Carrageenans from Nuclear Phases of Subantartic Mazzaella Laminarioides
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J Appl Phycol DOI 10.1007/s10811-015-0641-2 Carrageenans from nuclear phases of subantartic Mazzaella laminarioides (Gigartinales, Rhodophyta) and graft copolymerization of alkali-modified carrageenan with acrylamide 1 3,4 1 2 1 Franco Arias & Andrés Mansilla & Betty Matsuhiro & Jorge Pavez & Rodrigo Torres & Mauricio Yáñez-Sánchez1 Received: 5 March 2015 /Revised and accepted: 1 June 2015 # Springer Science+Business Media Dordrecht 2015 Abstract Aqueous extraction of cystocarpic, tetrasporic, and Keywords Carrageenans .Mazzaellalaminarioides .Nuclear vegetative individuals of Mazzaella laminariodes collected in phases . Copolymerization the Magellan Ecoregion, Southern Chile, afforded sulfated galactans. Analysis by Fourier transform infrared spectros- copy (FT-IR) and by 1H and 13C NMR spectroscopy indicated Introduction that purified extracts from cystocarpic (CP) and vegetative individuals (VP) were mixtures of κ/ι-type carrageenans and Carrageenans are a family of polysaccharides extracted with their precursors μ- and ν-carrageenans, whereas the aqueous hot water from several species of red algae. They are com- extract from tetrasporic individuals was a λ-type carrageenan. posed of alternating 3-linked β-D-galactopyranosyl (unit G) Alkaline treatment of CP and VP polysaccharides produced and 4-linked α-D-galactopyranosyl (unit D) or 3,6- modified polysaccharides with lower content of sulfate groups anhydro-α-D-galactopyranosyl (unit DA) residues, substitut- and with a concomitant increase of 3,6-anhydrogalactose con- ed by sulfate groups. Members of this family differ in 3,6- tent; analysis by 2D 1H and 13C NMR indicated the presence anhydro-D-galactose content and the substitution pattern of of hybrid κ-ι-carrageenans. Copolymerization of alkali- sulfate groups (Rees 1963; Dolan and Rees 1965; Anderson treated CP carrageenan with acrylamide was achieved by et al. 1968, 1973; Penman and Rees 1973; Painter 1983; Usov using ceric ammonium nitrate as the initiator; studies by 1998; 2011; Lahaye 2001). Different carrageenans are desig- NMR spectroscopy indicated grafting of polyacrylamide at nated by Greek letters according to Knutsen et al. (1994). The position C-6 of β-galactopyranosyl residues. Reaction of most commercially important carrageenans are the gelling modified CP with acrylamide assisted by microwave irradia- κ-(G4S-DA) and ι-(G4S-DA2S) carrageenans, and the non- tion in the presence of ammonium persulfate afforded a co- gelling λ-carrageenan (G2S-D2S,6S) (Prajapati et al. 2014). polymer grafted at position C-2 of β-galactopyranosyl Waaland (1975), McCandless et al. (1975, 1983), and Doty residues. and Santos (1978) established that different generations of members of the Gigartinaceae produce different types of car- rageenan; according to the authors, tetrasporic plants produce * Betty Matsuhiro the λ-type carrageenan, whereas the gametophytic plants pro- [email protected] duce κ-type carrageenans. However, chemical and spectro- scopic studies of the polysaccharides extracted from different 1 Departamento de Ciencias del Ambiente, Universidad de Santiago de nuclear phases of carrageenophytes indicated the presence of Chile, Av. L. B. O’Higgins 3363, Santiago, Chile complex mixtures of carrageenans (Ayal and Matsuhiro 1987; 2 Departamento de Química de Los Materiales, Facultad de Química y Matulewicz et al. 1989; Matsuhiro and Urzúa 1992; Falshaw Biología, Universidad de Santiago de Chile, Av. L. B. O’Higgins and Furneaux 1994, 1995; Usov 2011). 3363, Santiago, Chile Carrageenans are widely used in food and pharmaceutical 3 Laboratorio de Macroalgas Antárticas y Subantárticas, Universidad industries due to their physicochemical properties, such as de Magallanes, Av. Bulnes 1465, Punta Arenas, Chile gelling, thickening, emulsifying, and stabilizing properties 4 Instituto de Ecología y Biodiversidad, Santiago, Chile (Campo et al. 2009). Carrageenans also present interesting J Appl Phycol biological properties, including antioxidant, anticoagulant, an- D2O, after isotopic exchange (3×0.75 mL) at 70 °C on a Bruker titumor, and immunomodulatory activities (Campo et al. Avance DRX 400 spectrometer (Bruker, UK) using the sodium 2009; Wijesekara et al. 2011; Barahona et al. 2011, 2012; salt of 3-(trimethylsilyl)-1-propionic-2,2,3,3-d4 acid as internal De Araujo et al. 2013; Prajapati et al. 2014). Recently, studies reference. The 2D experiments were performed using a pulse on the application of carrageenans in drug delivery systems field gradient incorporated into an NMR pulse sequence. The have gained much interest (Nanaki et al. 2010; Rana et al. number of scans in each experiment was dependent on the 2011; Li et al. 2013, 2014). On the other hand, copolymeriza- sample concentration (30–45 mg mL−1). Gas–liquid chroma- tion of carrageenans with vinyl monomers produces graft co- tography (GLC) was performed on a Shimadzu GC-14B chro- polymers with new properties and applications (Prasad et al. matograph (Shimadzu, Japan) equipped with a flame ionization 2006; Meena et al. 2006; Pourjavadi et al. 2008; Hosseinzadeh detector using a SP 2330 column (0.25 mm×30 m) and carried 2009; Mishra et al. 2010; Sand et al. 2012; Yadav et al. 2012; out with an initial 5 min hold at 150 °C and then at 5 °C min−1 Verma et al. 2014). to 210 °C for 10 min. The helium flow was 20 mL min−1. Mazzaella laminarioides (Gigartinales, Rhodophyta) was Nitrogen content was determinedbymicroanalysisinFacultad formerly placed in the genus Iridaea and was transferred to de Química, Universicdad Católica de Chile, Chile. Mazzaella by Hommersand et al. (1993, 1994). The first study on the characterization of sulfated galactans from unsorted Extraction and purification The dried ground seaweed Iridaea laminarioides collected in central Chilean coast (32° (100 g) was heated with distilled water (2 L) at 85 °C for 58′ 00″ S, 71° 29′ 00″ W) was performed in 1976; fraction- 3 h, cooled to 45 °C, and centrifuged at 3500×g. The super- ation of the aqueous extract using increasing concentration of natant was dialyzed against distilled water for 48 h using KCl showed the presence of a mixture of sulfated galactans Spectra/Por membrane (MWCO 3500), concentrated in vacuo (Matsuhiro and Zanlungo 1976). More recently, Jouanneau to ~200 mL and poured over 1 L of ethyl alcohol. The precip- et al. (2011)conductedtheenzymaticdegradationof itate was separated by centrifugation, dissolved in the mini- phycocolloids from commercial unsorted samples of Chilean mum volume of distilled water, and poured over five times its M. laminarioides; they reported the presence of κ- and ι-car- volume of 2-propanol. After 24 h, the precipitate was separat- rageenan, and their precursors μ- and ν-carrageenan in the ed by centrifugation, dissolved in distilled water, and freeze- same galactan chain indicating the hybrid nature of the carra- dried. The residue of the extraction was re-extracted twice geenan systems. M. laminarioides grows abundantly in the with water in the same conditions. Magellan Ecoregion, southernmost Chile, and it is not exploited for industrial purposes (Mansilla et al. 2012). The Chemical analysis Total sugars were determined by the aim of this work is the structural characterization of the phenol-sulfuric acid method using D-galactose as standard phycocolloids from two nuclear phases and vegetative indi- (Chaplin 1986). The content of 3,6-anhydrogalactose was de- viduals of M. laminarioides collected in the Magellan termined according to Matsuhiro and Zanlungo (1983) using Ecoregion and the preparation of a glycocopolymers by con- D-fructose as standard (Matsuhiro et al. 2014). The amount of jugation of a modified κ-ι-carrageenan with acrylamide. sulfate was determined by a modification of Dodgson and Price method (Barahona et al. 2012). Materials and methods Total hydrolysis The polysaccharide (0.005 g) was heated with 0.75 mL of 2 M trifluoroacetic acid for 2 h at Tetrasporic, cystocarpic, and vegetative plants of 120 °C, and the acid was removed by co-evaporations M. laminarioides (Bory de Saint-Vicent) Fredericq were col- with distilled water. The product of hydrolysis was dis- lected in Fuerte Bulnes (53° 37′ S, 70° 55′ W), Magellan solved in 2 mL of distilled water, stirred with sodium Ecoregion. Specimens were deposited in Herbario de borohydride (0.0012 g) for 2 h, acidified with Amberlite Criptógamos, Universidad de Magallanes, Punta Arenas, Chile. IR-120 resin, and filtered. The filtrate was concentrated in Analytical grade solvents were from Merck (Germany); reagent vacuo, and the white solid was removed by repeated co- grade chemicals were from Sigma (USA). Absorbance was evaporations with methyl alcohol. The resulting syrup measured in a Genesys 5 Thermospectronic spectrophotometer was acetylated with acetic anhydride in dry pyridine and (Thermo Fisher Scientific, USA). Fourier transform infrared analyzed by GLC. Per-O-acetates of D-galactitol, D- spectroscopy (FT-IR) spectra in KBr pellets of seaweeds and glucitol, D-mannitol, L-rhamnitol, L-fucitol, and D- polysaccharides were sampled in the 4000–400 cm−1 region xylitol were used as standards. using a Bruker IFS 66v instrument (Bruker, UK) according to Leal et al. (2008); all the samples were previously milled under Alkaline treatment Native polysaccharides from vegetative liquid nitrogen and dried for 8 h in vacuo at 56 °C. 1HNMR and cystocarpic plants were alkali modified according to the (400.13 MHz) and 13C(100.62MHz)spectrawererecordedin procedure published by Ciancia et al. (1993).