Article pubs.acs.org/JAFC
Molecular Characterization and Anti-inflammatory Activity of Galactosylglycerides and Galactosylceramides from the Microalga Isochrysis galbana Carolina de los Reyes,† María J. Ortega,† Azahara Rodríguez-Luna,§ Elena Talero,§ Virginia Motilva,§ and Eva Zubía*,† †
Departamento de Quı ́mica Orgánica, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510 Puerto Real (Cádiz), Spain § Departamento de Farmacologı ́a, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain S Supporting Information *
ABSTRACT: Isochrysis galbana is a marine microalga rich in PUFAs that is widely used as feed in aquaculture and more recently investigated for its potential in food applications and as source of bioactive compounds. In this study, the biomass obtained from cultures of I. galbana has been investigated to determine its content in glycosylglycerides and glycosylceramides. By using NMR, UPLC-MS/MS, and fatty acid profiles, the structures of ten monogalactosyldiacylglycerols (MGDGs 1−10) and nine digalactosyldiacylglycerols (DGDGs 11−19) have been established. Two distinctive features of the galactosylglycerides from I. galbana are the wide presence of highly unsaturated acyl chains derived from stearidonic acid (18:4Δ6Z,9Z,12Z,15Z) and octadecapentaenoic acid (18:5Δ3Z,6Z,9Z,12Z,15Z), as well as the unusual coexistence of αβ-DGDGs and ββ-DGDGs. Three new galactosylceramides, isogalbamides A-C (20−22), have also been isolated and characterized by NMR and MS/MS. These metabolites, which are the first galactosylceramides described from microalgae, derive from unprecedented tetraolefinic sphingoid bases. In anti-inflammatory assays, the MGDG and DGDG mixtures and the isolated DGDGs 11 and 12 showed significant activity as inhibitors of the production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide-stimulated human THP-1 macrophages, while the galactosylceramides showed moderated activity. KEYWORDS: microalgae, Isochrysis galbana, galactosylglycerides, MGDG, DGDG, galactosylceramides, anti-inflammatory activity, TNF-α inhibition
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INTRODUCTION Over the past decades, microalgae have been the focus of a growing interest among researchers, manufacturers, and entrepreneurs in the fields of food and nutrition because of the capacity of certain microalgal species to produce high-value products, in particular, long-chain ω-3 polyunsaturated fatty acids (PUFAs) and carotenoids.1−4 The high growth rates of microalgal cultures, the possibility of enhancing the synthesis or accumulation of the desired metabolites by the culture conditions, and the feasibility of setting large-scale cultures in various climates and nonarable lands make microalgae even more attractive as resources for the production of bioactive compounds.1,2,5,6 However, only a few species of microalgae are currently on the market,1,2 and new classes of microalgal metabolites deserve to be explored. In particular, polar lipids of microalgae such as the glycolipids have scarcely been studied from the points of view of both structure and bioactivity.7−12 In microalgae, similar to macroalgae and seed plants, the predominant class of glycolipids is formed by the galactosylglycerides, which form the thylakoid membranes of the chloroplasts where the oxygenic photosynthesis takes place.13,14 The two main classes of galactosylglycerides are the monogalactosyldiacylglycerols (MGDGs) and the digalactosyldiacylglycerols (DGDGs), which may contain a large portion of the ω-3 PUFAs of the microalga.13,15 Interestingly, several studies have shown that various galactolipids from vegetables and edible © 2016 American Chemical Society
macroalgae possess antitumor-promoting and anti-inflammatory properties, suggesting the possible contribution of this class of metabolites to the beneficial effects of a diet rich in vegetables and fruits, as well as the potential of the galactosylglycerides for the development of nutraceuticals.16−18 Another class of bioactive polar lipids is formed by the monohexosylceramides, also known as cerebrosides.14,19 These are sphingolipid derivatives consisting of a long-chain amino alcohol, or sphingoid base, in which the amino group is amidelinked to a fatty acid and the primary hydroxy group is glycosidelinked to a hexose. The presence of this class of metabolites in microalgae is scarcely known, and only recently has the isolation of two glucosylceramides from a microalga of the genus Tetraselmis been reported.20 In this context, the aim of this research was to study the biomass derived from mass cultures of the microalga Isochrysis galbana to characterize its content in glycolipids, in particular, glycosylglycerides and glycosylceramides. I. galbana is a marine microalga widely used in aquaculture as feed for larvae of mollusks, crustaceans, and fishes because of its suitable cell features and its nutritional value, especially its PUFA composition.21 Received: Revised: Accepted: Published: 8783
September 2, 2016 October 26, 2016 October 26, 2016 October 27, 2016 DOI: 10.1021/acs.jafc.6b03931 J. Agric. Food Chem. 2016, 64, 8783−8794
Article
Journal of Agricultural and Food Chemistry
(9:1, v/v, 10 mL each) and then with MeOH (10 mL each). The methanolic solutions were combined and evaporated to yield the fraction I-MGDG (788 mg). Fraction C4 was suspended in MeOH/H2O (9:1, v/v, 1 mL), transferred onto an SPE-C18 cartridge (1 g) preconditioned with MeOH/H2O (9:1, v/v, 2 mL), and eluted with MeOH/H2O (9:1, v/v, 10 mL) and then with MeOH (10 mL). The methanolic solution was evaporated under reduced pressure to yield the fraction I-DGDG (113 mg). An aliquot of fraction I-DGDG (61 mg) was separated by reversed phase HPLC (MeOH/H2O, 98:2, v/v) to yield the digalactosyldiacylglycerols 11 (4.8 mg) and 12 (9.4 mg). Fraction C3 was suspended in MeOH/H2O (9:1, v/v, 50 mg/mL) and transferred onto SPE-C18 cartridges (2 × 1 g) preconditioned with MeOH/H2O (9:1, v/v, 2 mL) that were eluted with 15 mL of the same solvent and then with 15 mL of MeOH. The methanolic fraction was evaporated and repeatedly separated by reversed phase HPLC (MeOH/ CH3CN, 8:2, v/v) to yield the galactosylceramides 20 (tR 33.6 min, 15.2 mg), 21 (tR 30.1 min, 4.8 mg), and 22 (tR 35.5 min, 3.1 mg), together with a mixture of the oxidation compounds 23a/23b (tR 16.4 min, 3.1 mg). Basic Hydrolysis of Galactosylglycerides. A sample of fraction I-MGDG (35.1 mg) was disolved in 5 mL of a mixture 1:1, v/v, of MeOH and aqueous KOH (10%), and the mixture was stirred at room temperature for 2 h. After the addition of aqueous HCl (1%) to pH 5, the mixture was extracted with n-hexane (3 × 5 mL) to remove fatty acids. The residual aqueous layer was transferred onto an SPE-C18 cartridge (500 mg) and eluted with MeOH (5 mL). The evaporation of the solvent under reduced pressure yielded (2R)-1-O-β-D-galactopyranosylglycerol: [α]25D −3.0 (c 0.1, H2O), lit.8 [α]D −4.5 (c 0.17, H2O). Determination of MGDG and DGDG Molecular Species by UPLC-MS/MS. A sample of fraction I-MGDG and a sample of fraction I-DGDG were separately dissolved in MeOH at a concentration of 50 μg/mL and subjected to UPLC-MS. The analyses were performed on an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm) (Waters), flow rate = 0.8 mL/min, injection volume = 5.0 μL. The mobile phase was composed of (A) acetonitrile and (B) H2O + formic acid (0.1%). The gradient elution program was from 90% A at t = 0 to 95% A at t = 4 min and 100% A at t = 6 min. The mass spectrometer was operated in the positive electrospray ionization mode (ESI(+)), full scan acquisition. The parameters were as follows: source temperature, 120 °C; capillary voltage, 3.0 kV; cone voltage, 40 V (for I-MGDG) or 20 V (for I-DGDG); mass scan range m/z 100−1000 Da (for I-MGDG) or 200−1200 Da (for I-DGDG). MS/MS spectra were obtained using trap collision energies of 40 eV for I-MGDG and 45 eV for I-DGDG. Analysis of the Fatty Acid Composition of the MGDGs and DGDGs by GC-MS. A portion of each galactolipid fraction (34.2 mg for I-MGDG and 14.0 mg for I-DGDG) was treated with MeOH/HCl 10:1 (v/v) as described previously.27 The fatty acid methyl ester (FAME) mixtures thus obtained were dissolved in CH2Cl2 and subjected to GC-MS analysis using an Agilent J&W DB-5ms column (250 μm × 30 m, 0.25 μm film) (Agilent). An aliquot was analyzed on a QUATTRO Micro GC instrument with He as carrier gas, operating at 70 eV. The column temperature was elevated from 150 to 300 °C (8 °C/min) and held at 300 °C for 1 min. Another aliquot of the FAME mixture was analyzed on a high-resolution SYNAPT G2 instrument using atmospheric pressure ionization (API) in positive ionization mode. The column temperature was maintained at 90 °C for 1 min, then raised to 200 °C (20 °C/min) and to 300 °C (5 °C/min), and held at 300 °C for 2 min. Fatty acids were identified by comparison of retention time, molecular formula, and mass spectral data with FAME standards (Supelco-37) and a NIST library. UPLC-MS/MS Analysis of DGDGs 11 and 12. Analyses were performed on an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm) (Waters) at a flow rate of 0.8 mL/min and an injection volume of 5.0 μL. The mobile phase was composed of (A) acetonitrile and (B) H2O + formic acid (0.1%). The gradient elution program was from 85% A, 15% B at t = 0 to 100% A at t = 3 min. ESI(+) and ESI(−) mass spectra were collected in full scan acquisition. The parameters were as follows: source temperature, 150 °C; capillary voltage, 3 kV;
More recently, several studies have focused on the use of the biomass of I. galbana for food applications, in particular, to enrich biscuits and pasta products.22−24 In addition, the biomass of I. galbana could be a valuable resource for the extraction of bioactive compounds.2,25 Although a number of chemical analyses of I. galbana can be found in the literature, they mainly describe biochemical compositions and fatty acid profiles performed during studies on culture, extraction procedures, or applications of this microalga.21,25,26 Herein we describe the first characterization of the MGDGs, DGDGs, and glycosylceramides of I. galbana together with a study of their anti-inflammatory properties as inhibitors of tumor necrosis factor-α (TNF-α), a cell signaling protein involved in the systemic acute phase inflammatory response.
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MATERIALS AND METHODS
General. Optical rotations were measured on a PerkinElmer 341 polarimeter (PerkinElmer, Boston, MA, USA). 1H and 13C NMR spectra were recorded on an Agilent-500 (Agilent Technologies, Santa Clara, CA, USA) using CD3OD (Merck KGaA, Darmstadt, Germany) or CDCl3 (Sigma-Aldrich, St. Louis, MO, USA) as solvent. Chemical shifts were referenced using the corresponding solvent signals (δH 3.30 and δC 49.0 for CD3OD, δH 7.26 and δC 77.0 for CDCl3). COSY, HSQC, and HMBC experiments were performed using standard Agilent pulse sequences. UPLC-MS analyses were performed by using an Acquity UPLC H-CLASS system (Waters, Milford, MA, USA) coupled to an HRMS SYNAPT 2G (Waters) equipped with an electrospray interface (ESI) and a quadrupole-time-of-flight (QTOF) analyzer. GC-MS analyses were performed on a QUATTRO Micro GC instrument (Waters) and on an HRMS SYNAPT 2G (Waters) equipped with an APGC interface and a QTOF analyzer. Column chromatography was carried out on silica gel (Kiesegel 60, 63−200 μm) (Merck). SPE separations were performed on Supelco DSC18 cartridges (500 mg/3 mL or 1 g/6 mL) (Supelco, Bellefonte, PA, USA). HPLC separations were performed on a LaChrom-Hitachi apparatus (Merck) equipped with LiChrospher 100 RP-18 (250 × 10 mm, 10 μm) (Merck) and Kromasil 100-5C18 (250 × 10 mm, 5 μm) (Hichrom, Reading, UK) columns, using a differential refractometer RI-71 (Merck). Phorbol 12-myristate 13-acetate (PMA), dexamethasone, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All solvents were of HPLC grade. Biological Material. Dried biomass derived from mass cultures of I. galbana (seawater species, Phylum Haptophyta, Class Coccolithophyceae, Order Isochrysidales, Family Isochrysidaceae) was provided by the Department of Biotechnology of the Instituto Tecnológico de Canarias (Santa Lucı ́a, Gran Canaria, Spain). Stocks of I. galbana are maintained at the Microalgae Collection of the Instituto Tecnológico de Canarias under the code ITC-Iso-01. Maintenance of stocks, scale-up of inocula, and production of the biomass were performed using f/2 medium. Extraction, Obtention of MGDG and DGDG Fractions, and Isolation of Compounds. The dried biomass of I. galbana (60 g) was extracted with acetone/MeOH (1:1, v/v, 4.5 L) at room temperature. After filtration, the solvent was evaporated under reduced pressure (bath temperature = 35 °C), and the resulting extract (18.5 g) was subjected to silica gel column chromatography (30 × 5 cm) using as eluents n-hexane/Et2O 1:1, v/v (1.0 L), Et2O (1.0 L), CHCl3/MeOH 8:2, v/v (1.0 L), and MeOH (1.0 L). The fraction eluted with CHCl3/MeOH (8:2, v/v), namely, fraction C (3.0 g), was further separated by silica gel column chromatography (30 × 3 cm) using as eluents n-hexane/EtOAc mixtures (4:6 and 3:7, v/v, 0.4 L each), then EtOAc (0.4 L), CHCl3/ MeOH mixtures (9:1, 85:15, and 8:2, v/v, 0.4 L each), and finally MeOH (0.25 L). After TLC and 1H NMR analysis, the aliquots were pooled into fractions C1 (463 mg), C2 (1.1 g), C3 (420 mg), C4 (163 mg), C5 (249 mg), and C6 (347 mg). Fraction C2 was suspended in MeOH/H2O (9:1, v/v, 5 mL), transferred onto SPE-C18 cartridges (4 × 1 g) preconditioned with MeOH/ H2O (9:1, v/v, 2 mL each cartridge), and eluted with MeOH/H2O 8784
DOI: 10.1021/acs.jafc.6b03931 J. Agric. Food Chem. 2016, 64, 8783−8794
Article
Journal of Agricultural and Food Chemistry cone voltage, 50 V; trap collision energy, 20−40 eV; mass scan range, m/z 100−1200 Da. Characterization of DGDGs 11 and 12. Compound 11: [α]25D −2 (c 0.1, MeOH); HRESIMS(+) m/z 953.5262 [M + Na]+ (calcd for C51H78O15Na, 953.5238); ESI(+)-MS/MS m/z (rel int) 953 (40), 679 (100), 677 (18); ESI(−)-MS/MS m/z (rel int) 929 (5), 673 (40), 415 (36), 397 (100), 275 (66), 273 (24). Compound 12: [α]25D +30 (c 0.1, MeOH); HRESIMS(+) m/z 931.5397 [M + Na]+ (calcd for C49H80O15Na, 931.5395); ESI(+)-MS/MS m/z (rel int) 931 (33), 677 (13), 657 (100); ESI(−)-MS/MS m/z (rel int) 907 (2), 651 (40), 415 (30), 397 (100), 273 (22), 253 (36). 1H NMR (CD3OD, 500 MHz) and 13 C NMR (CD3OD, 125 MHz) data of compounds 11 and 12 can be found in Table 2. UPLC-MS/MS Analysis of Isogalbamides A−C (20−22). Analyses were performed on an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm) (Waters), at a flow rate of 0.6 mL/min and an injection volume of 5.0 μL. The mobile phase was composed of (A) H2O + formic acid (0.1%), (B) acetonitrile, and (C) methanol. The gradient elution program was from 10% A, 45% B, 45% C at t = 0 to 95% B, 5% C at t = 9 min. The mass spectrometer was operated in ESI(+) mode, in full scan acquisition. The parameters were as follows: source temperature, 150 °C; capillary voltage, 0.7 kV; cone voltage, 20 V; trap collision energy, 25 eV. The elution of compounds from the column was simultaneously monitored by a photodiode array (PDA) detector (Waters) at λ 250−300 nm. Characterization of Galactosylceramides 20−23. Isogalbamide A (20): [α]25D +2.2 (c 0.1, MeOH); HRESIMS(+) m/z 806.6157 [M + H]+ (calcd for C47H84NO9, 806.6146); ESI(+)-MS/MS m/z (rel int), 788 (15), 644 (3), 626 (100), 608 (65), 336 (19), 273 (32). Isogalbamide B (21): [α]25D +1.7 (c 0.1, MeOH); HRESIMS(+) m/z 792.6013 [M + H]+ (calcd for C46H82NO9, 792.5990); ESI(+)-MS/MS m/z (rel int) 792 (15), 774 (51), 630 (11), 612 (100), 594 (62), 336 (22), 259 (11). Isogalbamide C (22): [α]25D +4.3 (c 0.1, MeOH); HRESIMS (+) m/z 794.6148 [M + H]+(calcd for C46H84NO9, 794.6146); ESI(+)-MS/MS m/z (rel int): 776 (51), 632 (23), 614 (100), 596 (23), 456 (8), 452 (21), 356 (24), 259 (17), 258 (17). Mixture of 23a/23b: [α]25D +2.3 (c 0.1, MeOH); HRESIMS (+) m/z 838.6047 [M + H]+(calcd for C47H84NO11, 838.6044). 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) of compounds 20−23 can be found in Table 4. Acid Methanolysis of Isogalbamide A (22). Compound 22 (7.8 mg) was dissolved in 2 mL of MeOH/HCl (10:1, v/v) and refluxed for 15 h. After cooling, the reaction was extracted with n-hexane (3 × 3 mL). The hexane layers were combined, rinsed with brine (3 mL), and dried over MgSO4. After filtration and evaporation of the solvent under reduced pressure, the methyl ester 24 was obtained (3.4 mg). (2R,3E)-2Hydroxydocosa-3-enoic acid methyl ester (24): [α]25D −41.7 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 5.88 (dddd, 1, J = 15.2, 6.8, 6.8, and 1.5 Hz, H-4), 5.50 (ddt, 1, J = 15.2, 6.2, and 1.5 Hz, H-3), 4.60 (br d, 1, J = 6.2 Hz, H-2), 3.80 (s, 3, OMe), 2.06 (dt, 2, J = 7.0 and 7.0 Hz, H2-5), 1.25−1.40 (m, 32, H2-6 to H2-21), 0.88 (t, 3, J = 7.0 Hz, Me-22); HRESIMS(+) m/z 351.3304 [M + H − H2O]+ (calcd for C23H43O2, 351.3263). Cell Culture. The THP-1 human monocytic leukemia cell line was obtained from the American Type Culture Collection (TIB-202, ATCC, USA) and cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Anti-inflammatory Assays. Cytotoxicity Assay. The viability of THP-1 cells upon exposure to fractions I-MGDG and I-DGDG (6.25, 12.5, 25, 50, and 100 μg/mL) and to compounds 11, 12, and 20−22 (6.25, 12.5, 25, 50, and 100 μM) was determined by the sulforhodamine B (SRB) assay as previously described.27 Determination of TNF-α Production. For differentiation into macrophages, THP-1 cells (105 cells/mL) were incubated with PMA (0.2 μM) for 3 days in 96-well plates as previously described.27 Then, the medium was removed, and the cells were washed with PBS, and incubated for 1 h with fractions I-MGDG and I-DGDG (12.5 and 25 μg/mL each), DGDGs 11 and 12 (12.5 and 25 μM each), and galactosylceramides 20−22 (6.25 μM) (the viability of cells was >95% throughout the experiment). Dexamethasone (1 μM) was used as
positive reference compound. All of the solutions were prepared by dilution of stock solutions in DMSO with the appropriate amounts of fresh medium. The highest concentration of each treatment used in the assay contained 0.1%, v/v, DMSO. Subsequently, the inflammatory response was induced by addition of lipopolysaccharide (LPS, 1 μg/mL). Controls contained medium with equivalent amounts of solvent compared to treatments and were incubated with and without LPS. After 24 h of incubation, supernatant fluids were collected and stored at −80 °C until TNF-α measurement. A commercial ELISA kit (Diaclone GEN-PROBE, Besançon cedex, France) was used to quantify TNF-α according to the manufacturer’s protocol.
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RESULTS AND DISCUSSION Obtention of MGDG and DGDG Fractions from the Biomass of I. galbana. After extraction of the biomass with acetone/MeOH and subsequent silica gel column chromatography of the lipid extract, the glycolipids were in the fraction eluted with CHCl3/MeOH (8:2, v/v). Two consecutive separation steps of this fraction by silica gel column chromatography and SPE on C18 cartridges led to the obtention of a fraction containing MGDGs (I-MGDG) and a fraction containing DGDGs (I-DGDG). The efficiency of the separation procedure and the type of glycosylglycerides contained in these fractions were unambiguously confirmed from their 1H NMR spectra (Figure 1). Thus, the 1H NMR spectrum of fraction I-MGDG (Figure 1A) exhibited between 3.4 and 5.3 ppm the signals of a galactose residue linked to a glycerol unit. In particular, the signal at δ 3.82 (dd, J = 3.3 and 1.1 Hz), typical of the equatorial proton H-4′ of a galactose moiety, and the signal at δ 4.23 (d, J = 7.7 Hz), due to the axial anomeric proton H-1′ of a β-glycosidic bond, were especially diagnostic. The remaining signals of the spectrum, between 0.8 and 1.0 ppm, between 1.2 and 3.0 ppm, and between 5.3 and 5.6 ppm, were due to the terminal methyl groups, the methylene groups, and the olefinic protons, respectively, of the acyl chains. Basic hydrolysis of fraction I-MGDG yielded a compound identified as (2R)-1-O-β-D-galactopyranosylglycerol,8 thus supporting the S configuration at C-2 on the glycerol moiety and the D configuration of the galactose residue. The 1H NMR spectrum of fraction I-DGDG (Figure 1B) exhibited between 3.4 and 5.3 ppm numerous signals attributable to two galactose residues linked to a glycerol unit. In particular, the signal at δ 4.86 (d, J = 3.7 Hz) was typical of the equatorial anomeric proton H-1″ of the terminal galactose of DGDGs exhibiting the usual configuration Gal[α1″→6′]Gal (I in Figure 1B). Interestingly, the spectrum also showed a signal at δ 4.31 (d, J = 7.3 Hz) attributable to the axial anomeric proton H-1″ of DGDGs containing the terminal galactose residue bound in β-glycosidic linkage (II in Figure 1B).28 The configuration at C-2 of the glycerol moiety in the DGDGs was proposed to be the same as that found in the MGDGs based on biosynthetic grounds, by taking into account that DGDGs derive from MGDGs by addition of a galactose residue either from UDPgalactose or from another MGDG molecule to form αβ- and ββ-DGDGs, respectively.29 To the best of our knowledge, this is the first description of DGDGs exhibiting the unusual configuration Gal[β1″→6′]Gal in microalgae. Moreover, only a few papers have described the presence of this type of DGDG in higher plants, in particular, leguminous seeds.30 Determination of MGDG Molecular Species by UPLC-MS/MS. Fraction I-MGDG was subjected to analysis by UPLC-ESI(+)MS and, after a series of trials, suitable conditions were found for the chromatographic separation of the 8785
DOI: 10.1021/acs.jafc.6b03931 J. Agric. Food Chem. 2016, 64, 8783−8794
Article
Journal of Agricultural and Food Chemistry
Figure 1. 1H NMR spectra (500 MHz, CD3OD) of fractions (A) I-MGDG and (B) I-DGDG obtained from the biomass of the microalga Isochrysis galbana. Numbers above the signals correspond to the protons of the galactose and glycerol moieties.
Figure 2. Total ion current (TIC) UPLC-ESI(+)MS chromatogram of fraction I-MGDG obtained from the extract of I. galbana.
compounds in
