al. 1990, Furcellaria fastigiata, Polysiphonia 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 Brongniartella 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 Laurencia 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 red algae 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 2009/70 Article in press - uncorrected proof 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 (Sunriseீ, 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 Ceramiales Ceramiaceae Callot June 2007 H.M. Crouan) Halos 2 Asparagopsis armata Harvey Bonnemaisoniales Bonnemaisoniaceae Anse de Melon March 2007 3 Brongniartella byssoides (Goodenough et Ceramiales Rhodomelaceae Callot June 2007 Woodward) F. Schmitz 4 Calliblepharis jubata (Goodenough et Woodward) Gigartinales 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 Ceramium ciliatum (J. Ellis) Ducluzeau Ceramiales Ceramiaceae Anse de Melon May 2007 9 Cystoclonium purpureum (Hudson) Batters Gigartinales Cystocloniaceae Anse de Melon May 2007 10 Dilsea carnosa (Schmidel) Kuntze Gigartinales Dumontiaceae Bertheaume July 2007 11 Dumontia 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