Oxidative damage is initiated by reactive oxygen species (ROS) and influences the pathogenesis of various disorders, such as atherosclerosis, neurodegenerative diseases, inflammatory diseases and aging. The mechanism of oxidative stress is an imbalance between ROS production and the antioxidant defense capacities of the cell, leading to reactions between those oxidative molecules and lipids, proteins and DNA [1
Endothelial dysfunction is recognized as an early marker of atherogenic risk [3
]. Physiologically, the respiratory chain in mitochondria produces small amounts of ROS. Under these conditions, free radicals are involved in the regulation of several processes, such as signaling pathways and gene expression, but they are also involved in the proliferation, migration and apoptosis of vascular cells [5
]. In an overproduction of ROS (hypertension, hypercholesterolemia, ischemia/reperfusion, hyper oxygenation, stress, pollution, radiation, etc
.), the physiological mechanisms of control and protection are overwhelmed, and ROS interact with nitric oxide (NO) to form peroxynitrite (ONOO−
), a highly toxic compound. The bioavailability of endothelial NO is then reduced, and its vasoprotective effect is disrupted [6
Antioxidant molecules, especially carotenoids, play an important role in the control of the oxidative process. These antioxidant molecules (carotenoids) possess a strong antioxidant power due to their double-bonded structure, allowing the delocalization of impaired electrons [9
]. There is a growing interest in astaxanthin (3,3′-dihydroxy-β-β′-carotene-4,4′-dione), from the carotenoids’ family. It is a xanthophyll, meaning that, unlike β-carotene and lycopene, it is a polar molecule that is able to scavenge free oxygen radicals. The polyene chain in astaxanthin crosses the cell membrane, allowing the polar ends of the molecule to be exposed to the cytoplasm and external sides of the cell in the meantime [1
]. This disposition facilitates the electron transfer from the cytoplasm to the outer part of the cell. The terminal ring of the astaxanthin seems to be the final scavenger of the ROS. The astaxanthin by its provision could also have a synergistic effect with vitamin C that would recharge astaxanthin once it has scavenged ROS [12
]. One of the major applications of astaxanthin is its use as feed additive for salmonid fish species. The European Food Safety Authority recommends a dietary amount of synthetic astaxanthin of 100 mg/kg. This astaxanthin diet can be extended to other fish, ornamental fish and crustaceans at the same dose [13
]. Moreover, its antioxidant properties confer to astaxanthin an interesting therapeutic potential with applications, such as anticancer, anti-diabetic and anti-inflammatory agent [1
]. This molecule has shown promising results in animal, but also in human experiments, leading to a decrease of blood pressure and an increase of HDL rate [11
]. When used in pre-conditioning, astaxanthin lessened the extent of myocardial infarction in rats and rabbits and thus appears very promising for cardiovascular treatment [17
]. Oral administration of astaxanthin to ApoE and Low Density Lipoprotein-Receptor KO mice also reduced the occurrence of aortic atheroma [19
] and, when tested on HUVEC and murine platelets, showed a decreased production of ONOO−
and increased NO bioavailability [20
Natural astaxanthin may come from different origins, derived from algae, crustaceans or krill, and be extracted in many different ways [1
]. Thus, synthetic astaxanthin (AstaS) is a racemic mixture of three isomers (3-R
) and (3-S
], whereas the chemical composition of natural astaxanthin depends on the natural source and even on the extraction method applied. For instance, extracts of Haematococcus pluvialis
(a reference microalgae strain for the production of natural astaxanthin) contains a mixture of carotenoids where an astaxanthin isomer (3-S
) and its ester derivatives are the major compounds [11
]. Due to the lack of homogeneity in the composition of the natural source samples, a complete physicochemical study is mandatory. The purpose of this work was to carry out a comparative study between three astaxanthin samples: synthetic astaxanthin and natural astaxanthin from Haematococcus pluvialis
extracted by two different methods. First, a physicochemical characterization of samples by spectrophotometry and HPLC-MS was performed. Next, their antioxidant activities were evaluated by using the Trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays. Finally, a biological evaluation was carried out in order to assess their biocompatibility and their ability to inhibit intracellular stress in human endothelial cells for cardiovascular applications.
3. Experimental Section
3.1. Chemicals and Biological Reagents
Dimethyl sulfoxide (DMSO, Lot: SZBD1830V), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Isopropanol, 2′,7′-dichlorofluorescein 3′6′-diacetate (2′,7′-DCFH-DA, Lot: 092M4004V), tert-butyl hydroperoxide (t-BuOOH/Luperox Lot: BCBJ2885V), AAPH (2,2′-azobis(2-amidino-propane) dihydrochloride (Ref. 440914) and fluorescein (Ref. F6377) were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). The same firm also provided 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, lot: BCBJ8170V) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS, lot: 061M538V). Acetone (HPLC gradient grade) was supplied by Panreac (Barcelona, Spain).
Concerning the cell culture, minimum essential medium (MEM), phosphate buffered saline (PBS), fetal calf serum and penicillin-streptomycin-amphotericin (PSA) were provided by GIBCO (Life Technologies, Carlsbad, CA, USA). Stable glutamine was purchased from PAA (PAA laboratories GmbH, Pasching, Austria). Human umbilical vein endothelial cells (HUVEC) were purchased from ATCC (CRL 1730). The synthetic astaxanthin (AstaS) and echinenone standard were purchased from Ehrenstorfer Standards (LGC Standards). Canthaxanthin standard was purchased from Fluka (Buchs, Switzerland).
3.2. Algae Material
The powder (AstaP) and AstaCO2 oleoresin of Haematococcus pluvialis, a microalgae strain from the coast of Chile, were purchased from Pigmentos Naturales (Pigmentos Naturales, SA, Iquique Chile). The extraction of AstaCO2 oleoresin was prepared by NATECO2 (85283 Wolnzach, Germany) using supercritical CO2 as the solvent for the extraction. This AstaCO2 oleoresin is commercially available as 10% w/w AstaCO2/sunflower oil. All of those products were stored at −20 °C to avoid degradation of thermal compounds.
3.4. Spectrophotometric Measurements and Astaxanthin Concentration Calculations
Spectrophotometric measurements were made on a Perkin Elmer Lambda 12 Spectrophotometer in the UV-visible domain (200–800 nm), using a scan speed of 50 nm/min. The AstaS spectra show a maximum absorption at 490 nm. A calibration curve was obtained from AstaS.
The astaxanthin extinction coefficient (ε482 = 1.25 × 105 L·mol−1·cm−1) was obtained from the synthetic astaxanthin (AstaS) calibration curve in DMSO and used to calculate the astaxanthin contents in the extracts (AstaP and AstaCO2). Our stock solutions for the biological experiments are respectively 2.1 and 127 mM for AstaP and AstaCO2.
The antioxidant capacities of natural (AstaP), supercritical (AstaCO2
) and synthetic astaxanthin (AstaS) were realized by the TEAC method [27
]. Briefly, a stock solution of the free radical ABTS•+
(7 mM) was prepared by mixing vol/vol ABTS solution (7 mM) and potassium persulfate (K2
, 270.322 g/mol, colorless) solution (2.45 mM) in distilled water. The mixture was placed in the dark, at room temperature for 12 to 16 h before use. Then, we diluted the solution in PBS (1/1000) in order to have an absorbance of 0.7 ± 0.2 at 734 nm. The standard antioxidant used in this test was Trolox. Before use, solutions of Trolox and samples were freshly prepared at different concentrations in PBS 1×. Fifty microliters of these solutions were added to 1 mL ABTS•+
and incubated 1 h at 25 °C; then, absorbance was measured at 734 nm (UV-Visible Lambda 12, PerkinElmer Inc., Norwalk, CT, USA). The inhibition percentage was calculated with the following formula:
Then, the percentage of inhibition was plotted versus the antioxidant concentration. The TEAC corresponds to the Trolox concentration having the same antioxidant activity as the considered sample. To calculate TEAC, we plotted the curves of the inhibition degree of Trolox and of the sample, then we divided the slopes of the curves by the Trolox curve slope. The TEAC coefficient, being a slope ratio, is unitless. We can report this value for the initial mass of products, pure or natural mixtures, in mmol Trolox/g. The TEAC was evaluated at different times.
Antioxidant capacity of natural (AstaP), supercritical (AstaCO2
) and synthetic astaxanthin (AstaS) was performed by the ORAC method [43
]. Briefly, solutions of fluorescein (4 nM), AAPH (2,2′-azobis(2-amidino-propane) dihydrochloride, 160 μM) and Trolox (0–100 μM) were prepared in PBS. One hundred fifty microliters of fluorescein solution were added to each well (96-well microplate), and 25 μL of samples, blank (PBS) or standard (trolox) were placed; then, the reaction was started by adding AAPH (25 μL). Fluorescence was monitored at 37 °C every minute for 60 min (3 × 8 measurements par point) at wavelengths of excitation and emission, respectively, of 485 and 528 nm. The area under the curve (AUC) of relative fluorescence was calculated. ORAC values were expressed as Trolox equivalent in μM and calculated as the slope ratio from curves, AUCnet
the concentration of antioxidant and trolox, respectively (AUCnet
3.7. Chromatography: HPLC-DAD and HPLC-(APCI) Ion Trap MS
Characterization of AstaxP and AstaCO2 by HPLC-DAD and HPLC-(APCI+) ion trap MS in the positive ion mode was performed on an Agilent 1100 series (Agilent Technologies, Waldbronn, Germany). The analytical column was an Ultrabase C18 (5 μm, 250 mm × 4.6 mm i.d.) from AnalísisVínicos (Tomelloso, Spain) and kept in a column oven at 20 °C. The mobile phase consisted of an 80-min gradient from 83:17 to 98:2 (acetone/water) at a flow rate of 0.8 mL/min. The injection volume was set at 20 µL. The DAD scan range was from 200 to 800 nm. The MS parameters were: capillary voltage 3500 V, skimmer 40 V, source temperature 400 °C, drying gas 5 L·min−1 at 350 °C, nebulizer gas 60 psi, scan range 100–2200 m/z, maximal accumulation time 200 ms.
3.8. MTT Reduction Assay
HUVEC cells were cultured in low glucose (1 g/L) minimum essential medium (MEM) supplemented with 10% (v/v) fetal calf serum, 1% penicillin-streptomycin-amphotericin at 37 °C and 5% CO2. Experiments were performed with cells between Passages 10 and 15.
HUVEC cells were seeded in uncoated 24-well cell-culture plates (6 × 104
cells/well) with medium for 48 h. The cells were then treated with various concentrations of astaxanthin (1–15 µM). The final DMSO concentration in the assay in astaxanthin samples never exceeded 1% and had no influence on cell growth. Samples were added at the time of seeding in order to obtain concentrations of 0, 1, 5, 10 and 15 µM. In addition to the control group, we also cultured cells in a medium without sample, but containing 1% DMSO. After 48 h, a total of 1 mL of MTT was added in each well at a concentration of 0.5 mg/mL [46
]. The plates were stored for 3 h in the dark at room temperature. Then, MTT solution was removed from each well, and 300 µL of isopropanol were added. Plates were stored once again in the dark and overnight at 4 °C. Optical density (OD) was evaluated at 570 nm on the TECAN plate reader. The test met the acceptance criteria if the mean OD570
of blanks was approximately 0.2. If viability was reduced to <70% of the blank, it was considered as having cytotoxic potential [47
To evaluate cell morphology, HUVEC cells (104 cells/well) were cultured in Lab-Teck plates for 48 h. Then, cells were stained with Alexa Fluor-phalloidin (Molecular Probes) to highlight the cytoskeleton.
3.9. Cellular Antioxidant Activity Assay
To measure ROS production in HUVEC cells, we used the DCFH-DA, an oxidation-sensitive indicator. This probe is not fluorescent in its original form and can freely cross cell membranes. Into the living cells, two acetate groups (DA) are removed from the indicator forming DCFH, which is still not fluorescent. In the presence of ROS, DCFH is oxidized to fluorescent 2′,7′-dichlorofluorescein (DCF), which can be measured by fluorometry [48
HUVECs were cultured to 80% of confluence in low glucose-MEM medium. Then, cells were detached with trypsin and seeded at a density of 104
cells/well in 96-well cell-culture plates. After 24 h of incubation, the medium was washed and DCFH-DA was added for an in-well concentration of 10 µM. Plates were incubated for 1 h at 37 °C. After a full hour of exposition to the probe, the excess of indicator in the medium was washed with PBS, to make sure that only the intracellular oxidation was measured. After the PBS washing, astaxanthin samples were added. Concomitantly, we induced an in vitro
oxidative stress using the tert
-butyl hydroperoxide (Luperox, t
-BuOOH at 100 µM) [49
]. Stress controls (without the antioxidant samples and with Luperox) and blanks (without DCFH-DA) were performed on each plate. Fluorescence intensity was measured using a fluorescence spectrophotometer at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. A dose-response curve has been established.
The ROS scavenging activity was expressed as CAA (%) and calculated using the equation: CAA (%) = [(I
c − I
c] × 100, described previously [27
c is the intensity of cells exposed to Luperox without antioxidant sample, and I
s the intensity of cells exposed to Luperox and samples at different concentrations.
3.10. Statistical Analysis
Each experiment was tested three times to determine the reproducibility and to provide the mean ± standard deviation. A p-value <0.05 was considered significant. Data were analyzed for statistical significance using one-way analysis of variance (ANOVA), followed by Tukey’s HSD post hoc test using JMP software (Version 9; SAS Institute, Cary, NC, USA).
Astaxanthin has been demonstrated here to inhibit intracellular induced stress in human endothelial cells without any cytotoxicity and modification of the morphology up to 5 μM. However, it should be noted that astaxanthin was dissolved in DMSO, which has itself an influence on the viability of HUVEC cells, representing a limitation in this study. Complementary studies are required to solve the issue of vehicles and/or media for astaxanthin applications.
A comparative study among different astaxanthin sources, extraction methods and final formulations was performed. This study showed that astaxanthin from natural extracts from Haematococcus pluvialis had higher antioxidant activity than commercial synthetic astaxanthin. In this context, HPLC-ESI characterization of natural extracts (AstaP and AstaCO2) showed the presence of other carotenoids and astaxanthin mono- and di-esters in contrast with synthetic astaxanthin, which contains only a mixture of three isomers of free astaxanthin. Moreover, the final formulation may also have an influence on the antioxidant capacity of astaxanthin. In our study, both AstaCO2 and AstaP come from the same source, but they were extracted by using different methods (supercritical fluids and polar solvent, respectively) and presented under different formulations (in sunflower oil and DMSO, respectively). Further studies are necessary to understand the real activity of each fraction of natural extracts.
To conclude, our results strengthen the hypothesis that the antioxidant capacity of astaxanthin contributes to a decrease in the risk of oxidative stress-related diseases, such as cardiovascular diseases.