Carotenoid Extraction from the Gonad of the Scallop Nodipecten Nodosus
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Carotenoid extraction from the gonad of the scallop Nodipecten nodosus (Linnaeus, 1758) (Bivalvia: Pectinidae) Suhnel, S.a*, Lagreze, F.a, Ferreira, JF.a Campestrini, LH.b and Maraschin, M.b* aLaboratório de Moluscos Marinhos, Universidade Federal de Santa Catarina – UFSC Servidão dos Coroas, s/n, Barra da Lagoa, CEP 88061-600, Florianópolis, SC, Brazil bLaboratório de Morfogênese e Bioquímica Vegetal, Universidade Federal de Santa Catarina – UFSC Rod. Admar Gonzaga, 1346, Itacorubi, CEP 88040-900, Florianópolis, SC, Brazil *e-mail: [email protected], [email protected] Received November 14, 2007 – Accepted March 12, 2008 – Distributed February 28, 2009 (With 2 figures) Abstract In marine bivalve mollusks, unsaturated molecules called carotenoids are present in the natural diet and play an important role in different biological process, especially in reproduction. In order to gain more insights into these compounds in Nodipecten nodosus it was necessary to develop a suitable protocol for extraction of carotenoids from the gonads. Female gonads of cultured scallops (75 mm length) were lyophilized and macerated in liquid N2. To verify the effect of composition in organosolvents on the extracting solutions, two organic solvents were tested: acetone and hexane (Ac = O:Hex) at four ratios, 1:1, 1:3, 1:5, and 2:3, in four static extraction times: 0, 5, 10, and 15 minutes. Total carotenoids and astaxanthin contents were determined in the crude extracts by UV-visible spectrophotometry and high performance liquid chromatography (HPLC), respectively. Triplicate aliquots of 50 mg were used for each treatment. The results indicated that the best single extraction (0.312 ± 0.016 µg carotenoids/mg) was attained with Ac = O: Hex 1:3, for 15 minutes. Through exhaustive extraction methodology (10x), a superior yield (0.41 ± 0.001 µg carotenoids/mg) was obtained from a gonad sample in comparison to the highest value found for a single extraction. Astaxanthin content was reduced by 8.6% in carotenoid extract preservation assay, i.e., –18 °C, 26 days incubation, under N2 atmosphere. Keywords: carotenoids, Nodipecten nodosus, extraction, organic solvents, scallop. Extração de carotenóides da gônada da vieira Nodipecten nodosus (Bivalvia: Pectinidae) Resumo Em moluscos bivalves marinhos, carotenóides insaturados estão presentes na dieta natural, com um importante pa- pel em diversos processos biológicos, em especial na reprodução. A elucidação dos efeitos destes compostos em Nodipecten nodosus requer o desenvolvimento de um protocolo adequado para a extração de carotenóides das gônadas desses animais. Para isso, gônadas de vieiras cultivadas (75 mm de comprimento) foram liofilizadas e maceradas em N2 líquido. Amostras em triplicata com 50 mg foram coletadas para a utilização em cada tratamento. Os conteúdos de carotenóides totais e astaxantina foram determinados via espectrofotometria de luz UV-visível e cromatografia líquida de alta eficiência (CLAE), respectivamente. O efeito da composição em organosolventes das soluções de extração foi testado utilizando-se acetona (Ac = O) e hexano (Hex) em quatro proporções (Ac = O:Hex): 1:1, 1:3, 1:5, e 2:3; em quatro tempos de extração: 0, 5, 10, e 15 minutos. Os resultados mostraram que o melhor rendimento de extração (0,312 ± 0,016 µg carotenóides/mg) foi obtido com Ac = O:Hex, 1:3, por 15 minutos. Com a utilização de protocolo de extração exaustiva (10x), uma quantidade superior (0,41 ± 0,001 µg de carotenóides/mg) foi obtida de amostras de gônada, comparativamente aos valores obtidos em extrações únicas. O conteúdo de astaxantina foi reduzido em 8,6% em testes de preservação deste metabólito em extratos crus (–18 °C, 26 dias de incubação em atmosfera de N2). Palavras-chave: carotenóides, Nodipecten nodosus, extração, solventes orgânicos, vieiras. 1. Introduction Scallops are bivalve mollusks of the Pectinidae In aquatic organisms, particularly in mollusks, caro- family that are economically important in aquaculture. tenoids are responsible for tissue pigmentation (Meyers, According to FAO (2005), scallop world production 1994). In addition, some carotenoids are vitamin A pre- reached 1.27 million metric tones in 2005, with China as cursors (Miki et al., 1982; Farías-Molina, 2001) and are the major producer (81% of the world production). fundamental to the growth and maturation of gonads and Braz. J. Biol., 69(1): 209-215, 2009 209 Suhnel, S. et al. by increasing fertilization. The fecundity rate plays an Carotenoids extracts should be stored in the dark under important role in embryonic development and hatching N2 atmosphere (Oliver and Palou, 2000). (Petit et al., 1991; Olson, 1993; Menasveta et al., 1994; The most commonly used techniques for caroten- Mantiri et al., 1995; Farías-Molina, 2001), optimizing oids detection are UV-visible spectrophotometry, mass larval feasibility and olfactory perception and chem- spectrometry and hydrogen or carbon nuclear magnetic oreception (Farías-Molina, 2001). They are also effec- resonance spectroscopy (1H-NMR, 13C-NMR), coupled tive photo-protectors against UV radiation and, due to or not with chromatographic techniques (Rodriguez- their anti-oxidant action (Miki, 1991; Miki et al., 1994; Amaya, 2001; Schoefs, 2002). Regardless of the tech- Farías-Molina, 2001), enhance tolerance to environ- nique utilized, carotenoid extraction is highly influenced mental stress, such as high concentrations of ammonia by procedural variables such as type of sample, type and or low oxygen concentrations (Tacon, 1981; Torrissen, ratio of solvents, time of extraction, storage conditions, 1984; Menasveta et al., 1995; Olson and Owens, 1998). etc. Thus, taking into account the possibility of usage of Furthermore, they also stimulate cellular growth and several analytical techniques, the comparison of results Ca2+ transportation through the cell membrane (Farías- should be done with precaution as they may not be pre- Molina 2001), and have also been recognized as immu- cise. nostimulant substances (Liñan-cabelo et al. 2002; Cuzon Miki et al. (1982) extracted carotenoids from the go- et al., 2004). nads of four species of scallops, i.e., Chlamys nipponensis Studies to evaluate the influence of carotenoids in akazara, Pecten albicans, Chlamys nobilis, and mollusk reproduction are strongly important, in special Patinopecten yessoensis. The procedure included extrac- in scallops where these compounds help the immunologi- tion with acetone, followed by drying the samples with cal system (Schleder, 2007), the reproductive maturation anhydrous sodium sulfate. The extract was concentrated and provide gametes with energy to the larvae develop- under low pressure (rotary evaporator), partitioned in a ment. Due to a disconnection between the natural repro- liquid-liquid chromatographic system (benzene:water, ductive cycle and the best period for hatchery production 1:1) and recovered in the organic fraction. Carotenoids of larvae and seeds, in our case, for Nodipecten nodosus, were determined by thin-layer chromatography using laboratory maturation is a fundamental factor to the suc- acetone:benzene (1:9) and dichloromethane:ethyl ac- cess of the commercial production, and carotenoids play etate (4:1) as mobile phases, sequentially. Pectenolone, an important role in this process. pectenoxanthin, pectenol, astaxanthin and tetrol were Carotenoids are a family of natural compounds identified in the carotenoid fraction. Quantitatively, the where the structural variations (chemo-diversity) are de- Chlamys nipponensis akazara scallop presented the scribed and characterized from bacteria, algae, fungi and highest concentration of these secondary metabolites. superior plants, and the photosynthesizing organisms are Escarria et al. (1989) extracted carotenoids from responsible for their biosynthesis (Rodriguez-Amaya, fresh Argopecten circularis scallop gonads with petrole- et al., 1984). um ether:acetone:water (6:3:1), followed by saponifica- Carotenoids are cyclic or noncyclic tetraterpenes tion of the extracts. Carotenoids determination was done with properties and activities in biological systems relat- by column chromatography (CC) and thin-layer chro- ed to their structures. In the biosynthesis pathway, acetyl- matography (TLC) on silica gel with chloroform:ethyl CoA is the precursor, and two intermediate compounds acetate (4:1) as the mobile phase. The results revealed are formed: mevalonic acid and its pyrophosphate form. lutein and astaxanthin as major carotenoids in the gonads Both originate from the basic structural unit of these of this species. metabolites, constituted by five-carbon (C5H8) isoprene Similarly, Viarengo et al. (1995) exhaustively ex- units (Rodriguez-Amaya, 2001). The isoprene units are tracted (5x) carotenoids from fresh tissue of gills and assembled “head-to-tail”, except in the central posi- digestive glands of Adamussium colbecki and Pecten tion of the resulting compound, which is “tail-to-tail”, jacobaeus with acetone and petroleum ether. Total carote- producing a symmetric structure with reversion of the noids were determined spectrophotometrically at 451 nm symmetry plan at the centre of the molecule (Rodriguez- wavelength. The carotenoid yields in gills (0.005 µg Amaya, 1984; Rodrigues-Amaya, 1997; Padula, 1999; carotenoids/mg) and digestive glands (0.813 µg carote- Rodriguez-Amaya, 2001). They are highly unsaturated noids/mg) were higher in Adamussium colbecki than in molecules which are subject to oxidation, as the polyene Pecten jacobaeus (gills: 0.262 µg carotenoids/mg,