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Carotenoid composition of three bloom-forming algae species
Carotenoid Composition of three Bloom-forming Algae Species J. Deli, S. Gonda, L.Z.S. Nagy, I. Szab´o, G. Guly´as-Fekete, A. Ag´ocs, K. Marton, G. Vasas PII: DOI: Reference:
S0963-9969(14)00325-1 doi: 10.1016/j.foodres.2014.05.020 FRIN 5262
To appear in:
Food Research International
Received date: Revised date: Accepted date:
21 February 2014 30 April 2014 3 May 2014
Please cite this article as: Deli, J., Gonda, S., Nagy, L.Z.S., Szab´ o, I., Guly´ as-Fekete, G., Ag´ocs, A., Marton, K. & Vasas, G., Carotenoid Composition of three Bloom-forming Algae Species, Food Research International (2014), doi: 10.1016/j.foodres.2014.05.020
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ACCEPTED MANUSCRIPT Carotenoid Composition of three Bloom-forming Algae Species
Department of Pharmacognosy, University of Pécs, Medical School, H-7624 Pécs, Hungary
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J. Delia,b*, S. Gondac, L. ZS. Nagyc, I. Szabób, G. Gulyás-Feketea, A. Agócsb, K. Martond, G. Vasasc
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Department of Biochemistry and Medical Chemistry, University of Pécs, Medical School, H-7624 Pécs, Hungary c
Institute of Bioanalysis, University of Pécs, Medical School, H-7624 Pécs, Hungary
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Department of Botany, Division of Pharmacognosy University of Debrecen, H-4032 Debrecen, Hungary
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e-mail: [email protected]
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*Corresponding author at: Department of Pharmacognosy, University of Pécs, Medical School, Pécs, Hungary. Tel: +36-72 503-650/28833, Fax: +36-72 503-650/28826
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Keywords: Microalgae, Carotenoids, HPLC, HPLC–MS
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ACCEPTED MANUSCRIPT Abstract
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Algal mass productions are widespread events throughout the world. Most research deals with the environmental impact, ecology and toxicity of these phenomena, but the algae are also promising sources of bioactive natural products, and also are potential food additives. In the current study, we aimed to characterize the carotenoid composition of three non-toxic algal isolates with distinct taxonomical position, namely Dunaliella salina, Euglena sanguinea and a Nostoc strain. The strains were screened for carotenoid composition with HPLC-UV-APCI-MS with comparison with authentic standards. Carotenoids were purified with open coloumn chromatography, and characterized by LC-UV-MS and NMR. All three species contained a high amount of carotenoids. The composition of the carotenoid pattern was somewhat different compared to literature data. The Dunaliella carotenoid fraction contained lutein (52.1% of total carotenoid), -carotene (13.1%), violaxanthin (11.5%) and neoxanthin (6.2%) as chief compounds. The chief carotenoids in the Nostoc strain was echinenone (34.9% ), while main components of the Euglena isolate were diatoxanthin (39.0%), lutein (23.7%), an unidentified carotenoid (9.6%) and -carotene (5.4%). Diatoxanthin is identified by NMR spectroscopy. The carotenoid patterns of the examined strains are somewhat different from the patterns described in the literature. This can be the results of genetic or environmental differences, or combinations thereof. Despite these differences, our study shows the potency of these algae in production of carotenoids, and possibly, usage as food additives.
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ACCEPTED MANUSCRIPT 1. Introduction
Overproduction of photoautotrophic organisms, like algae and cyanobacteria, are well-
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known phenomena that have been found in many types of fresh and marine habitats over the
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past few decades (Reynolds & Walsby, 1975). Near to the several unpleasant accompanying incidences with health and economic consequences, spectacular discoloration of the habitats
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was also detected. Many cyanobacterial and algal strains can produce unique metabolites with diverse chemistry and bioactivity which may cause the phenomenon (Paerl & Huisman, 2008).
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To survive in a competitive environment could be a strategy of algae, why have developed compounds in a significant level of structural-chemical diversity. Over 15,000 novel
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compounds have been chemically assignated (Cardozo, Guaratini, Barros, Falcao, Tonon, Lopes, Campos, Torres, Souza, Colepicolo & Pinto, 2007). This suggests that algae are a promising group to furnish novel biochemically active substances, in addition the current
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application of metabolites isolated from diverse classes of algae is increasing. The
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exploration of these metabolites and organisms for pharmaceutical purposes for human nutrition and for other utilities is justified (Garson, 1989).
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Although many algae species known in the world (according to some estimates 40,000 species), only a few of them are able to build typical appearance of mass (Reynolds & Walsby, 1975). This natural mass of algae, called algal-bloom, is a cheap and adequate natural resource for exploring bioactive metabolites.
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Cyanobacteria or blue-green algae are the largest group of photosynthetic prokaryotes that exist in large diversity and distribution in the world. They occur in almost every habitat on the earth. Their photosynthetic system is closely similar to eucaryotes because they have chlorophyll a and photosystem II, and carry out oxygenic photosynthesis (Paerl, 2013).
Nostoc, a genus of cyanobacteria, is one of the most widespread phototrophic bacteria. They are filamentous and heterocystous cyanobacteria, commonly observed in both aquatic and terrestrial habitats. Species of the genus Nostoc are among the most widespread of all nitrogen-fixing cyanobacteria. Communities of Nostoc commune, in particular, are prominent in those terrestrial limestone environments of tropical, polar, and temperate regions which are subject to extremes of water availability (Whitton, Donaldson & Potts, 1979).
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ACCEPTED MANUSCRIPT Dunaliella salina (D. salina) is a green unicellular microalga that shows remarkable degree of adaptation to a wide range of salt concentrations (from 0.02% to salt saturation, about 35%). Its halotolerant property allows Dunaliella species to survive and grow in many
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marine habitats, especially in concentrated saline lake such as the Pink Lake in Western
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Australia (D. salina), the Great Salt Lake in Utah (D. viridis, 1000-250000 cell ml-1), and the Dead Sea in Israel (D. parva, 8800-15000 cell ml-1). D. salina can also be found in South
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Europe, North Africa, salty evaporating ponds in Mexico and the Solar salty pond in Australia. The genus Dunaliella, includes about 30 species of which 25 are found in brackish water and 5 in freshwater (Melkonian & Preisig, 1984; Leonard & Caceres, 1993). The
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optimal conditions for carotenogenesis are those that limit growth and include exposure to high light intensities and other stress factors, especially nutrient deprivation.
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Dunaliella salina has 90% of β-carotene and 10% of other carotenoids. Carotenoids are made up of α-carotene and xanthophylls like lutein, zeaxanthin, and cryptoxanthins similar to the ones found in food and vegetables (Gouveia & Emphis, 2003). These xanthophylls have a
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widespread application in the pharmaceutical industry and cosmetics as well as in animal
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feed (Venkatesan, Swamy, Senthil, Bhaskar & Rengasamy, 2013). The microalga Dunaliella salina is one of the richest sources of natural -carotene, which is a lipid-soluble orange
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pigment that is used as a colorant in food and feed. Under standard growth conditions D. salina contains approximately 5–10 mg β-carotene per gram dry weight, which is similar to other green algae (Del Campo, García-Gonzáles & Guerrero, 2007). The β-carotene content can rise to as much as 10% of dry weight when D. salina is subjected
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to stress conditions such as high salinity, high light intensity, nutrient deprivation and extreme temperatures (Ben-Amotz, Katz & Avron, 1982; Ben-Amotz & Avron, 1983; Borowitzka, M.A., Borowitzka, L.J. & Kessly, 1990; Shaish, Avron, Pick & Ben-Amotz, 1993; Ben-Amotz, 1996; Krol, Maxwell & Huner, 1997; Kleinegris, Janssen, Brandenburg & Wijffels, 2009).
Euglena sanguinea is an ubiquitous algal species found in many shallow, eutrophic freshwater systems. This species of Euglenophyte is commonly cause surface bloom with intensive red color. The carotenoids of Euglenophyceae have been studied previously (Liaaen-Jensen, 1977; Liaaen-Jensen, 1978; Goodwin, 1980a; Rowan, 1989), and comprise -carotene, diatoxanthin, diadinoxanthin, heteroxanthin and neoxanthin. A quantitative carotenoid analysis of a natural see water bloom of Euglena sanguinea Ehrenberg (Grung &
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ACCEPTED MANUSCRIPT Liaaen-Jensen, 1993) revealed the presence of -carotene (1% of total carotenoids), monoesters of (3S)-adonirubin (3%), diesters of (3S,3'R)-adonixanthin (13%), diesters of (3S,3'S)-astaxanthin (75%), 19-monoester of (3R,3'R,6R)-loroxanthin (1%), (3R,3'R)-
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diatoxanthin (6%), diadinoxanthin (1%) and neoxanthin (trace).
More than 750 structurally defined carotenoids are reported from nature; land plants,
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algae, bacteria including cyanobacteria and photosynthetic bacteria, archaea, fungus and animals. The main goal of our study is to analyse and identify the carotenoid composition of three bloom-forming algal species and may draw attention to the importance of the natural
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algal-blooms as alternative sources of active substances like carotenoids.
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2. Materials and Methods 2.1 Chemicals
HPLC and analytical grade solvents were used. The authenthic samples were taken from
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our collection (-carotene, -carotene, -carotene 5,6-epoxide, -carotene 5,8-epoxide,
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lutein, zeaxanthin, neoxanthin, violaxanthin, antheraxanthin) and CaroteNature GmbH (-
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carotene, echinenone, canthaxanthin, diatoxanthin).
2.2 Collection and culture conditions The mass occurring cells of Dunaliella salina were observed in Lacul Băilor Cojocna
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(N46°44.90652'; E23°50.44080'), Cojocna (Kolozs). 1 L sample were collected for isolation of the strain caused discoloration of the water. The isolated Dunaliella salina was grown in modified Johnson medium (Borowitzka, 1988) with 60 g/L NaCl content. The cultures were kept in glass flasks thermostatically maintained at 28 oC and illuminated with cool white fluorescent light (80 μmol·photons m-2 s-1). Aeration and mixing was achieved by bubbling with sterile air. For collecting dry mass, samples were centrifuged (6.000 × g, 5 min), and the pellets were lyophilized. Nostoc sp. was collected from Hortobágy, Szákahalom on the 13th of May, 2011. The Nostoc strain was isolated from the environmental sample. Nostoc sp. was grown in liquid nitrogen free medium of Allen (Allen). The cultures were kept in glass flasks thermostatically maintained at 28 oC and illuminated with cool white fluorescent light (80 μmol·photons m-2 s1
). Aeration and mixing was achieved by bubbling with sterile air. For collecting dry mass,
samples were centrifuged (10.000 × g, 5 min), and the pellets were lyophilized.
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ACCEPTED MANUSCRIPT Euglena sanguinea bloom occurred in Kurca-river, Szentes, Hungary. The bloom was sampled on the 13th of July, 2012. Samples were taken from the water surface at the center of the pond, where the cells were associated into mass and covered the water surface in thick
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layer. Ten L net samples were collected for harvesting of the water-bloom-causing species.
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The collected species was identified as Euglena sanguinea. Strain was harvested by
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centrifuge at 8,000 rpm for 10 min, and the pellet was lyophilized.
2.3 Extraction of the carotenoids
Lyophilized samples (1-3 g) were extracted twice with acetone and once with Et2O. After
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evaporation the residue of acetonic extracts was dissolved in Et2O. The ethereal solutions were combined and this total extract was saponified at room temperature in heterogeneous
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phase (30% KOH/MeOH) overnight. The reaction mixture was washed with water ten times, dried over anhydrous Na2SO4, evaporated to dryness in vacuo, and the residue was dissolved in benzene. The saponified pigments were stored in benzene solution at -20 °C, under
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nitrogen in darkness until the preparation of HPLC samples.
2.4 Iodine-catalyzed photoisomerization of carotenoids
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A solution of the carotenoid (1 mg) in benzene (50 ml) was isomerized at room temperature under N2 in scattered daylight in the presence of I2 (ca. 0.02 equiv.) (Molnár & Szabolcs 1993). The isomerization was monitored by UV/VIS spectroscopy. When the thermodynamic equilibrium was reached after ca. 40 min, the mixture was washed with 5%
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Na2S2O3 solution to remove I2 , and after the usual workup, was submitted to HPLC. 2.5. Open column chromatography of Nostoc The saponified extract was subjected to open column chromatography (CaCO3, Biogal, Hungary, toluene/n-hexane, from 20:80 to 30:70). After development three fractions were visible. Fraction 1: 2 mm length pink band (canthaxanthin); Fraction 2: 5 mm length pink band echinenone ; Fraction 3: 10 mm length yellow band (-carotene, -carotene 5,6epoxide, -carotene-5,8-epoxide). After processing, which consisted in cutting the column packing into sections and extracting each section, fractions 1−3 were obtained, which were submitted to HPLC analysis. 2.6 Open column chromatography of the Euglena saponified extract
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ACCEPTED MANUSCRIPT The saponified extract was subjected to open column chromatography (CaCO3, Biogal, Hungary, toluene/n-hexane, from 20:80 to 30:70). After development five fractions were visible. Fraction 1: 4 mm lenght ochre band (mixture of polar carotenoids); Fraction 2: 3 mm
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lenght pink band (mixture); Fraction 3: 3 mm lenght yellow band (lutein); Fraction 4: 10 mm
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lenght ochre band (diatoxanthin); Fraction 5: 20 mm lenght yellow band (-carotene). After processing, which consisted in cutting the column packing into sections and extracting each
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section, Fractions 1−5 were obtained, which were submitted to HPLC analysis, and in addition Fraction 4 was submitted to 1H-NMR analysis.
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2.7 Open column chromatography of the Euglena non-saponified extract The non-saponified extract was subjected to open column chromatography (MgO-celite
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1:1 from n-hexane to toluene/n-hexane 20:80). After development three fractions were visible. Fraction 1: 10 mm lenght brown band (non esterified carotenoids); Fraction 2: 25 mm lenght red band (monoesters); Fraction 3: 50 mm lenght red band (diesters); After processing,
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which consisted in cutting the column packing into sections and extracting each section,
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Fractions 1-3 were submitted to HPLC-DAAD analysis.
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2.8 Equipments
HPLC-DAD: gradient pump Dionex P680; detector: Dionex PDA-100; = 450 nm; T: 22o data acquisition was performed by Chromeleon 6.70 software.
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HPLC-MS: Agilent 6350 Accurate-Mass Q-TOF LC/MS, data acquisition was performed Agilent MassHunter Qualitative Analysis B.04.00. For LC-(APCI)MS the positive ion mode was used, with TIC, scanning range 200-1500 m/z, corona voltage 2.6 kV, fragmentor voltage 150 V, skimmer 60V, Oct 1RF Vpp 750 V. The flow rate of the dried nitrogen as nebulizer gas 240 l/h and the vaporizer temperature was 400 °C. Column: 250 x 4.6 mm i.d.; YMC C30, 3µm. Eluents: A: 81% MeOH, 15% tert-butylmethyl-ether (tBME), 4% H2O, B: 6% MeOH, 90% tBME, 4% H2O. The chromatograms were performed in linear gradient: 0’ 100% A – 90’ 100% B eluent. Flow rate: 1.00 cm3/min. The 1H (500 MHz) NMR spectrum of diatoxanthin was measured with a Bruker DRX Avance II spectrometer. Chemical shifts are referenced to the residual solvent signals. 2.9 NMR and MS data of isolated diatoxanthin MALDI-TOF MS: 548 (M-H2O), 566 (M+), 589 (M+Na+). 1H-NMR (CDCl3, 500 MHz): δ (ppm) 1.07, s, Me(16’), Me(17’); 1.14, s, Me(16); 1.20, s, Me(17); 1.45, m, Hax-C(2); 1.48,
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ACCEPTED MANUSCRIPT m, Hax-C(2’); 1.74, s, Me(18’); 1.77, m, Heq-C(2’); 1.84, m, Heq-C(2); 1.92, s, Me(18); 1.95, s, Me(20); 1.96, s, Me(20’); 1.97, s, Me(19’); 2.00, s, Me(20); 2.42, dd, J = 5.4 Hz, 16.9 Hz, Heq-C(4); 4.00, m, H-C(3) & H-C(3’); 6.10-6.15, m, H-C(7’) & H-C(8’); 6.15, d, J = 11.4 Hz,
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H-C(10’); 6.25, d, J = 9.8 Hz, H-C(14’); 6.27, d, J = 9.0 Hz, H-C(14); 6.36, d, J = 15.1 Hz,
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H-C(12’); 6.45, d, J = 12.0 Hz, H-C(10), H-C(10); 6.61-6.71, m, H-C(15) & H-C(11’) & H-
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C(15’).
3.Results and Discussion
Spectrophotometric methods (Schiedt & Liaaen-Jensen 1995) were used to determine the
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total carotenoid content of the samples. The algae samples contained different amounts of carotenoids: Dunaliella 6.3 mg/g; Nostoc: 0.46 mg/g; and Euglena: 2.16 mg/g of the freezed-
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dried sample.
HPLC-DAD and HPLC-MS analysis of the saponified and non-saponified carotenoid extracts was used to identify free carotenoids and carotenoid esters among the three samples.
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Based on their UV-VIS and mass spectrum as well as co-chromatography with authentic
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standards, several main and minor carotenoids were identified. The 9Z- and 13Z-isomers were identified by comparison of the retention times and the UV-VIS spectra in the mixture
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of the individual carotenoids obtained by I2 catalyzed stereomutation. The three investigated algae species collected in eastern Hungary showed three different
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carotenoid compositions, as detailed below.
3.1 Dunaliella salina
The chromatogram is shown in Figure 1, the retention times UV-VIS data, molecular mass and identifications of carotenoids are shown in Table 1. Since LC-APCI-MS was performed in positive ionization mode, pigments were detected as the quasimolecular ion [M+H]+, except for lutein (peak 10), whose [M+H-H2O]+ ion was obtained as the main fragment. Dunaliella is a green micro algae, thus the carotenoid profile was characterizing for chloroplast pigments. Dunaliella species is known as a good source of -carotene, surprisingly, our carotenoid fraction contained only 13% -carotene, the main carotenoid was lutein (52%). Similar carotenoid composition was described by Fu, Guðmundsson, Paglia, Herjólfsson, Andrésson, Palsson & Brynjólfsson (2013).
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ACCEPTED MANUSCRIPT Other major peaks were identified as violaxanthin (11%) and (9Z)-neoxanthin (6%). In addition, neoxanthin, neochrome, luteoxanthin, antheraxanthin, zeaxanthin, - and carotene, (9Z)- and (13Z)--carotene and (9Z)--carotene were detected as minor carotenoids.
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The chromatogram of the non-saponified sample of Dunaliella extract showed an essentially
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same carotenoid fingerprint than that of saponified extracts. We could not detect carotenoid esters, and the four new peaks, that appeared in the unsaponified extracts belong the different
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chlorophyll isomers (Retention time 4.64 min, absorption max 464 nm, composition 4.1%; Retention time 19.34 min, absorption max 430 nm, composition 13.2%; Retention time 20.94 min, absorption max 430 nm, composition 0.9%; Retention time 42.64 min: absorption max
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436 nm, composition 3.1%).
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The species of the Chlorophyceae taxon usually contain - and -carotene, violaxanthin, neoxanthin, as major carotenoids; and low amounts of zeaxanthin, loroxanthin, siphonaxanthin can also be present (Takaichi, 2011). Dunaliella salina is often described as a
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species, that has the ability to accumulate large amounts of -carotene (Ben-Amotz & Avron
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1983; Fu, Guðmundsson, Paglia, Herjólfsson, Andrésson, Palsson & Brynjólfsson 2013). In our particular sample, however, lutein made up 52% of the total carotenoid composition.
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The pattern shows no major biosynthetic difference from the carotenoid pattern described for D. salina, qualitatively our strain resembles those presented in the literature. Most of the detected carotenoids are members of the simple -carotene derivates (-carotene itself, and
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lutein), or the simple -carotene derivates (violaxanthin, neoxanthin, zeaxanthin and carotene itself). Of the biosynthetic path involving -carotene, about half of the -carotene is converted on to other metabolites. Most carotenoids enter the metabolic route starting with carotene, and most molecules end up as accumulated lutein. The cause of this shift may be either genetic, or may be the result of environmental conditions (Fu, Guðmundsson, Paglia, Herjólfsson, Andrésson, Palsson & Brynjólfsson 2013). It is the high carotenoid content that can make these algae functional foods. The main carotenoid of our sample, lutein and zeaxanthin were tested for many pharmacological effects with success, which include but are not limited to the following. The yellow colour of these carotenoids allow them to operate as „natural sunglass” absorbing blue light in the eye, thus reducing damage done to the optical photoreceptor layer (Krinsky, Landrum & Bone, 2003). These carotenoids are exclusively of dietary origin in humans, and a study has revealed, that increase in lutein uptake increases macular pigment density within 4 weeks (Berendschot,
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ACCEPTED MANUSCRIPT Goldbohm, Klöpping, van de Kraats, van Norel & van Norren 2000). Despite clinical trials resulted in somewhat conflicting results about the effects of lutein on visual performance, these carotenoids are considered useful moelcules of the diet. Other positive results include
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chemopreventive potential, i. e. high dietary intake of lutein has been associated with reduced
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risks of some types of cancers, including endometrian and ovarian cancers (Bertone, Hankinson, Newcomb, Rosner, Willett, Stampfer & Egan, 2001). Trying to establish a link
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with other cancer types, however, yielded rather mixed results, which needs further study. Carotenoids including lutein were also found to be effective against early atherosclerosis in a study (Dwyer, Paul-Labrador, Fan, Shircore, Bairey & Dwyer, 2004). The many studies
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about these disease risks need further evaluation because of the sometimes conflicting evidences. The background behind both atherosclerosis prevention and cancer prevention are
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both based on the potent antioxidant potential of these molecules. Carotenoids are especially effective at scavenging singlet O2, a property that has been shown in many studies. In this property however, lutein is much less effective, than -carotene. (Cantrell, McGarvey,
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Truscott, Rancan & Böhm, 2003).
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Despite not being a source of extremely high amounts of -carotene, we think, that the presented algal strain can be used as a nutritional source of bioactive natural products, such as
3.2 Nostoc sp.
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lutein, -carotene and violaxanthin.
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Figure 2 shows an elution profile of HPLC for the saponified pigments extracted from the isolated Nostoc strain. The absorption maxima of the main carotenoid (peak 20) in the HPLCchromatogram were 450, and 476 nm, and the spectral fine structure of %III/II was low. It had a relative molecular mass of 536, and hence peak 20 carotenoid was identified as -carotene. The absorption spectra of the carotenoid peaks 19 and 21 were compatible with that of the (9Z)and (13Z)--carotene obtained by I2 catalyzed stereomutation of -carotene. Their relative molecular mass was 536, thus the peaks were identified as (9Z)- and (13Z)--carotene, respectively. The carotenoid peaks 8 and 15 showed broad absorption spectra in the HPLC eluent, and their absorption maxima were around 474 and 464 nm, respectively. They had relative molecular masses of 564 and 550, respectively, and hence these peak 8 and 15 carotenoids were identified as canthaxanthin and echinenone, respectively.
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ACCEPTED MANUSCRIPT The I2 catalyzed stereomutation of echinenone produced four Z-isomers, namely (9Z)-, (9’Z)-, (13Z)- and (13’Z)-echinenone. These isomers could be separated well on the C30 column, and could be identified by their UV-VIS spectrum without differentiation of 9- and
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9’ or 13 and 13’ isomers. Comparison of the retention time, the UV-VIS spectra and
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molecular mass of these compounds allowed the identification of peak 11, 12 and 17, 18 as Z-isomers of echinenone. With a similar method, (9Z)-canthaxanthin was identified, while
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(13Z)-canthaxanthin could not be detected in this sample.
The absorption maxima of the peak 14 carotenoid in HPLC eluent were 429 (shoulder), 445, and 472 nm, and the spectral fine structure of %III/II was 73; this is the ratio of the peak
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heights of the longest and the middle wavelength absorption bands from the trough between the two peaks (Britton 1995). It had a relative molecular mass of 552, and hence this peak 14
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carotenoid was identified as -carotene 5,6-epoxide.
The saponified extract of Nostoc was submitted to open column chromatography on CaCO3 adsorbent. Three bands were obtained by elution with toluene-hexane 30-70. The first
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pink band consisted of the most polar unidentified carotenoids, canthaxanthin and cis isomers
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of echinenone. The second band contained echinenone. The third yellow band contained carotene, its isomers, and -carotene epoxides. This fraction was further chromatographed on
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CaCO3 column with n-hexane for separation of -carotene and -carotene 5,6- and 5,8epoxide. The purified -carotene 5,6-epoxide was compared to the semisynthetic (mixture of 5R,6S and 5S,6R) -carotene 5,6-epoxide on chiral HPLC column. In this way the peak 14
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was identified as (5R,6S)--carotene 5,6-epoxide. The non-saponified sample of Nostoc extract, similarly to Dunaliella, showed the same carotenoid composition as the saponified extracts. We could not detect carotenoid esters, and the 4 new peaks appeared in non-saponified extracts belonged the different chlorophyll isomers (Retention time 13.23 min: absorption max 430 and 664 nm, composition 4.4%; Retention time 13.92 min, absorption max 419 and 655 nm, composition 1.9%; Retention time 19.43 min, absorption max 432 and 665 nm, composition 1.1%; Retention time 34.58 min, absorption max 408 and 666 nm, composition 2.5%). Our result demonstrated that our Nostoc isolate contained echinenone (35%) and -carotene (36%) as major compounds similarly to earlier published results (Goodwin, stb). The minor carotenoids were canthaxanthin, -carotene 5,6-epoxide, -carotene 5,8-epoxide, (9Z)- and (13Z)--carotene. Unfortunately some peaks remained unidentified, the identification of these minor compounds is in progress.
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ACCEPTED MANUSCRIPT In Nostoc species, several carotenoids have been described so far. Of the biosynthetic pathways described in (Takaichi, Maoka & Mochimaru, 2009) for Nostoc commune, a clear subpath dominated active in our isolate, namely the “keto pathway” leading to formation of
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echinenone and canthaxanthin from -carotene. The carotenoid composition in our isolate is
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also quite similar to those described in (Takaichi, Maoka & Mochimaru, 2009), with a few exceptions. In our isolate, nostoxanthin and caloxanthin have not been identified, but they are
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present at rather high amount (11%, and 5%, respectively) in the Japanese isolate. We also did not detect fucosides, carotenoid glycosides (higher m/z). The main similarity was consistent with results of the previous studies: the main carotenoid of the Nostoc sample was
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-carotene, along with the astaxanthin precursor echinenone
Just like the major carotenoids described in the Dunaliella section, -carotene also has
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been investigated by many research groups for biological functions. However, as the intake of -carotene is multi-correlated with many bioactive phytochemicals, evaluation of its effect alone a is challenging task in epidemiological studies. The molecule failed to show cancer
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prevention ability, in fact, supplementation increased the risk of some cancer types according
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to a recent meta-analysis study (Druesne‐Pecollo, Latino‐Martel, Norat, Barrandon, Bertrais, Galan & Hercberg, 2010). Also, no clear effect on cardiovascular protection could be
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observed in a meta-analysis on effects of vitamins and antioxidants on cardiovascular events (Myung, Ju, Cho, Oh, Park, Koo & Park, 2013). Despite -carotene has antioxidant activity, that is often regarded a pharmacological effect, several other clinical applications have failed
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to produce clear advantages of supplementation. Further research is required to assess the clinically relevant in vivo protective effects of -carotene supplementation against major diseases. There are no clinical data available on effects of echinenone in humans. The presented Nostoc strain can still be used as a source of -carotene, echinenone, or a supplement source material.
3.3 Euglena sanguinea The chromatogram of the saponified extract of Euglena sanguinea is shown in Figure 3, the retention times, UV-VIS spectrum data, molecular mass and identifications of carotenoids are shown in Table 3. -Carotene and it’s 9Z- and 13Z-isomers (peak 15-17) were identified as we described above in the case of Nostoc species. The absorption maxima of the main peak (peak 10) carotenoid in the HPLC eluent were 450, and 478 nm, and the spectral fine structure of %III/II was 44. It had a relative molecular
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ACCEPTED MANUSCRIPT mass of 566 which corresponded to the formula of C40H54O2. Co-chromatography with authentic diatoxanthin sample suggested that peak 10 was diatoxanthin. Open column chromatography of the saponified extract resulted in five fractions. Fraction 4
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contained peak 10 carotenoid (~ 0.5 mg), after processing, was submitted to MALDI-TOF
IP
and 1H NMR analysis. The MALDI-TOF of this compound showed a molecular ion at m/z
hydrogen atoms probably in the polyene chain.
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566 which confirmed the formula C40H54O2 which was in agreement with missing two Owing to the small amount of sample, 1H,1H-COSY and
13
C NMR data could not be
recorded. Thus, NMR analysis was restricted to proton measurement of the peak 10
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carotenoid. The 1H NMR chemical shifts were compared with those of published Haugan & Liaaen-Jensen (1994) earlier. Although some minor contamination was present in the sample,
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most of the proton-signals known in the literature (Haugan & Liaaen-Jensen, 1994) were possible to be identified (see experimental section). Consequently, the NMR spectra confirmed the identity of peak 10 being diatoxanthin. Peak 8 was tentatively identified as
D
(13Z)- or (13’Z)-diatoxanthin by it’s UV-VIS spectrum and molecular mass. Unfortunately,
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other Z-isomers of diatoxanthin could not be detected. We suppose, that this isomers were overlapped by other components, and they could be detected in mixed peaks.
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The absorption maxima of the peak 11 carotenoid in the HPLC eluent were 440, and 469 nm (%III/II = 68) and it had a relative molecular mass of 582, which corresponded to the formula of C40H54O3. Based on these results peak 11 carotenoid was identified as diadinoxanthin (5,6-epoxy-diatoxanthin).
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In the UV−Vis spectra of the other main component peak 6 had 445, 474, nm as maxima and the [M+H-H2O]+ ion (551) was detected with MS as the main fragment. These were in accordance with above reported data for lutein. Three other minor compounds were identified successfully. Peak 3 carotenoid had the 412, 435, 463 nm maxima and relative molecular mass of 600. The co-chromatography with authentic sample confirmed the identity of peak 3 carotenoid with (9Z)-neoxanthin. Two other minor components were peak 18 and 19 carotenoids with 472, 499 nm and 468, 495 adsorption maxima respectively in the HPLC eluent. The HPLC-MS investigation showed 528 relative molecular mass for both compounds. Because of the lack of authentic samples and based on the findings of Fiksdahl & Liaaen-Jensen (1988) peak 18 was tentatively identified as 3,4,7,8,3’,4',7',8'-octadehydro-,-carotene and peak 19 as (9Z)3,4,7,8,3’,4',7',8'-octadehydro-,-carotene.
13
ACCEPTED MANUSCRIPT The non-saponified extract of E. sanguinea was studied similarly. In contrast with the composition of non-saponified Dunaliella and Nostoc extracts, the Euglena contained larger amounts of mono and diesters as is shown in Fig. 4. Carotenoids, represented in both free
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forms and as esters, were determined in the non-saponified extracts as presented in Table 4.
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Molecular weights (MW) of all possible combinations of fatty acids and carotenoids (monoand di-esters) were calculated, knowing that carotenoid esters are formed by the esterification
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of the carotenoid hydroxyl group. To make the identification of the esters easier, a small amount of non-saponified extract was separated by open column chromatography and we obtained three fractions: non-esterified carotenoids, monoesters and diesters. After the
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saponification of the mono and diester fractions practically, only free diatoxanthin was obtained, which indicated that lutein were present only in non-esterified form. The HPLC-MS
MA
investigation showed the presence of C16:0-, C17:0- and C18:0-monoesters of diatoxanthin and C15:0,15:0- (or C14:0,16:0-), C15:0,16:0-, C16:0,16:0-, C16:0,17:0- and C17:0,17:0- (or C16:0,18:0-), diesters of diatoxanthin. It should be noted, the occurrence of C15:0 and C17:0 carboxylic acids is very
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rare.
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In contrast to the observations of Grung & Liaaen-Jensen (1993), who found astaxanthin as main component in see-water Euglena sanguinea, in the freshwater species lutein and
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diatoxanthin were the main compounds. In addition (9Z)-neoxanthin, diatoxanthin, (9Z)- and (all-E)--carotene were identified as minor carotenoids. Species of the Euglenophyta usually contain the following main carotenoids: -carotene
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and diadinoxanthin, with the minor carotenoids zeaxanthin, neoxanthin, diatoxanthin, loroxanthin and siphonaxanthin also being present in some species (Takaichi, 2011). The species of current interest, E. sanguinea, was shown to contain
diesters of (3S,3′S)-
astaxanthin as chief carotenoids in one study (Grung & Liaaen-Jensen 1993), as well as other diesters. In contrast, our study revealed no astaxanthin carotenoid derivate (measured as aglyca). Other studies (Gerber & Häder, 1994) have found -carotene, astaxanthin diester, and
diatoxanthin as main carotenoids in the same species, with many other minor
compounds as well. Biosynthetically, the carotenoid pattern of the scecies is dominated by diatoxanthin and lutein, the former being a product of the pathway involving -carotene, the latter a derivate of -carotene. There is no such information in the literature on different genotypes of E. sanguinea, but so far, it is worth to mention that members of the astaxanthin pathway, i.e. astaxanthin, canthaxanthin, echinenone (Takaichi, 2011) were not detected from our isolate.
14
ACCEPTED MANUSCRIPT Biosynthesis was rather shifted towards formation of a common -carotene pathway product, lutein. This phenomenon remains to be studied in later work. The carotenoid composition makes this isolate a potential source of dietary supplement
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production as well. The lutein pharmacological data has been briefly described previously.
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Diatoxanthin and alloxanthin and their cis-isomers suppress the expression of enzymes (COX-2, NOS) and molecules (cytokines, interleukines) participating in inflammatory
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Hosokawa, Sashina, Maoka & Miyashita, 2008).
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processes much more effectively than other carotenoids studied previously (Kobishi,
4. Conclusions
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Three non-toxic bloom-forming species were characterized for carotenoid composition. The three taxonomically distinct species contain high amounts of biologically active carotenoids. Of species interest, a lutein-accumulating Euglena sanguinea isolate was
D
presented, along with a high -carotene and echinenone containing Nostoc isolate, and a
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Dunaliella salina strain capable of accumulating lutein, violaxanthin and different carotenes. These species can be used as industrial sources of the presented carotenoids, and after further
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research, may potentially serve as functional food supplement raw materials.
Acknowledgments
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We thank Mrs. Judit Rigó, Ms. Zsuzsanna Götz and Mr. Roland Lukács for their assistance, Dr. Katalin Böddi for the MALDI-TOF measurement. This study was supported by the grant OTKA K 83898 (Hungarian National Research Foundation) and TÁMOP-4.2.2.A11/1/KONV-2012-0065 project. We also thank CaroteNature Gmbh (Switzerland) for providing us with carotenoid standards.
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Berendschot, T. T., Goldbohm, R. A., Klöpping, W. A., van de Kraats, J., van Norel, J., &
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Gerber, S., & Häder, D. P. (1994). Effects of enhanced UV-B irradiation on the red coloured freshwater flagellate Euglena sanguinea. FEMS Microbiology Ecology, 13(3), 177-184.
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Goodwin, T. W. (1980). The Biochemistry of the Carotenoids, Vol. I, Chapman and Hall, London.
Gouveia, L., & Emphis, J. (2003). Relative stability of microalgal carotenoids in microalgal
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Liaaen-Jensen, S. (1977). Algal carotenoids and chemosystematics. In Faulkner, D. J. &
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Molnár, P., & Szabolcs, J. (1993). (Z/E)-Photoisomerization of C40-carotenoids by iodine. J. Chem. Soc., Perkin Trans. 2 261-266.
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ACCEPTED MANUSCRIPT Takaichi, S. (2011). Carotenoids in algae: distributions, biosynthesis and functions. Marine drugs, 9, 1101-1118. Takaichi, S., Maoka, T., & Mochimaru, M. (2009). Unique Carotenoids in the terrestrial
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Name
%
UV-VIS
(min)
IP
Ret.Time
m/z
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Peak No.
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Table 1. Carotenoid composition of saponified Dunaliella salina.
(nm)
+
[M+H ]
6.65
neochrome
0.45
405, 428, 446
601
2
7.19
neoxanthin
0.38
414, 439, 468
601
3
7.99
violaxanthin
11.49
415, 437, 467
601
4
8.79
(9Z)-neoxanthin
6.23
411, 435, 463
601
5
9.64
luteoxanthin
2.96
420, 446
601
6
11.37
antheraxanthin
1.34
447, 471
585
7
11.72
(13Z)-neoxanthin*
1.08
413, 436, 463
601
8
11.98
(13’Z)-neoxanthin*
1.56
418,437, 463
601
9
12.98
(13Z)-violaxanthin*
1.28
415, 437, 464
601
10
13.91
lutein
52.11
443, 471
551**
11
16.09
zeaxanthin
2.06
449, 476
569
12
17.47
(9Z or 9’Z)-lutein
0.48
439, 466
551
13
30.28
(13Z)--carotene
0.98
338, 443, 467
537
14
31.93
-carotene
0.53
444, 472
537
15
35.53
-carotene
13.10
450, 476
537
16
37.85
9Z-carotene
1.52
445,471
537
17
51.94
-carotene
0.44
459, 488
537
18
52.83
(9Z)--carotene
1.96
438, 460, 490
537
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CE P
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NU
1
*changeable, ** [M+H+-H2O]
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Name
IP
Ret.Time
%
(min)
m/z
UV-VIS
SC R
Peak No.
T
Table 2. Carotenoid composition of saponified Nostoc sp.
(nm)
+
[M+H ]
6.28
unidentified cis
0.83
271, 448
543
2
7.61
unidentified cis
0.28
287, 421
567
3
9.13
unidentified red
0.42
465
567
4
10.48
unidentified red
1.06
438
557
5
15.04
unidentified red
1.16
455
551
6
15.96
unidentified red
0.54
471
565
7
16.93
unidentified red
1.01
470
565
8
17.93
canthaxanthin
4.73
474
565
9
20.27
mixture red
0.37
455
551
10
20.92
(9Z)-canthaxanthin
0.68
469
565
11
21.58
(13Z/13’Z)-echinenone
1.26
355, 455
551
12
21.88
(13’Z/13Z)-echinenone
1.10
355, 455
551
13
23.66
unidentified
0.36
450, 477
551
14
25.33
-carotene 5,6-epoxide
1.92
445, 472
553
15
26.41
echinenone
34.92
464
551
16
27.93
-carotene 5,8-epoxide
0.71
427, 452
553
17
28.87
(9Z/9’Z)-echinenone
3.95
454
551
18
29.33
(9’Z/9Z)-echinenone
2.33
452
551
19
30.27
(13Z)--carotene
1.94
443, 467
537
20
35.63
-carotene
36.48
450, 476
537
21
37.98
(9Z)--carotene
3.94
445, 471
537
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D
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NU
1
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ACCEPTED MANUSCRIPT
Name
%
UV-VIS
(min)
(nm)
5.27
unidentified
3.97
2
5.85
unidentified
0.42
3
8.89
(9Z)-neoxanthin
4
12.03
5
m/z
+
[M+H ]
450
583
451
583
1.77
412, 435, 463
601
unidentified
0.30
417, 438, 466
583
13.29
unidentified
0.51
337, 439, 467
583
6
13.80
lutein
23.67
445, 474
551*
7
16.00
unidentified
2.57
433, 457
583
8
16.52
(13Z)-diatoxanthin
2.63
340, 454, 470
567
9
16.99
mixture
10
19.10
diatoxanthin
11
19.75
12
22.57
13
24.25
14
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D
MA
NU
1
IP
Ret.Time
SC R
Peak No.
T
Table 3. Carotenoid composition of saponified Euglena sanguiena
3.36
430, 456 583, 607 450, 478
567
diadinoxanthin
1.29
440, 469
583
unidentified
1.81
451, 479
591
unidentified
9.58
476
591
25.10
unidentified
0.60
347, 445, 472
591
15
30.07
(13Z)--carotene
0.50
338, 443, 468
537
16
35.42
-carotene
5.36
450, 476
537
17
37.78
(9Z)--carotene
0.96
421, 446, 472
537
18
42.21
unidentified
1.13
472, 498
529
19
45.94
unidentified
0.54
468, 495
529
AC
CE P
39.01
* [M+H+-H2O]
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D
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IP
0.79 1.85 6.27 5.73 0.69 13.43 2.93 15.77 0.87 0.54 2.11 0.95 0.79 3.59 3.59 1.59 0.77 2.98 3.51 0.36 0.91 4.12 1.08 5.82 1.44 10.65 0.95 5.89
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(9Z)-neoxantin unidentified lutein not carotenoid not carotenoid unidentified unidentified diatoxantin diadinoxanthin unidentified unidentified. unidentified chlorophyll pheophytine C17-diatoxanthin unidentified unidentified C18-diatoxanthin unidentified C17-diatoxanthin unidentified. C18-diatoxanthin unidentified. C15,C15-diatoxanthin C15,C16-diatoxanthin C16,C16-diatoxanthin C16,C17-diatoxanthin C17,C17-diatoxanthin
UV-VIS (nm) 412, 435, 463 460 445,474 465 464 477 428, 456 450, 478 419, 440, 469 480 454, 478 445, 472 435, 652 409 451, 476 470 446, 471 478 479 480 471 479 470 470 480 479 479 479
SC R
%
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Name
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Ret.Time (min) 8.80 11.14 13.66 14.03 15.40 15.88 16.87 18.89 19.57 20.40 22.33 24.94 32.10 34.78 35.59 36.73 37.91 38.83 39.59 41.48 42.13 43.53 45.91 51.94 53.21 54.48 55.75 57.00
AC
Peak No.
T
Table 4. Carotenoid composition of non-saponified Euglena sanguiena.
m/z + [M+H ] 601 565 551 595 not detected 583, 595 583 567 583 595 593 377 885 805, 871 805 819 805 833 805 819 805 833 813 1015 1029 1043 1057 1071
* [M+H+-H2O]
23
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2 000
SC R
IP
T
Figure 1
mAU
1 750
10
NU
1 500
WVL:450 nm
MA
1 250
1 000
D
750
TE
3
500
4 12
-200
9
10.0
11
12
20.0
13 14
16
17
18
min 30.0
40.0
50.0
60.0
70.0
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0.0
5 678
CE P
250
15
HPLC chromatogram of Dunaliella salina (peak identification in Table 1.)
24
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mAU
IP
1 200
T
Figure 2
WVL:450 nm
SC R
20
1 000
15
NU
800
MA
600
400
200 2
3 4
5
7
6
9
10 11
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1
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8
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5.0
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0
10.0
15.0
17 1213
14
16
21
19 18
min 20.0
25.0
30.0
35.0
40.0
45.0
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HPLC chromatogram of the saponified extract of Nostoc sp. (peak indentification in Table 2.)
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900
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Figure 3
mAU
750
SC R
10
WVL:450 nm
6
NU
625
500
250
MA
375
1 11 8 7 9 45
-100 0,0
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2
10,0
16
12 14
TE
3
13
D
125
20,0
15
17
18
19
min 30,0
40,0
50,0
60,0
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HPLC chromatogram of saponified extract of Euglena sanguinea (peak identification in Table 3.)
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mAU
IP
8
6
NU
300
200
3
100
MA
4
2
0.0
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-50
10
10.0
20.0
24
26 28
19
15 14
18
22 25
11
5
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9
D
7
1
WVL:450 nm
SC R
400
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Figure 4.
13
12
1617
21 20
23
27
min 30.0
40.0
50.0
60.0
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HPLC chromatogram of non-saponified extract of Euglena sanguinea (peak identification in Table 4.)
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ACCEPTED MANUSCRIPT Highlights Characterziation of the carotenoids of a lutein-rich Dunaliella salina strain.
Characterziation of the carotenoids of an echinenone-rich Nostoc strain.
Characterziation of the carotenoids of a diatoxanthin-rich Euglena sanguinea strain.
Three non-toxic algae were shown to be excellent natural sources of carotenoids.
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S0963-9969(14)00325-1 doi: 10.1016/j.foodres.2014.05.020 FRIN 5262
To appear in:
Food Research International
Received date: Revised date: Accepted date:
21 February 2014 30 April 2014 3 May 2014
Please cite this article as: Deli, J., Gonda, S., Nagy, L.Z.S., Szab´ o, I., Guly´ as-Fekete, G., Ag´ocs, A., Marton, K. & Vasas, G., Carotenoid Composition of three Bloom-forming Algae Species, Food Research International (2014), doi: 10.1016/j.foodres.2014.05.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Carotenoid Composition of three Bloom-forming Algae Species
Department of Pharmacognosy, University of Pécs, Medical School, H-7624 Pécs, Hungary
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J. Delia,b*, S. Gondac, L. ZS. Nagyc, I. Szabób, G. Gulyás-Feketea, A. Agócsb, K. Martond, G. Vasasc
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Department of Biochemistry and Medical Chemistry, University of Pécs, Medical School, H-7624 Pécs, Hungary c
Institute of Bioanalysis, University of Pécs, Medical School, H-7624 Pécs, Hungary
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Department of Botany, Division of Pharmacognosy University of Debrecen, H-4032 Debrecen, Hungary
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e-mail: [email protected]
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*Corresponding author at: Department of Pharmacognosy, University of Pécs, Medical School, Pécs, Hungary. Tel: +36-72 503-650/28833, Fax: +36-72 503-650/28826
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Keywords: Microalgae, Carotenoids, HPLC, HPLC–MS
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ACCEPTED MANUSCRIPT Abstract
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Algal mass productions are widespread events throughout the world. Most research deals with the environmental impact, ecology and toxicity of these phenomena, but the algae are also promising sources of bioactive natural products, and also are potential food additives. In the current study, we aimed to characterize the carotenoid composition of three non-toxic algal isolates with distinct taxonomical position, namely Dunaliella salina, Euglena sanguinea and a Nostoc strain. The strains were screened for carotenoid composition with HPLC-UV-APCI-MS with comparison with authentic standards. Carotenoids were purified with open coloumn chromatography, and characterized by LC-UV-MS and NMR. All three species contained a high amount of carotenoids. The composition of the carotenoid pattern was somewhat different compared to literature data. The Dunaliella carotenoid fraction contained lutein (52.1% of total carotenoid), -carotene (13.1%), violaxanthin (11.5%) and neoxanthin (6.2%) as chief compounds. The chief carotenoids in the Nostoc strain was echinenone (34.9% ), while main components of the Euglena isolate were diatoxanthin (39.0%), lutein (23.7%), an unidentified carotenoid (9.6%) and -carotene (5.4%). Diatoxanthin is identified by NMR spectroscopy. The carotenoid patterns of the examined strains are somewhat different from the patterns described in the literature. This can be the results of genetic or environmental differences, or combinations thereof. Despite these differences, our study shows the potency of these algae in production of carotenoids, and possibly, usage as food additives.
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ACCEPTED MANUSCRIPT 1. Introduction
Overproduction of photoautotrophic organisms, like algae and cyanobacteria, are well-
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known phenomena that have been found in many types of fresh and marine habitats over the
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past few decades (Reynolds & Walsby, 1975). Near to the several unpleasant accompanying incidences with health and economic consequences, spectacular discoloration of the habitats
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was also detected. Many cyanobacterial and algal strains can produce unique metabolites with diverse chemistry and bioactivity which may cause the phenomenon (Paerl & Huisman, 2008).
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To survive in a competitive environment could be a strategy of algae, why have developed compounds in a significant level of structural-chemical diversity. Over 15,000 novel
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compounds have been chemically assignated (Cardozo, Guaratini, Barros, Falcao, Tonon, Lopes, Campos, Torres, Souza, Colepicolo & Pinto, 2007). This suggests that algae are a promising group to furnish novel biochemically active substances, in addition the current
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application of metabolites isolated from diverse classes of algae is increasing. The
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exploration of these metabolites and organisms for pharmaceutical purposes for human nutrition and for other utilities is justified (Garson, 1989).
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Although many algae species known in the world (according to some estimates 40,000 species), only a few of them are able to build typical appearance of mass (Reynolds & Walsby, 1975). This natural mass of algae, called algal-bloom, is a cheap and adequate natural resource for exploring bioactive metabolites.
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Cyanobacteria or blue-green algae are the largest group of photosynthetic prokaryotes that exist in large diversity and distribution in the world. They occur in almost every habitat on the earth. Their photosynthetic system is closely similar to eucaryotes because they have chlorophyll a and photosystem II, and carry out oxygenic photosynthesis (Paerl, 2013).
Nostoc, a genus of cyanobacteria, is one of the most widespread phototrophic bacteria. They are filamentous and heterocystous cyanobacteria, commonly observed in both aquatic and terrestrial habitats. Species of the genus Nostoc are among the most widespread of all nitrogen-fixing cyanobacteria. Communities of Nostoc commune, in particular, are prominent in those terrestrial limestone environments of tropical, polar, and temperate regions which are subject to extremes of water availability (Whitton, Donaldson & Potts, 1979).
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ACCEPTED MANUSCRIPT Dunaliella salina (D. salina) is a green unicellular microalga that shows remarkable degree of adaptation to a wide range of salt concentrations (from 0.02% to salt saturation, about 35%). Its halotolerant property allows Dunaliella species to survive and grow in many
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marine habitats, especially in concentrated saline lake such as the Pink Lake in Western
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Australia (D. salina), the Great Salt Lake in Utah (D. viridis, 1000-250000 cell ml-1), and the Dead Sea in Israel (D. parva, 8800-15000 cell ml-1). D. salina can also be found in South
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Europe, North Africa, salty evaporating ponds in Mexico and the Solar salty pond in Australia. The genus Dunaliella, includes about 30 species of which 25 are found in brackish water and 5 in freshwater (Melkonian & Preisig, 1984; Leonard & Caceres, 1993). The
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optimal conditions for carotenogenesis are those that limit growth and include exposure to high light intensities and other stress factors, especially nutrient deprivation.
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Dunaliella salina has 90% of β-carotene and 10% of other carotenoids. Carotenoids are made up of α-carotene and xanthophylls like lutein, zeaxanthin, and cryptoxanthins similar to the ones found in food and vegetables (Gouveia & Emphis, 2003). These xanthophylls have a
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widespread application in the pharmaceutical industry and cosmetics as well as in animal
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feed (Venkatesan, Swamy, Senthil, Bhaskar & Rengasamy, 2013). The microalga Dunaliella salina is one of the richest sources of natural -carotene, which is a lipid-soluble orange
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pigment that is used as a colorant in food and feed. Under standard growth conditions D. salina contains approximately 5–10 mg β-carotene per gram dry weight, which is similar to other green algae (Del Campo, García-Gonzáles & Guerrero, 2007). The β-carotene content can rise to as much as 10% of dry weight when D. salina is subjected
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to stress conditions such as high salinity, high light intensity, nutrient deprivation and extreme temperatures (Ben-Amotz, Katz & Avron, 1982; Ben-Amotz & Avron, 1983; Borowitzka, M.A., Borowitzka, L.J. & Kessly, 1990; Shaish, Avron, Pick & Ben-Amotz, 1993; Ben-Amotz, 1996; Krol, Maxwell & Huner, 1997; Kleinegris, Janssen, Brandenburg & Wijffels, 2009).
Euglena sanguinea is an ubiquitous algal species found in many shallow, eutrophic freshwater systems. This species of Euglenophyte is commonly cause surface bloom with intensive red color. The carotenoids of Euglenophyceae have been studied previously (Liaaen-Jensen, 1977; Liaaen-Jensen, 1978; Goodwin, 1980a; Rowan, 1989), and comprise -carotene, diatoxanthin, diadinoxanthin, heteroxanthin and neoxanthin. A quantitative carotenoid analysis of a natural see water bloom of Euglena sanguinea Ehrenberg (Grung &
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ACCEPTED MANUSCRIPT Liaaen-Jensen, 1993) revealed the presence of -carotene (1% of total carotenoids), monoesters of (3S)-adonirubin (3%), diesters of (3S,3'R)-adonixanthin (13%), diesters of (3S,3'S)-astaxanthin (75%), 19-monoester of (3R,3'R,6R)-loroxanthin (1%), (3R,3'R)-
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diatoxanthin (6%), diadinoxanthin (1%) and neoxanthin (trace).
More than 750 structurally defined carotenoids are reported from nature; land plants,
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algae, bacteria including cyanobacteria and photosynthetic bacteria, archaea, fungus and animals. The main goal of our study is to analyse and identify the carotenoid composition of three bloom-forming algal species and may draw attention to the importance of the natural
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algal-blooms as alternative sources of active substances like carotenoids.
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2. Materials and Methods 2.1 Chemicals
HPLC and analytical grade solvents were used. The authenthic samples were taken from
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our collection (-carotene, -carotene, -carotene 5,6-epoxide, -carotene 5,8-epoxide,
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lutein, zeaxanthin, neoxanthin, violaxanthin, antheraxanthin) and CaroteNature GmbH (-
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carotene, echinenone, canthaxanthin, diatoxanthin).
2.2 Collection and culture conditions The mass occurring cells of Dunaliella salina were observed in Lacul Băilor Cojocna
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(N46°44.90652'; E23°50.44080'), Cojocna (Kolozs). 1 L sample were collected for isolation of the strain caused discoloration of the water. The isolated Dunaliella salina was grown in modified Johnson medium (Borowitzka, 1988) with 60 g/L NaCl content. The cultures were kept in glass flasks thermostatically maintained at 28 oC and illuminated with cool white fluorescent light (80 μmol·photons m-2 s-1). Aeration and mixing was achieved by bubbling with sterile air. For collecting dry mass, samples were centrifuged (6.000 × g, 5 min), and the pellets were lyophilized. Nostoc sp. was collected from Hortobágy, Szákahalom on the 13th of May, 2011. The Nostoc strain was isolated from the environmental sample. Nostoc sp. was grown in liquid nitrogen free medium of Allen (Allen). The cultures were kept in glass flasks thermostatically maintained at 28 oC and illuminated with cool white fluorescent light (80 μmol·photons m-2 s1
). Aeration and mixing was achieved by bubbling with sterile air. For collecting dry mass,
samples were centrifuged (10.000 × g, 5 min), and the pellets were lyophilized.
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ACCEPTED MANUSCRIPT Euglena sanguinea bloom occurred in Kurca-river, Szentes, Hungary. The bloom was sampled on the 13th of July, 2012. Samples were taken from the water surface at the center of the pond, where the cells were associated into mass and covered the water surface in thick
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layer. Ten L net samples were collected for harvesting of the water-bloom-causing species.
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The collected species was identified as Euglena sanguinea. Strain was harvested by
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centrifuge at 8,000 rpm for 10 min, and the pellet was lyophilized.
2.3 Extraction of the carotenoids
Lyophilized samples (1-3 g) were extracted twice with acetone and once with Et2O. After
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evaporation the residue of acetonic extracts was dissolved in Et2O. The ethereal solutions were combined and this total extract was saponified at room temperature in heterogeneous
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phase (30% KOH/MeOH) overnight. The reaction mixture was washed with water ten times, dried over anhydrous Na2SO4, evaporated to dryness in vacuo, and the residue was dissolved in benzene. The saponified pigments were stored in benzene solution at -20 °C, under
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nitrogen in darkness until the preparation of HPLC samples.
2.4 Iodine-catalyzed photoisomerization of carotenoids
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A solution of the carotenoid (1 mg) in benzene (50 ml) was isomerized at room temperature under N2 in scattered daylight in the presence of I2 (ca. 0.02 equiv.) (Molnár & Szabolcs 1993). The isomerization was monitored by UV/VIS spectroscopy. When the thermodynamic equilibrium was reached after ca. 40 min, the mixture was washed with 5%
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Na2S2O3 solution to remove I2 , and after the usual workup, was submitted to HPLC. 2.5. Open column chromatography of Nostoc The saponified extract was subjected to open column chromatography (CaCO3, Biogal, Hungary, toluene/n-hexane, from 20:80 to 30:70). After development three fractions were visible. Fraction 1: 2 mm length pink band (canthaxanthin); Fraction 2: 5 mm length pink band echinenone ; Fraction 3: 10 mm length yellow band (-carotene, -carotene 5,6epoxide, -carotene-5,8-epoxide). After processing, which consisted in cutting the column packing into sections and extracting each section, fractions 1−3 were obtained, which were submitted to HPLC analysis. 2.6 Open column chromatography of the Euglena saponified extract
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ACCEPTED MANUSCRIPT The saponified extract was subjected to open column chromatography (CaCO3, Biogal, Hungary, toluene/n-hexane, from 20:80 to 30:70). After development five fractions were visible. Fraction 1: 4 mm lenght ochre band (mixture of polar carotenoids); Fraction 2: 3 mm
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lenght pink band (mixture); Fraction 3: 3 mm lenght yellow band (lutein); Fraction 4: 10 mm
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lenght ochre band (diatoxanthin); Fraction 5: 20 mm lenght yellow band (-carotene). After processing, which consisted in cutting the column packing into sections and extracting each
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section, Fractions 1−5 were obtained, which were submitted to HPLC analysis, and in addition Fraction 4 was submitted to 1H-NMR analysis.
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2.7 Open column chromatography of the Euglena non-saponified extract The non-saponified extract was subjected to open column chromatography (MgO-celite
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1:1 from n-hexane to toluene/n-hexane 20:80). After development three fractions were visible. Fraction 1: 10 mm lenght brown band (non esterified carotenoids); Fraction 2: 25 mm lenght red band (monoesters); Fraction 3: 50 mm lenght red band (diesters); After processing,
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which consisted in cutting the column packing into sections and extracting each section,
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Fractions 1-3 were submitted to HPLC-DAAD analysis.
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2.8 Equipments
HPLC-DAD: gradient pump Dionex P680; detector: Dionex PDA-100; = 450 nm; T: 22o data acquisition was performed by Chromeleon 6.70 software.
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HPLC-MS: Agilent 6350 Accurate-Mass Q-TOF LC/MS, data acquisition was performed Agilent MassHunter Qualitative Analysis B.04.00. For LC-(APCI)MS the positive ion mode was used, with TIC, scanning range 200-1500 m/z, corona voltage 2.6 kV, fragmentor voltage 150 V, skimmer 60V, Oct 1RF Vpp 750 V. The flow rate of the dried nitrogen as nebulizer gas 240 l/h and the vaporizer temperature was 400 °C. Column: 250 x 4.6 mm i.d.; YMC C30, 3µm. Eluents: A: 81% MeOH, 15% tert-butylmethyl-ether (tBME), 4% H2O, B: 6% MeOH, 90% tBME, 4% H2O. The chromatograms were performed in linear gradient: 0’ 100% A – 90’ 100% B eluent. Flow rate: 1.00 cm3/min. The 1H (500 MHz) NMR spectrum of diatoxanthin was measured with a Bruker DRX Avance II spectrometer. Chemical shifts are referenced to the residual solvent signals. 2.9 NMR and MS data of isolated diatoxanthin MALDI-TOF MS: 548 (M-H2O), 566 (M+), 589 (M+Na+). 1H-NMR (CDCl3, 500 MHz): δ (ppm) 1.07, s, Me(16’), Me(17’); 1.14, s, Me(16); 1.20, s, Me(17); 1.45, m, Hax-C(2); 1.48,
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ACCEPTED MANUSCRIPT m, Hax-C(2’); 1.74, s, Me(18’); 1.77, m, Heq-C(2’); 1.84, m, Heq-C(2); 1.92, s, Me(18); 1.95, s, Me(20); 1.96, s, Me(20’); 1.97, s, Me(19’); 2.00, s, Me(20); 2.42, dd, J = 5.4 Hz, 16.9 Hz, Heq-C(4); 4.00, m, H-C(3) & H-C(3’); 6.10-6.15, m, H-C(7’) & H-C(8’); 6.15, d, J = 11.4 Hz,
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H-C(10’); 6.25, d, J = 9.8 Hz, H-C(14’); 6.27, d, J = 9.0 Hz, H-C(14); 6.36, d, J = 15.1 Hz,
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H-C(12’); 6.45, d, J = 12.0 Hz, H-C(10), H-C(10); 6.61-6.71, m, H-C(15) & H-C(11’) & H-
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C(15’).
3.Results and Discussion
Spectrophotometric methods (Schiedt & Liaaen-Jensen 1995) were used to determine the
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total carotenoid content of the samples. The algae samples contained different amounts of carotenoids: Dunaliella 6.3 mg/g; Nostoc: 0.46 mg/g; and Euglena: 2.16 mg/g of the freezed-
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dried sample.
HPLC-DAD and HPLC-MS analysis of the saponified and non-saponified carotenoid extracts was used to identify free carotenoids and carotenoid esters among the three samples.
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Based on their UV-VIS and mass spectrum as well as co-chromatography with authentic
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standards, several main and minor carotenoids were identified. The 9Z- and 13Z-isomers were identified by comparison of the retention times and the UV-VIS spectra in the mixture
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of the individual carotenoids obtained by I2 catalyzed stereomutation. The three investigated algae species collected in eastern Hungary showed three different
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carotenoid compositions, as detailed below.
3.1 Dunaliella salina
The chromatogram is shown in Figure 1, the retention times UV-VIS data, molecular mass and identifications of carotenoids are shown in Table 1. Since LC-APCI-MS was performed in positive ionization mode, pigments were detected as the quasimolecular ion [M+H]+, except for lutein (peak 10), whose [M+H-H2O]+ ion was obtained as the main fragment. Dunaliella is a green micro algae, thus the carotenoid profile was characterizing for chloroplast pigments. Dunaliella species is known as a good source of -carotene, surprisingly, our carotenoid fraction contained only 13% -carotene, the main carotenoid was lutein (52%). Similar carotenoid composition was described by Fu, Guðmundsson, Paglia, Herjólfsson, Andrésson, Palsson & Brynjólfsson (2013).
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ACCEPTED MANUSCRIPT Other major peaks were identified as violaxanthin (11%) and (9Z)-neoxanthin (6%). In addition, neoxanthin, neochrome, luteoxanthin, antheraxanthin, zeaxanthin, - and carotene, (9Z)- and (13Z)--carotene and (9Z)--carotene were detected as minor carotenoids.
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The chromatogram of the non-saponified sample of Dunaliella extract showed an essentially
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same carotenoid fingerprint than that of saponified extracts. We could not detect carotenoid esters, and the four new peaks, that appeared in the unsaponified extracts belong the different
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chlorophyll isomers (Retention time 4.64 min, absorption max 464 nm, composition 4.1%; Retention time 19.34 min, absorption max 430 nm, composition 13.2%; Retention time 20.94 min, absorption max 430 nm, composition 0.9%; Retention time 42.64 min: absorption max
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436 nm, composition 3.1%).
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The species of the Chlorophyceae taxon usually contain - and -carotene, violaxanthin, neoxanthin, as major carotenoids; and low amounts of zeaxanthin, loroxanthin, siphonaxanthin can also be present (Takaichi, 2011). Dunaliella salina is often described as a
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species, that has the ability to accumulate large amounts of -carotene (Ben-Amotz & Avron
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1983; Fu, Guðmundsson, Paglia, Herjólfsson, Andrésson, Palsson & Brynjólfsson 2013). In our particular sample, however, lutein made up 52% of the total carotenoid composition.
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The pattern shows no major biosynthetic difference from the carotenoid pattern described for D. salina, qualitatively our strain resembles those presented in the literature. Most of the detected carotenoids are members of the simple -carotene derivates (-carotene itself, and
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lutein), or the simple -carotene derivates (violaxanthin, neoxanthin, zeaxanthin and carotene itself). Of the biosynthetic path involving -carotene, about half of the -carotene is converted on to other metabolites. Most carotenoids enter the metabolic route starting with carotene, and most molecules end up as accumulated lutein. The cause of this shift may be either genetic, or may be the result of environmental conditions (Fu, Guðmundsson, Paglia, Herjólfsson, Andrésson, Palsson & Brynjólfsson 2013). It is the high carotenoid content that can make these algae functional foods. The main carotenoid of our sample, lutein and zeaxanthin were tested for many pharmacological effects with success, which include but are not limited to the following. The yellow colour of these carotenoids allow them to operate as „natural sunglass” absorbing blue light in the eye, thus reducing damage done to the optical photoreceptor layer (Krinsky, Landrum & Bone, 2003). These carotenoids are exclusively of dietary origin in humans, and a study has revealed, that increase in lutein uptake increases macular pigment density within 4 weeks (Berendschot,
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chemopreventive potential, i. e. high dietary intake of lutein has been associated with reduced
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risks of some types of cancers, including endometrian and ovarian cancers (Bertone, Hankinson, Newcomb, Rosner, Willett, Stampfer & Egan, 2001). Trying to establish a link
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with other cancer types, however, yielded rather mixed results, which needs further study. Carotenoids including lutein were also found to be effective against early atherosclerosis in a study (Dwyer, Paul-Labrador, Fan, Shircore, Bairey & Dwyer, 2004). The many studies
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about these disease risks need further evaluation because of the sometimes conflicting evidences. The background behind both atherosclerosis prevention and cancer prevention are
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both based on the potent antioxidant potential of these molecules. Carotenoids are especially effective at scavenging singlet O2, a property that has been shown in many studies. In this property however, lutein is much less effective, than -carotene. (Cantrell, McGarvey,
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Truscott, Rancan & Böhm, 2003).
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Despite not being a source of extremely high amounts of -carotene, we think, that the presented algal strain can be used as a nutritional source of bioactive natural products, such as
3.2 Nostoc sp.
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lutein, -carotene and violaxanthin.
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Figure 2 shows an elution profile of HPLC for the saponified pigments extracted from the isolated Nostoc strain. The absorption maxima of the main carotenoid (peak 20) in the HPLCchromatogram were 450, and 476 nm, and the spectral fine structure of %III/II was low. It had a relative molecular mass of 536, and hence peak 20 carotenoid was identified as -carotene. The absorption spectra of the carotenoid peaks 19 and 21 were compatible with that of the (9Z)and (13Z)--carotene obtained by I2 catalyzed stereomutation of -carotene. Their relative molecular mass was 536, thus the peaks were identified as (9Z)- and (13Z)--carotene, respectively. The carotenoid peaks 8 and 15 showed broad absorption spectra in the HPLC eluent, and their absorption maxima were around 474 and 464 nm, respectively. They had relative molecular masses of 564 and 550, respectively, and hence these peak 8 and 15 carotenoids were identified as canthaxanthin and echinenone, respectively.
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ACCEPTED MANUSCRIPT The I2 catalyzed stereomutation of echinenone produced four Z-isomers, namely (9Z)-, (9’Z)-, (13Z)- and (13’Z)-echinenone. These isomers could be separated well on the C30 column, and could be identified by their UV-VIS spectrum without differentiation of 9- and
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9’ or 13 and 13’ isomers. Comparison of the retention time, the UV-VIS spectra and
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molecular mass of these compounds allowed the identification of peak 11, 12 and 17, 18 as Z-isomers of echinenone. With a similar method, (9Z)-canthaxanthin was identified, while
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(13Z)-canthaxanthin could not be detected in this sample.
The absorption maxima of the peak 14 carotenoid in HPLC eluent were 429 (shoulder), 445, and 472 nm, and the spectral fine structure of %III/II was 73; this is the ratio of the peak
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heights of the longest and the middle wavelength absorption bands from the trough between the two peaks (Britton 1995). It had a relative molecular mass of 552, and hence this peak 14
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carotenoid was identified as -carotene 5,6-epoxide.
The saponified extract of Nostoc was submitted to open column chromatography on CaCO3 adsorbent. Three bands were obtained by elution with toluene-hexane 30-70. The first
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pink band consisted of the most polar unidentified carotenoids, canthaxanthin and cis isomers
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of echinenone. The second band contained echinenone. The third yellow band contained carotene, its isomers, and -carotene epoxides. This fraction was further chromatographed on
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CaCO3 column with n-hexane for separation of -carotene and -carotene 5,6- and 5,8epoxide. The purified -carotene 5,6-epoxide was compared to the semisynthetic (mixture of 5R,6S and 5S,6R) -carotene 5,6-epoxide on chiral HPLC column. In this way the peak 14
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was identified as (5R,6S)--carotene 5,6-epoxide. The non-saponified sample of Nostoc extract, similarly to Dunaliella, showed the same carotenoid composition as the saponified extracts. We could not detect carotenoid esters, and the 4 new peaks appeared in non-saponified extracts belonged the different chlorophyll isomers (Retention time 13.23 min: absorption max 430 and 664 nm, composition 4.4%; Retention time 13.92 min, absorption max 419 and 655 nm, composition 1.9%; Retention time 19.43 min, absorption max 432 and 665 nm, composition 1.1%; Retention time 34.58 min, absorption max 408 and 666 nm, composition 2.5%). Our result demonstrated that our Nostoc isolate contained echinenone (35%) and -carotene (36%) as major compounds similarly to earlier published results (Goodwin, stb). The minor carotenoids were canthaxanthin, -carotene 5,6-epoxide, -carotene 5,8-epoxide, (9Z)- and (13Z)--carotene. Unfortunately some peaks remained unidentified, the identification of these minor compounds is in progress.
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ACCEPTED MANUSCRIPT In Nostoc species, several carotenoids have been described so far. Of the biosynthetic pathways described in (Takaichi, Maoka & Mochimaru, 2009) for Nostoc commune, a clear subpath dominated active in our isolate, namely the “keto pathway” leading to formation of
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echinenone and canthaxanthin from -carotene. The carotenoid composition in our isolate is
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also quite similar to those described in (Takaichi, Maoka & Mochimaru, 2009), with a few exceptions. In our isolate, nostoxanthin and caloxanthin have not been identified, but they are
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present at rather high amount (11%, and 5%, respectively) in the Japanese isolate. We also did not detect fucosides, carotenoid glycosides (higher m/z). The main similarity was consistent with results of the previous studies: the main carotenoid of the Nostoc sample was
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-carotene, along with the astaxanthin precursor echinenone
Just like the major carotenoids described in the Dunaliella section, -carotene also has
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been investigated by many research groups for biological functions. However, as the intake of -carotene is multi-correlated with many bioactive phytochemicals, evaluation of its effect alone a is challenging task in epidemiological studies. The molecule failed to show cancer
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prevention ability, in fact, supplementation increased the risk of some cancer types according
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to a recent meta-analysis study (Druesne‐Pecollo, Latino‐Martel, Norat, Barrandon, Bertrais, Galan & Hercberg, 2010). Also, no clear effect on cardiovascular protection could be
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observed in a meta-analysis on effects of vitamins and antioxidants on cardiovascular events (Myung, Ju, Cho, Oh, Park, Koo & Park, 2013). Despite -carotene has antioxidant activity, that is often regarded a pharmacological effect, several other clinical applications have failed
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to produce clear advantages of supplementation. Further research is required to assess the clinically relevant in vivo protective effects of -carotene supplementation against major diseases. There are no clinical data available on effects of echinenone in humans. The presented Nostoc strain can still be used as a source of -carotene, echinenone, or a supplement source material.
3.3 Euglena sanguinea The chromatogram of the saponified extract of Euglena sanguinea is shown in Figure 3, the retention times, UV-VIS spectrum data, molecular mass and identifications of carotenoids are shown in Table 3. -Carotene and it’s 9Z- and 13Z-isomers (peak 15-17) were identified as we described above in the case of Nostoc species. The absorption maxima of the main peak (peak 10) carotenoid in the HPLC eluent were 450, and 478 nm, and the spectral fine structure of %III/II was 44. It had a relative molecular
12
ACCEPTED MANUSCRIPT mass of 566 which corresponded to the formula of C40H54O2. Co-chromatography with authentic diatoxanthin sample suggested that peak 10 was diatoxanthin. Open column chromatography of the saponified extract resulted in five fractions. Fraction 4
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contained peak 10 carotenoid (~ 0.5 mg), after processing, was submitted to MALDI-TOF
IP
and 1H NMR analysis. The MALDI-TOF of this compound showed a molecular ion at m/z
hydrogen atoms probably in the polyene chain.
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566 which confirmed the formula C40H54O2 which was in agreement with missing two Owing to the small amount of sample, 1H,1H-COSY and
13
C NMR data could not be
recorded. Thus, NMR analysis was restricted to proton measurement of the peak 10
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carotenoid. The 1H NMR chemical shifts were compared with those of published Haugan & Liaaen-Jensen (1994) earlier. Although some minor contamination was present in the sample,
MA
most of the proton-signals known in the literature (Haugan & Liaaen-Jensen, 1994) were possible to be identified (see experimental section). Consequently, the NMR spectra confirmed the identity of peak 10 being diatoxanthin. Peak 8 was tentatively identified as
D
(13Z)- or (13’Z)-diatoxanthin by it’s UV-VIS spectrum and molecular mass. Unfortunately,
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other Z-isomers of diatoxanthin could not be detected. We suppose, that this isomers were overlapped by other components, and they could be detected in mixed peaks.
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The absorption maxima of the peak 11 carotenoid in the HPLC eluent were 440, and 469 nm (%III/II = 68) and it had a relative molecular mass of 582, which corresponded to the formula of C40H54O3. Based on these results peak 11 carotenoid was identified as diadinoxanthin (5,6-epoxy-diatoxanthin).
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In the UV−Vis spectra of the other main component peak 6 had 445, 474, nm as maxima and the [M+H-H2O]+ ion (551) was detected with MS as the main fragment. These were in accordance with above reported data for lutein. Three other minor compounds were identified successfully. Peak 3 carotenoid had the 412, 435, 463 nm maxima and relative molecular mass of 600. The co-chromatography with authentic sample confirmed the identity of peak 3 carotenoid with (9Z)-neoxanthin. Two other minor components were peak 18 and 19 carotenoids with 472, 499 nm and 468, 495 adsorption maxima respectively in the HPLC eluent. The HPLC-MS investigation showed 528 relative molecular mass for both compounds. Because of the lack of authentic samples and based on the findings of Fiksdahl & Liaaen-Jensen (1988) peak 18 was tentatively identified as 3,4,7,8,3’,4',7',8'-octadehydro-,-carotene and peak 19 as (9Z)3,4,7,8,3’,4',7',8'-octadehydro-,-carotene.
13
ACCEPTED MANUSCRIPT The non-saponified extract of E. sanguinea was studied similarly. In contrast with the composition of non-saponified Dunaliella and Nostoc extracts, the Euglena contained larger amounts of mono and diesters as is shown in Fig. 4. Carotenoids, represented in both free
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forms and as esters, were determined in the non-saponified extracts as presented in Table 4.
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Molecular weights (MW) of all possible combinations of fatty acids and carotenoids (monoand di-esters) were calculated, knowing that carotenoid esters are formed by the esterification
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of the carotenoid hydroxyl group. To make the identification of the esters easier, a small amount of non-saponified extract was separated by open column chromatography and we obtained three fractions: non-esterified carotenoids, monoesters and diesters. After the
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saponification of the mono and diester fractions practically, only free diatoxanthin was obtained, which indicated that lutein were present only in non-esterified form. The HPLC-MS
MA
investigation showed the presence of C16:0-, C17:0- and C18:0-monoesters of diatoxanthin and C15:0,15:0- (or C14:0,16:0-), C15:0,16:0-, C16:0,16:0-, C16:0,17:0- and C17:0,17:0- (or C16:0,18:0-), diesters of diatoxanthin. It should be noted, the occurrence of C15:0 and C17:0 carboxylic acids is very
D
rare.
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In contrast to the observations of Grung & Liaaen-Jensen (1993), who found astaxanthin as main component in see-water Euglena sanguinea, in the freshwater species lutein and
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diatoxanthin were the main compounds. In addition (9Z)-neoxanthin, diatoxanthin, (9Z)- and (all-E)--carotene were identified as minor carotenoids. Species of the Euglenophyta usually contain the following main carotenoids: -carotene
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and diadinoxanthin, with the minor carotenoids zeaxanthin, neoxanthin, diatoxanthin, loroxanthin and siphonaxanthin also being present in some species (Takaichi, 2011). The species of current interest, E. sanguinea, was shown to contain
diesters of (3S,3′S)-
astaxanthin as chief carotenoids in one study (Grung & Liaaen-Jensen 1993), as well as other diesters. In contrast, our study revealed no astaxanthin carotenoid derivate (measured as aglyca). Other studies (Gerber & Häder, 1994) have found -carotene, astaxanthin diester, and
diatoxanthin as main carotenoids in the same species, with many other minor
compounds as well. Biosynthetically, the carotenoid pattern of the scecies is dominated by diatoxanthin and lutein, the former being a product of the pathway involving -carotene, the latter a derivate of -carotene. There is no such information in the literature on different genotypes of E. sanguinea, but so far, it is worth to mention that members of the astaxanthin pathway, i.e. astaxanthin, canthaxanthin, echinenone (Takaichi, 2011) were not detected from our isolate.
14
ACCEPTED MANUSCRIPT Biosynthesis was rather shifted towards formation of a common -carotene pathway product, lutein. This phenomenon remains to be studied in later work. The carotenoid composition makes this isolate a potential source of dietary supplement
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production as well. The lutein pharmacological data has been briefly described previously.
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Diatoxanthin and alloxanthin and their cis-isomers suppress the expression of enzymes (COX-2, NOS) and molecules (cytokines, interleukines) participating in inflammatory
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Hosokawa, Sashina, Maoka & Miyashita, 2008).
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processes much more effectively than other carotenoids studied previously (Kobishi,
4. Conclusions
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Three non-toxic bloom-forming species were characterized for carotenoid composition. The three taxonomically distinct species contain high amounts of biologically active carotenoids. Of species interest, a lutein-accumulating Euglena sanguinea isolate was
D
presented, along with a high -carotene and echinenone containing Nostoc isolate, and a
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Dunaliella salina strain capable of accumulating lutein, violaxanthin and different carotenes. These species can be used as industrial sources of the presented carotenoids, and after further
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research, may potentially serve as functional food supplement raw materials.
Acknowledgments
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We thank Mrs. Judit Rigó, Ms. Zsuzsanna Götz and Mr. Roland Lukács for their assistance, Dr. Katalin Böddi for the MALDI-TOF measurement. This study was supported by the grant OTKA K 83898 (Hungarian National Research Foundation) and TÁMOP-4.2.2.A11/1/KONV-2012-0065 project. We also thank CaroteNature Gmbh (Switzerland) for providing us with carotenoid standards.
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Berendschot, T. T., Goldbohm, R. A., Klöpping, W. A., van de Kraats, J., van Norel, J., &
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Gerber, S., & Häder, D. P. (1994). Effects of enhanced UV-B irradiation on the red coloured freshwater flagellate Euglena sanguinea. FEMS Microbiology Ecology, 13(3), 177-184.
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Goodwin, T. W. (1980). The Biochemistry of the Carotenoids, Vol. I, Chapman and Hall, London.
Gouveia, L., & Emphis, J. (2003). Relative stability of microalgal carotenoids in microalgal
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Acta Chem. Scand. 48, 899-904. Kleinegris, D., Janssen, M., Brandenburg, W.A., & Wijffels, R.H., (2009). The selectivity of milking of Dunaliella salina. Marine Biotechnology 12, 14–23. Kobishi, I., Hosokawa, M., Sashima, T., Maoka, T., Miyashita, K. (2008) Suppressive effects of alloxanthin and diatoxanthin from Halocynthia roretzi on LPS-induced expression pf pro-inflammatory genes in RAW264.7 cells. Journal of Oleo Science 57, 181-189. Krinsky, N.I., Landrum, J.T., & Bone, R.A. (2003). Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye. Annual Reviews in Nutrition 23, 171-201. Krol, M., Maxwell, D.P., & Huner, N.P.A. (1997). Exposure of Dunaliella salina to low temperature mimics the high light-induced accumulation of carotenoids and the carotenoid binding protein (Cbr). Plant and Cell Physiology 38, 213–216.
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Liaaen-Jensen, S. (1977). Algal carotenoids and chemosystematics. In Faulkner, D. J. &
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Molnár, P., & Szabolcs, J. (1993). (Z/E)-Photoisomerization of C40-carotenoids by iodine. J. Chem. Soc., Perkin Trans. 2 261-266.
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ACCEPTED MANUSCRIPT Takaichi, S. (2011). Carotenoids in algae: distributions, biosynthesis and functions. Marine drugs, 9, 1101-1118. Takaichi, S., Maoka, T., & Mochimaru, M. (2009). Unique Carotenoids in the terrestrial
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Name
%
UV-VIS
(min)
IP
Ret.Time
m/z
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Peak No.
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Table 1. Carotenoid composition of saponified Dunaliella salina.
(nm)
+
[M+H ]
6.65
neochrome
0.45
405, 428, 446
601
2
7.19
neoxanthin
0.38
414, 439, 468
601
3
7.99
violaxanthin
11.49
415, 437, 467
601
4
8.79
(9Z)-neoxanthin
6.23
411, 435, 463
601
5
9.64
luteoxanthin
2.96
420, 446
601
6
11.37
antheraxanthin
1.34
447, 471
585
7
11.72
(13Z)-neoxanthin*
1.08
413, 436, 463
601
8
11.98
(13’Z)-neoxanthin*
1.56
418,437, 463
601
9
12.98
(13Z)-violaxanthin*
1.28
415, 437, 464
601
10
13.91
lutein
52.11
443, 471
551**
11
16.09
zeaxanthin
2.06
449, 476
569
12
17.47
(9Z or 9’Z)-lutein
0.48
439, 466
551
13
30.28
(13Z)--carotene
0.98
338, 443, 467
537
14
31.93
-carotene
0.53
444, 472
537
15
35.53
-carotene
13.10
450, 476
537
16
37.85
9Z-carotene
1.52
445,471
537
17
51.94
-carotene
0.44
459, 488
537
18
52.83
(9Z)--carotene
1.96
438, 460, 490
537
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CE P
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D
MA
NU
1
*changeable, ** [M+H+-H2O]
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Name
IP
Ret.Time
%
(min)
m/z
UV-VIS
SC R
Peak No.
T
Table 2. Carotenoid composition of saponified Nostoc sp.
(nm)
+
[M+H ]
6.28
unidentified cis
0.83
271, 448
543
2
7.61
unidentified cis
0.28
287, 421
567
3
9.13
unidentified red
0.42
465
567
4
10.48
unidentified red
1.06
438
557
5
15.04
unidentified red
1.16
455
551
6
15.96
unidentified red
0.54
471
565
7
16.93
unidentified red
1.01
470
565
8
17.93
canthaxanthin
4.73
474
565
9
20.27
mixture red
0.37
455
551
10
20.92
(9Z)-canthaxanthin
0.68
469
565
11
21.58
(13Z/13’Z)-echinenone
1.26
355, 455
551
12
21.88
(13’Z/13Z)-echinenone
1.10
355, 455
551
13
23.66
unidentified
0.36
450, 477
551
14
25.33
-carotene 5,6-epoxide
1.92
445, 472
553
15
26.41
echinenone
34.92
464
551
16
27.93
-carotene 5,8-epoxide
0.71
427, 452
553
17
28.87
(9Z/9’Z)-echinenone
3.95
454
551
18
29.33
(9’Z/9Z)-echinenone
2.33
452
551
19
30.27
(13Z)--carotene
1.94
443, 467
537
20
35.63
-carotene
36.48
450, 476
537
21
37.98
(9Z)--carotene
3.94
445, 471
537
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CE P
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D
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NU
1
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ACCEPTED MANUSCRIPT
Name
%
UV-VIS
(min)
(nm)
5.27
unidentified
3.97
2
5.85
unidentified
0.42
3
8.89
(9Z)-neoxanthin
4
12.03
5
m/z
+
[M+H ]
450
583
451
583
1.77
412, 435, 463
601
unidentified
0.30
417, 438, 466
583
13.29
unidentified
0.51
337, 439, 467
583
6
13.80
lutein
23.67
445, 474
551*
7
16.00
unidentified
2.57
433, 457
583
8
16.52
(13Z)-diatoxanthin
2.63
340, 454, 470
567
9
16.99
mixture
10
19.10
diatoxanthin
11
19.75
12
22.57
13
24.25
14
TE
D
MA
NU
1
IP
Ret.Time
SC R
Peak No.
T
Table 3. Carotenoid composition of saponified Euglena sanguiena
3.36
430, 456 583, 607 450, 478
567
diadinoxanthin
1.29
440, 469
583
unidentified
1.81
451, 479
591
unidentified
9.58
476
591
25.10
unidentified
0.60
347, 445, 472
591
15
30.07
(13Z)--carotene
0.50
338, 443, 468
537
16
35.42
-carotene
5.36
450, 476
537
17
37.78
(9Z)--carotene
0.96
421, 446, 472
537
18
42.21
unidentified
1.13
472, 498
529
19
45.94
unidentified
0.54
468, 495
529
AC
CE P
39.01
* [M+H+-H2O]
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D
TE
IP
0.79 1.85 6.27 5.73 0.69 13.43 2.93 15.77 0.87 0.54 2.11 0.95 0.79 3.59 3.59 1.59 0.77 2.98 3.51 0.36 0.91 4.12 1.08 5.82 1.44 10.65 0.95 5.89
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(9Z)-neoxantin unidentified lutein not carotenoid not carotenoid unidentified unidentified diatoxantin diadinoxanthin unidentified unidentified. unidentified chlorophyll pheophytine C17-diatoxanthin unidentified unidentified C18-diatoxanthin unidentified C17-diatoxanthin unidentified. C18-diatoxanthin unidentified. C15,C15-diatoxanthin C15,C16-diatoxanthin C16,C16-diatoxanthin C16,C17-diatoxanthin C17,C17-diatoxanthin
UV-VIS (nm) 412, 435, 463 460 445,474 465 464 477 428, 456 450, 478 419, 440, 469 480 454, 478 445, 472 435, 652 409 451, 476 470 446, 471 478 479 480 471 479 470 470 480 479 479 479
SC R
%
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Name
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Ret.Time (min) 8.80 11.14 13.66 14.03 15.40 15.88 16.87 18.89 19.57 20.40 22.33 24.94 32.10 34.78 35.59 36.73 37.91 38.83 39.59 41.48 42.13 43.53 45.91 51.94 53.21 54.48 55.75 57.00
AC
Peak No.
T
Table 4. Carotenoid composition of non-saponified Euglena sanguiena.
m/z + [M+H ] 601 565 551 595 not detected 583, 595 583 567 583 595 593 377 885 805, 871 805 819 805 833 805 819 805 833 813 1015 1029 1043 1057 1071
* [M+H+-H2O]
23
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2 000
SC R
IP
T
Figure 1
mAU
1 750
10
NU
1 500
WVL:450 nm
MA
1 250
1 000
D
750
TE
3
500
4 12
-200
9
10.0
11
12
20.0
13 14
16
17
18
min 30.0
40.0
50.0
60.0
70.0
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0.0
5 678
CE P
250
15
HPLC chromatogram of Dunaliella salina (peak identification in Table 1.)
24
ACCEPTED MANUSCRIPT
mAU
IP
1 200
T
Figure 2
WVL:450 nm
SC R
20
1 000
15
NU
800
MA
600
400
200 2
3 4
5
7
6
9
10 11
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1
D
8
-200 0.0
5.0
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0
10.0
15.0
17 1213
14
16
21
19 18
min 20.0
25.0
30.0
35.0
40.0
45.0
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HPLC chromatogram of the saponified extract of Nostoc sp. (peak indentification in Table 2.)
25
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900
T
Figure 3
mAU
750
SC R
10
WVL:450 nm
6
NU
625
500
250
MA
375
1 11 8 7 9 45
-100 0,0
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2
10,0
16
12 14
TE
3
13
D
125
20,0
15
17
18
19
min 30,0
40,0
50,0
60,0
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HPLC chromatogram of saponified extract of Euglena sanguinea (peak identification in Table 3.)
26
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mAU
IP
8
6
NU
300
200
3
100
MA
4
2
0.0
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10.0
20.0
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26 28
19
15 14
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22 25
11
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HPLC chromatogram of non-saponified extract of Euglena sanguinea (peak identification in Table 4.)
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ACCEPTED MANUSCRIPT Highlights Characterziation of the carotenoids of a lutein-rich Dunaliella salina strain.
Characterziation of the carotenoids of an echinenone-rich Nostoc strain.
Characterziation of the carotenoids of a diatoxanthin-rich Euglena sanguinea strain.
Three non-toxic algae were shown to be excellent natural sources of carotenoids.
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