Bioaccessibility, Cellular Uptake, and Transport of Astaxanthin

Article Cite This: J. Agric. Food Chem. 2017, 65, 10223-10232

pubs.acs.org/JAFC

Bioaccessibility, Cellular Uptake, and Transport of Astaxanthin Isomers and their Antioxidative Effects in Human Intestinal Epithelial Caco‑2 Cells Cheng Yang,†,‡,§,∥ Hua Zhang,‡ Ronghua Liu,‡ Honghui Zhu,‡ Lianfu Zhang,*,†,§,∥,⊥ and Rong Tsao*,‡ †

State Key Laboratory of Food Science and Technology, §School of Food Science and Technology, ∥National Engineering Research Center for Functional Food, and ⊥Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China ‡ Guelph Research and Development Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada S Supporting Information *

ABSTRACT: The bioaccessibility, bioavailability, and antioxidative activities of three astaxanthin geometric isomers were investigated using an in vitro digestion model and human intestinal Caco-2 cells. This study demonstrated that the trans−cis isomerization of all-E-astaxanthin and the cis−trans isomerization of Z-astaxanthins could happen both during in vitro gastrointestinal digestion and cellular uptake processes. 13Z-Astaxanthin showed higher bioaccessibility than 9Z- and all-Eastaxanthins during in vitro digestion, and 9Z-astaxanthin exhibited higher transport efficiency than all-E- and 13Z-astaxanthins. These might explain why 13Z- and 9Z-astaxanthins are found at higher concentrations in human plasma than all-E-astaxanthin in reported studies. All three astaxanthin isomers were effective in maintaining cellular redox homeostasis as seen in the antioxidant enzyme (CAT, SOD) activities ; 9Z- and 13Z- astaxanthins exhibited a higher protective effect than all-E-astaxanthin against oxidative stress as demonstrated by the lower cellular uptake of Z-astaxanthins and lower secretion and gene expression of the pro-inflammatory cytokine IL-8 in Caco-2 cells treated with H2O2. We conclude, for the first time, that Z-astaxanthin isomers may play a more important role in preventing oxidative stress induced intestinal diseases. KEYWORDS: all-E-astaxanthin, 9Z-astaxanthin, 13Z-astaxanthin, cis-isomers, in vitro digestion, bioaccessibility, cellular uptake, Caco-2 cells

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problem of a lack of pure Z-astaxanthins.6 Furthermore, effects of pH and metal ions on the stability of Z-astaxanthins were also investigated to better understand how these isomers can be applied to functional foods and even pharmaceutical formulations. However, despite these findings, the higher concentration of Z-astaxanthins in human plasma after administration of all-E-astaxanthin-rich food is still not explained. The uptake mechanism inside the human GI tract and the metabolic fate of both all-E-astaxanthin and its Zisomers are still unknown,1,5,7,8 although some naturally occurring stereoisomers of astaxanthin have been studied in vitro and in vivo.9,10 It is not clear how and where the Zgeometric isomers of astaxanthin are formed in vivo, and whether or not the higher antioxidant activity of Z-astaxanthin isomers observed in vitro using a chemical-based method, and cell-based assays will also work on the inhibition of oxidative stress in vivo.6 Furthermore, questions related to the fate, structural features, and stability of Z-astaxanthins as opposed to all-E-astaxanthin in the human gastrointestinal (GI) tract and their cellular uptake remain to be answered before people

INTRODUCTION Astaxanthin (3,3′-dihydroxy-4,4′-dione- β,β′-carotene) is a wellknown xanthophyll widely found in marine seafood such as shrimp, lobster, and crab, and especially in their shell portions, and several microorganisms such as Phaf f ia rhodozyma and certain algaes.1−3 Salmon and rainbow trout are particularly rich dietary sources of astaxanthin for humans.4 The geometrical structure of naturally occurring astaxanthin is all-trans (all-E)astaxanthin. Because of its powerful antioxidant activity, many other biologic functions and potential health-promoting effects of all-E astaxanthin have been examined.2,3 A recent study showed that two of its cis-geometric isomers, 9Z- and 13Zastaxanthins were selectively absorbed into human plasma over all-E-astaxanthin after oral administration of an all-E-astaxanthin-rich meal.1 More interestingly, 9Z-astaxanthin has been found to exhibit higher antioxidant potential in vitro than all-Eastaxanthin by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and lipid peroxidation assays.5 On the other hand, a recent study showed that 13Z-astaxanthin had higher antioxidant activity than all-Eand 9Z-astaxanthins in oxygen radical absorbing capacity assays for lipophilic compounds (ORAC-L), photochemiluminescence, and cellular antioxidant activity assays.6 These results have instigated further interest in the health benefits and application of these Z-astaxanthins in functional foods. A rapid isomerization method of all-E-astaxanthin to produce Zastaxnathins was developed in a previous study to solve the © 2017 American Chemical Society

Received: Revised: Accepted: Published: 10223

September 12, 2017 October 24, 2017 October 30, 2017 October 30, 2017 DOI: 10.1021/acs.jafc.7b04254 J. Agric. Food Chem. 2017, 65, 10223−10232

Article

Journal of Agricultural and Food Chemistry

(Agilent, Waldbronn, Germany) system consisted of a degasser, a thermostated autosampler, a binary gradient pump, a diode array detector (DAD) and the ChemStation software (OpenLAB CDS, Version: Rev. C.01.07 SR1 [110]). Separation was performed on a Kinetex Biphenyl column (100 mm × 4.6 mm, 2.6 μm) (Phenomenex Inc., Torrance, CA, USA). The column temperature was set at 25 °C; the injection volume was 10 μL, and the flow rate was 0.7 mL/min for a total run time of 26 min. The other conditions were the same as described before.6 Quantification of astaxanthin isomers was performed by calculating their peak areas against a calibration curve of all-E-astaxanthin standard. The peak areas of 9Z- and 13Zastaxanthins were normalized on the basis of extinction coefficients at 470 nm to get the quantity in all-E- astaxanthin equivalents according to references.16,17 Simulated in Vitro Digestion. An in vitro digestion model was prepared according to the method of Li et al. with minor modifications.18−20 Briefly, a freshly prepared DMSO solution (18 μL) of purified astaxanthin isomers (0.6 mM) was added to 6 mL of Hanks’ balanced salt solution (HBSS) buffer containing α-amylase (final concentration: 0.2 mg/mL). The mixtures were incubated in a water bath shaker at 200 rpm for 10 min at 37 °C. After that, 10 mL of phosphate-buffered saline (PBS) with pH re-adjusted to 2.0 by 2 M HCl containing porcine pepsin (2950 U/mg protein, final concentration: 0.42 mg/mL) was added.19 The pH of the mixture was adjusted to pH 2.0 by 2 M HCl, and the mixture was incubated for 30 min under the same conditions. For studying the effect of the intestinal digestion, 10 mL of PBS was added to the mixture, and the pH of the mixture was adjusted to 6.5 with 1 M NaOH; then, pancreatin (8× USP, final concentration: 0.25 mg/mL), CaCl2 (final concentration: 4 mM), and bile salt (final concentration: 3.0 mg/mL) were added to the mixture.18,19 Subsequently, the digesta was incubated in a shaking water bath at 37 °C for another 2 h. A blank without astaxanthin isomers was incubated under the same conditions for correcting the interferences from the digestive enzymes and buffers. For monitoring possible isomerization or degradation of different astaxanthin isomers, aliquots of digesta were collected from the starting material, saliva, and gastric and small intestinal phases during simulated digestion and centrifuged at 2500g for 10 min to separate the micelle (aqueous) fraction from the enzymes (5810R, Brinkman Instruments Inc., Westbury, NY).21 Then, the supernatant was extracted twice with 500 μL of DCM. An aliquot of 800 μL of extract was transferred into a 2.0 mL tube and blow-dried under nitrogen gas. The dried samples were redissolved in 40 μL of dichloromethane:methanol (1:1, v/v) and centrifuged at 10000 rpm for 15 min before HPLC analysis. Carotenoid bioaccessibility was defined as the amount of ingested carotenoid(s) available for absorption in the gut after digestion.22 Therefore, bioaccessibility of astaxanthin isomers was expressed as the percent of the amount of astaxanthin isomers in digesta to the original amount added in the digestion model. All experiments were done in triplicate. Cell Culture. The Caco-2 human intestinal cell line was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Caco-2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen Life Technologies Inc., Burlington, ON, Canada) with 20% fetal bovine serum (FBS; HyClone, Invitrogen Life Technologies Inc., Burlington, ON, Canada) and 50 units/mL of penicillin-streptomycin (Invitrogen Life Technologies Inc., Burlington, ON, Canada) and incubated at 37 °C in 5% CO2 with fresh media replaced every 2−3 days. Cells used in this study were between passages 40 and 60. Uptake and Transport of Astaxanthin Isomers by Caco-2 Cells. Caco-2 human intestinal cells were seeded in a 24-well plate with 12 polyester (PET) membrane permeable support inserts (6.5 mm, 0.4 μM pore size, Corning Inc., Corning, NY, USA) at a density of 8 × 104 cells/well. Only differentiated enterocytes displaying unchanged transepithelial electrical resistance (TEER, Ω cm2) values were used in the experiment. TEER values were measured twice weekly using a Millicell-ERS Volt-Ohm Meter (Millipore, Bedford, MA, USA).

attempt to explain the observed physiological and biological phenomenon of Z-astaxanthins. The primary objective of this study was to systematically investigate the stability, profile changes, and transport efficiency of common geometric astaxanthin isomers during in vitro digestion and cellular uptake and transport. The second objective was to determine possible cellular metabolisms of the three astaxanthin isomers and explain the favorable accumulation of Z-astaxanthins in human plasma. The last was to study the protective effects of astaxanthin isomers against H2O2-induced oxidative stress in Caco-2 cells. Results of this study will help better understand the mechanism of action of different astaxanthin isomers and broaden the application of astaxanthin in functional foods.

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MATERIALS AND METHODS

Chemicals and Reagents. 3S,3′S-All-E-astaxanthin standard (HPLC purity >97%), α-amylase, pancreatin (8× USP), porcine pepsin, bile salts, dimethyl sulfoxide (DMSO), and formic acid were purchased from Sigma−Aldrich (Oakville, ON, Canada). HPLC-grade solvents, including acetonitrile, ethyl acetate, methanol, and dichloromethane (DCM) were purchased from EMD Chemicals (Gibbstown, NJ, USA). Acids were of ACS (American Chemical Society) grade and supplied by Fisher Scientific (Nepean, ON, Canada). Deionized water was obtained in house from a Thermo Scientific Barnstead Nanopure ultrapure water purification system (Ottawa, ON, Canada). Isomerization of All-E-astaxanthin. Isomerization was conducted based on previously reported methods.6,11 Briefly, all-Eastaxanthin in ethyl acetate solution containing iodine-doped titanium dioxide (I-TiO2) catalyst was incubated in a water bath for 2 h at 70 °C. After the reaction, the catalyst was removed by centrifugation at 10,000 rpm for 10 min. The reaction mixture was blown dry under a nitrogen stream and stored at −80 °C before further isolation and purification. Proportions of Z-astaxanthins were detected by HPLC. Isolation and Purification of Z-Astaxanthin Isomers. The dry mixture was dissolved in dichloromethane:methanol (1:1, v/v), filtered through a 0.22 μm membrane, and separated on a Kinetex C18 semipreparative column (5 μm, 250 mm × 10 mm) (Phenomenex Inc., Torrance, CA, USA) as described before.6 The flow rate was 3.0 mL/min. Under these conditions, fractions containing 9Z-astaxanthin and 13Z-astaxanthin were collected separately, blown dry under a nitrogen stream, and stored at −80 °C before use in different experiments. Identification of Astaxanthin Isomers by UHPLC-MS. Different astaxanthin isomers were identified by matching retention times, UV/vis spectra, and LC-MS data with those reported in the literature.6,7,12−15 LC-MS analysis of astaxanthin samples was carried out on a Thermo Scientific Q-Exactive Benchtop Orbitrap Mass Spectrometer connected with a Vanquish Flex Binary UPLC System (Massachusetts, US). The UHPLC system consists of a binary pump with vacuum degasser, autosampler, UV/vis photodiode array detector, and a column compartment. A Kinetex Biphenyl (100 mm × 4.6 mm, 2.6 μm) column (Phenomenex Inc., Torrance, CA, USA) was used for separation. A mobile phase consisting of solvent A (99.9% H2O + 0.1% formic acid) and solvent B (99.9% AcN + 0.1% formic acid) was used. The chromatographic elution condition was as follows: 0−20 min, 80 to 100% B; 20−23 min, 100% B; 23−24 min, 100 to 80% B; 24−30 min, 80% B. The column compartment was kept at 35 °C. The flow rate was set to 0.4 mL/min; the injection volume was 5 μL, and peaks were monitored at 470 nm. The positive heated-electrospray ionization (HESI) mode was used for data collection. The optimized HESI conditions were as follows: sheath gas, 50 arbitrary units (au); auxiliary gas, 13 au; sweep gas, 3 au; spray voltage, 3.5 kV; S-lens RF level, 50%; capillary temperature, 263 °C; and auxiliary heater temperature, 425 °C. The automatic gain control target and maximum injection time were 3e6 and 240 ms, respectively. Analysis of Astaxanthin Isomers. Samples containing astaxanthin isomers were analyzed using an Agilent HPLC series 1260 10224

DOI: 10.1021/acs.jafc.7b04254 J. Agric. Food Chem. 2017, 65, 10223−10232

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Figure 1. Structural changes and bioaccesssibility of astaxanthin isomers in different phases during in vitro digestion: (A) all-E-astaxanthin, (B) 9Zastaxanthin, and (C) 13Z-astaxanthin. (D) Bioaccessibility of astaxanthin isomers during in vitro digestion. Bioaccessibility of astaxanthin isomers is expressed as the percent of the amount of astaxanthin isomers in digesta to the original amount added in the mixture at the beginning. Mean ± SEM of three independent experiments. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to analyze the data in (D). Different letters in the same group indicate significant (p < 0.05) differences between means. Uptake and transport experiments were carried out according to previously described procedures with modifications.4,23 Briefly, Caco-2 cells were washed twice with DMEM media without FBS; then, 250 μL of freshly prepared astaxanthin isomer (dissolved in DMSO) containing DMEM media (final concentration: 0.6 μM) without FBS was added to inserts and incubated at 37 °C for different time intervals (1, 3, 6, 12, and 24 h). DMEM medium (750 μL) without FBS was added in the basolateral side. After incubation, TEER values were detected to ensure integrity of the monolayer; then, media from both sides of the insets were collected, and monolayers were washed twice with ice-cold PBS buffer. Cells were collected in 200 μL of PBS buffer, sonicated by a Qsonica Sonicator Q500 (Fisher Scientific), and extracted twice with 500 μL of DCM. An aliquot of 800 μL of the extract was transferred into a 2.0 mL tube and blow-dried under nitrogen gas. The dried samples were stored at −80 °C for a maximum of two days before HPLC analysis. A blank with the treatment with 0.1% DMSO was conducted under the same conditions to correct the interferences from the media. Cell viability was detected showing that none of the three astaxanthin isomers was cytotoxic at the working concentration (data not shown). Absorption (cellular uptake) efficiency and transport (basolateral secretion) efficiency were expressed as the percentage of astaxanthin isomer amount detected inside Caco-2 cells and that in the basolateral compartment to the originally added astaxanthin isomer in the apical

side. Cell studies were performed in triplicate and on three different days. Induced Oxidative Stress. The oxidative stress in Caco-2 cells was induced by 2 mM H2O2 according to reported protocols.24 Caco-2 cells were cultured in 24-well (with 1 mL medium) or 48-well (with 0.5 mL medium) culture plates (Corning Costar) at 2 × 105 cells/mL and grown for 4−6 days to reach 90% confluence.25 After washing twice with HBSS, cells were preincubated with 0.6 μM purified astaxanthin isomers in DMEM without FBS for 1 h before the treatment of 2 mM H2O2 for 1 h. After the treatment of H2O2, cells were washed twice and post-treated with astaxanthin isomercontaining medium for a further 22 h for interleukin-8 (IL-8) secretion, antioxidant enzyme activities, and IL-8 expression determination.24,26 The supernatants of the negative control (NC, cells not treated with H2O2), positive control (PC, cells treated with H2O2 alone), and sample-treated cells were collected and stored at −80 °C before measuring IL-8 release. Protein concentrations of supernatant and cell lysate were determined by DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) using bovine serum albumin (BSA) as a standard. Interleukin-8 Immunoassay. Treatment with H 2O2 was performed in a 48-well plate as mentioned above. The level of IL-8 in the culture supernatants was determined using an ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Quantification of IL-8 was accomplished 10225


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Journal of Agricultural and Food Chemistry by calibrating against the standard curve and expressed as IL-8% relative to PC. RNA Isolation and Real-Time RT-PCR. IL-8 was analyzed by real time-polymerase chain reaction (RT-PCR). After incubation for a total of 24 h, cells were rinsed twice with PBS and lysed by lysis buffer. The total RNA was extracted and purified using the Perfect Pure RNA Cultured Cell Kit (5 Prime, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The A260 and A280 were measured to quantify the RNA (NanoDrop ND-1000; Thermo Scientific, Wilmington, DE, USA). One microgram of total RNA was reverse transcribed into cDNA by a qScript cDNA Synthesis Kit (Quanta Biosciences, Inc., Gaithersburg, MD, USA). RT-PCR was carried out using iQ SYBR Green Supermix (Quanta Biosciences) on Applied Biosystems 7500 Fast and 7500 RTPCR system (Thermo Scientific, Burlington, ON, Canada) using the following conditions: 45 cycles of denaturization at 95 °C for 15 s and annealing and extension at 60 °C for 1 min using the primers listed in Table S1. The gene expression levels were calculated relative to the expression of the gene of 18S rRNA (18SrRNA) as the reference gene using the 2−ΔΔCt method.25 Results were expressed as fold change of IL-8 expression relative to that of the NC group. Determination of Intracellular Antioxidant Enzyme Activity. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) activities were measured using corresponding assay kits (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Briefly, Caco-2 cells in a 24-well plate were preincubated with 0.6 μM astaxanthin isomers for 1 h and then washed twice by FBS-free DMEM. H2O2 was added into wells to reach a final concentration of 2 mM and incubated for 1 h. After the post-treatment of astaxanthin isomers for 22 h, cells in each well were washed twice with PBS, collected, and sonicated in 0.25 mL of the corresponding sample buffer provided in the enzyme assay kit.24 The activity of each enzyme was determined according to the respective instructions in the kit, and the results were reported as % of NC. Measurement of Total Intracellular Glutathione. Intracellular total glutathione (GSH) was detected by an enzyme assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instruction with slight modification. An aliquot of cell lysate (250 μL) from each well in a 24-well plate was loaded in 1.5 mL centrifuge tubes and centrifuged at 10000g for 15 min at 4 °C. An aliquot of supernatant from each sample or standard (50 μL) was loaded into the corresponding wells of a 96-well plate and mixed with 150 μL assay cocktail. The fluorescence intensity was measured kinetically at 414 nm. Concentration of GSH was calculated using a standard curve and expressed as % of the negative control. Statistical Analysis. The experiments were performed in triplicate unless otherwise specified, and the values were presented as means ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to analyze data as specified in the figure captions. Differences were considered significant at p < 0.05. All statistical analyses were performed using SPSS (version 18.0, Chicago, IL, USA).

Purity of 13Z-astaxanthin could only reach 62.1% with the remaining 37.9% to be all-E-astaxanthin due possibly to the instability and reverse isomerization of 13Z-astaxanthin during purification and storage. Digestive Stability and Bioaccessibility of Astaxanthin Isomers During in Vitro Digestion. The higher concentrations of Z-astaxanthins in human plasma after administration of all-E-astaxanthin-rich meal has prompted several assumptions as to how and when isomerization happens: (1) trans−cis isomerization during gastrointestinal digestion or cellular absorption and transportation, (2) preferential uptake of Zastaxanthins, and (3) selective retention of Z-isomers.1,28 To prove these hypotheses, the digestive stability and profile changes of different astaxanthin isomers during in vitro digestion were first investigated. As shown in Figure 1, all astaxanthin isomers, regardless of the geometric forms, were transformed to other isomers during digestion, especially when in the gastric phase. It was interesting to note that they all reverse-isomerized back to the original structure at the last step, i.e., the intestinal phase. For all-E-astaxanthin, it decreased from the original 97.4% to 68.1% of all isomers in the gastric phase and then recovered to 81.5% in the intestinal phase (Figure 1A). In the same experiment, the small percentages of 13Zastaxanthin and 9Z-astaxanthin increased from the initial 0.52 and 0.54% to 15.3 and 11.8% in the gastric juice, respectively, and finally dropped to 9.7 and 7.3%, respectively, in the intestinal phase (Figure 1A). 9Z-Astaxanthin was more drastically isomerized to other forms; its percentage dropped from the original 90.1% to 30.1 and 70.8% in the gastric and intestinal phases, respectively (Figure 1B). 9Z-Astaxanthin mainly isomerized to all-E-astaxanthin followed by a small portion of 13Z-astaxanthin. The newly formed all-E- and 13Zastaxanthin increased from 5.5 and 4.4% (original) to 49.4 and 11.1% in the gastric and then decreased to 19.3 and 5.7% in the intestinal phase, respectively (Figure 1B). 13Z-Astaxanthin showed a slightly different pattern; only all-E-astaxanthin was newly formed during the gastrointestinal digestion (Figure 1C). In this experiment, the proportion of 13Z-astaxanthin decreased from the initial 62.1 to 23.6% and recovered to 46.2%; it was mainly isomerized to all-E-astaxanthin, which increased from 18.2 to 48.1% and then decreased to 36.3% at the initial gastric and intestinal phases, respectively. Both of the Z-astaxanthins were more readily isomerized to all-E-astaxanthin than to the other Z-isomer, although all-E-astaxanthin tended to be isomerized to 13Z-astaxanthin slightly more readily than to 9Z-astaxanthin. HPLC chromatograms of different astaxanthin isomer profiles in different phases are shown in Figure S1. The isomerization of all-E-astaxanthin to Z-astaxanthins in the saliva phase might be merely the result from incubation temperature (37 °C).29 It has been found that pH played a more important role than metal ions in the structure changes of astaxanthin isomers. At pH 2.0, 13Z- and 9Z-astaxanthins were prone to reverse isomerization to all-E-astaxanthin.6 This can be used to explain the similar observations of the present study, i.e., 13Zand 9Z-astaxanthins were more readily isomerized to all-Eastaxanthin in very acidic gastric phase (pH 2.0) (Figure 1). The similarity in isomer stability between previous (pH 2.0 buffered solution) and present (pH 2.0 buffered solution with digestive enzymes) studies suggests that pH might play more important roles in the stability and isomerization of astaxanthin isomers than digestive enzymes such as pepsin.6,29 All-Eastaxanthin and its 9Z- and 13Z-astaxanthins were found to be relatively stable in buffered solution at pH > 3.0.6 However, the

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RESULTS AND DISCUSSION Isomerization of All-E-astaxanthin. In the presence of ITiO2 catalyst, 9Z- and 13Z-astaxanthins reached the maximum at 22.7% and 16.9% in 2 h.6 Purified astaxanthin isomers were stored as powder in sealed amber tubes filled with nitrogen and kept at −80 °C. Isolation and Identification of Astaxanthin Isomers. Astaxanthin isomers were tentatively identified by matching chromatographic retention time and UV/vis spectra as shown in a previous report and further identified by UHPLC-MS. The protonated molecular ion of each astaxanthin isomer was 597.4 [M + H]+, which was in agreement with the other report.27 The purity of all-E-astaxanthin was 97.4%. 9Z-Astaxanthin was relatively stable, and its chromatographic purity was 90.1%. 10226


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Figure 2. (A) Absorption efficiency of astaxanthin isomers across differentiated Caco-2 cells during 24 h incubation; (B) transport efficiency of astaxanthin isomers across differentiated Caco-2 cells at indicated time points, and (C) isomeric profiles of each astaxanthin isomer in media and in cells at 24 h. Absorption efficiency and transport efficiency are expressed as the percentage of astaxanthin isomer amount detected inside Caco-2 cells or in the basolateral side of that originally added to the apical side. Cell studies were performed in triplicate and on three different days. Data are mean ± SEM; one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to analyze data in (B) and (C). Different letters indicate significant (p < 0.05) differences between means. 13Z-AST, all-E-AST, and 9Z-AST represent 13Z-astaxanthin, all-Eastaxanthin, and 9Z-astaxanthin, respectively. *p < 0.05; **p < 0.01; ***p < 0.001 compared to their counterpart in media.

astaxanthin had the highest bioaccessibility (37.1%) at the end of the gastrointestinal digestion, followed by 9Zastaxanthin (28.6%) and all-E-astaxanthin (19.7%), indicating that 13Z- and 9Z- astaxanthins had higher bioaccessibility than that of all-E-astaxanthin. Previously, Z-astaxanthins were found to have higher solubility in the solvent mixtures used in antioxidant assays.6 The higher solubility might have also aided better micellization of Z-isomers with bile salts and pancreatin in the intestinal phase of the present study, resulting in increased bioaccessibility.37 Sy et al. showed that the hydrophobicity of carotenoids was a key factor affecting their bioaccessibility in naturally produced mixed micelles.38 Both our observation and those reported in the literature therefore support the hypothesis that higher bioaccessibility of the Zastaxanthins come from the higher solubility over all-Eastaxanthin.29 Chitchumroonchokchai et al. also reported higher micellarization of Z-astaxanthins in uncooked fillets from wild and aquacultured salmon than that of all-Eastaxanthin.4 The bioaccessibility of all-E-astaxanthin in the present study was less than that reported by Sy et al. (49.7%) possibly due to the different delivery systems or extraction methods used.38

present study not only suggests that they may be changed to other isomeric forms at the low pH gastric phase, but they can be reverse-isomerized back to the original form and be stable in the intestinal phase where pH is near neutral and ready to be absorbed (Figure 1). This could simply be caused by the electron transition between the isomers and the ions or proteins in the digesta as a result of pH change.30,31 To our knowledge, the present work is the first bioaccessibility study on Z-astaxanthins albeit in a simulated upper gut digestion model. Isomerization of other carotenoids from all-trans to the cis geometrical configuration has been reported in vitro and in vivo.32−34 cis-Lycopene was reported in gastric milieu and the stomachs of ferrets.32,33 Low trans−cis isomerization of lutein and zeaxanthin was found in an in vitro digestion model by Granado-Lorencio et al.34 On the other hand, others such as Richelle et al. reported that isomerization of lycopene did not happen in the gastrointestinal lumen.35 The discrepancy among those different digestion models might be caused by the higher pH (pH 2.0−4.0) or other conditions of the different in vitro digestion models or detecting methods.36 Using a simulated digestion model, the present study showed that, among all three astaxanthin isomers tested, 13Z10227


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Figure 3. Possible mechanism for the passive diffusion process of three astaxanthin isomers.

astaxanthin.43 The initial uptake trend of all-E-astaxanthin before 3 h (Figure 2A) might be explained by a quick passive diffusion of all-E-astaxanthin owing to its linear structure that makes it much easier to permeate through the cell membrane than the sterically bulky 9Z-astaxanthin and 13Z-astaxanthin in the beginning (Figure 3).16 Further studies on the effect of carotenoid transporter proteins on the cellular uptake of different astaxanthin isomers are necessary to help illustrate the uptake mechanism. No significant metabolism occurred to the all-E-astaxanthin during cross-membrane uptake and transport. The profiles or ratios of the Z-isomers taken up in the Caco-2 cells were significantly different (p < 0.05) from those in the culture media after 24 h of incubation, especially for 13Z-astaxanthin (Figure 2C). The cis−trans isomerization rate from Zastaxanthins to all-E-astaxanthin was higher than the trans−cis isomerization from all-E-astaxanthin during cellular uptake. For example, the percentage of 13Z-astaxanthin decreased by 16.8% in the cells as compared to the medium, whereas that of all-Eastaxanthin increased by 22.5% during the cellular uptake of 13Z-astaxanthin. A similar cis−trans isomerization pattern was observed for 9Z-astaxanthin, although the magnitude was significantly lower. The rate of trans−cis isomerization of all-Eastaxanthin was even lower at 2−5% (Figure 2C). Similar results have been reported for other carotenoids. Isomerization of all-E-lycopene was reported to take place within enterocytes (Caco-2, Caco-2 SM, Caco-2 HTB37) during absorption.35 However, different results were found with β-carotene isomers in Caco-2 cells incubated with Dunaliella salina supplement. No change in isomeric profile was observed. Factors such as vehicles used might be attributed to such difference.40 Results of the present study on astaxanthin isomers and findings by others on plant-based carotenoids may help explain the higher concentration of Z-astaxanthin isomers in human plasma after oral administration of all-E-astaxanthin-rich diet. Four critical processes are proposed here: (1) the isomerization of all-E-astaxanthin during in vitro digestion (Figure 1A), (2) the relatively high stability of three astaxanthin isomers and higher bioaccessibility of the two Z-astaxanthin isomers during in vitro digestion (Figure 1D), (3) higher cross-membrane transport efficiency of the Z-astaxanthins (Figure 2B), and (4) relative stability of all-E-astaxanthin and the two Z-astaxanthin isomers during uptake and transport across intestinal cells (Figure 2C). Effects of Astaxanthin Isomers on H2O2-Induced IL-8 Secretion in Caco-2 Cells. The results presented above demonstrate that the three astaxanthin isomers could survive the GI tract and make contact with cells in the intestinal epithelium, i.e., the luminal surface or lining of both the small and large intestine of the GI tract, thus further modulating local

Although the bioaccessibilities of all three astaxanthin isomers were relatively low (20−37%) at the end of the gastrointestinal digestion (Figure 1D), the isomeric profiles of astaxanthin isomers in the digesta were similar to the respective mixtures in intestinal phase as shown in Figure 1A−C, suggesting that all three isomers are able to reach the small intestine and contact intestinal epithelial cells. Uptake and Transport of Astaxanthin Isomers by Caco-2 Cells. Caco-2 cells are widely used as an in vitro model to study intestinal epithelial physiology and interactions between food nutrients and the gut. In this study, the Caco-2 cell line was used to examine and compare cellular uptake, transport efficiency, and the isomeric profile of all-E-, 9Z-, and 13Z-astaxanthins during their cellular transport across differentiated Caco-2 cell monolayers. The results are shown in Figure 2. Cellular uptake began significantly at 3 h, and the absorption efficiency increased drastically at 12 h for all three isomers of astaxanthin and continued to increase until 24 h (Figure 2A). The absorption efficiency of all-E-astaxanthin was 8.30% followed by 9Z-astaxanthin and 13Z-astaxanthin at 7.12 and 4.54% in 24 h, respectively. The trend was generally in a time-dependent manner (Figure 2A). DMSO has been successfully used as a carrier for delivering carotenoids to cell cultures.23,39 Delivering vehicles used in the cell model system and cell line types might also have a significant effect on cellular uptake of carotenoid isomers.40 Chitchumroonchokchai et al. observed higher uptake of astaxanthin in enriched krill oil (17.5%) over that from wild salmon (14.3) using the micelle fraction.4,41 In the present study, the initial uptake at 1 h was fastest for all-E-astaxanthin, although it dropped at 3 h before steady kinetics was shown for all three isomers (Figure 2A). A similar uptake trend of all-E-lycopene was found in LNCap human prostate cells.42 The transmembrane absorption of astaxanthin isomers was assessed using a Caco-2 cell monolayer model and expressed as transport efficiency, and because of the detection limit of HPLC in the present study, astaxanthin isomers were only detected in the basolateral side after 12 h (Figure 2B). 9ZAstaxanthin exhibited higher transport efficiencies of 0.26 and 0.68% at 12 and 24 h, respectively, followed by 13Z-astaxanthin (0.12 and 0.28%) and all-E-astaxanthin (0.12 and 0.21%) (Figure 2B). Metabolism and transport of food bioactives, such as astaxanthin, across the intestinal membrane are a multifaceted and dynamic process involving different transport mechanisms. Two diffusion processes including simple diffusion and a facilitated process mediated by scavenger receptor class B type 1 protein and CD36 have been proposed for explaining crossmembrane transportation of other carotenoids instead of 10228


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not statistically significant (p > 0.05). Meanwhile, quantitative RT-PCR results showed that IL-8 gene expression after a total incubation time of 24 h was also significantly inhibited by the three isomers (Figure 4B), consistent with the IL-8 secretion (Figure 4A). Shorter treatment (3 h) also showed inhibited expression of the IL-8 gene over that in PC, but the effect was not significant (Figure S2). This might be caused by the low concentration of astaxanthin isomers or insufficient time for uptake. Other carotenoids including β-carotene and lutein have been reported to exhibit similar inhibitory effects on the H2O2induced increase in IL-8 expression in gastric epithelial AGS cells.44 Although treatment with 2 mM H2O2 caused the viability of Caco-2 cells (PC) to decrease by an acceptable ∼10% compared to normal cells (NC) from the water-soluble tetrazolium-1 (WST-1) proliferation assay, there was no significant difference between PC and astaxanthin isomertreated groups at 0.6 μM (Figure S3). Effects of Astaxanthin Isomers on Cellular Antioxidant Enzyme Activities in H2O2-Treated Caco-2 Cells. Antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) work together as an intrinsic antioxidant defense system of cells. SOD converts superoxide anions into H2O2 or oxygen, and CAT or GPx subsequently decomposes H2O2 into water and oxygen. GPx works together with GR to regulate the production of GSH, which plays an important role in antioxidant defense to protect cells from oxidative damage made by reactive oxygen species (ROS) such as hydrogen peroxide.25 Antioxidants such as astaxanthin can react directly with ROS to reduce cellular oxidative stress but can also modulate the redox state inside cells through regulating the activities of the above-mentioned different antioxidant enzymes. In the present study, the significantly reduced CAT activity by 2 mM H2O2 (PC) (85% of NC) was completely recovered and significantly (p < 0.05) increased by the two Z-astaxanthin isomers (0.6 μM, 24 h of exposure) (Figure 5A). The significantly increased CAT activity by 9Z-astaxanthin can also be interpreted as a result of higher absorption efficiency of 9Z-astaxanthin (Figure 2A) and its higher antioxidant activity.6 Similarly, compared with the PC group, SOD activity was also recovered or improved by all three astaxanthin isomers (Figure 5B). Among them, 13Z-astaxanthin exhibited the most significant upregulation of SOD activity followed by that of allE- astaxanthin (p < 0.05) and 9Z-astaxanthin. The degree of the modulatory effects of the three astaxanthin isomers on activities of SOD and CAT may be governed by the inter-related factors of the cellular antioxidant enzyme system as seen in other studies.46,47 Nevertheless, all three astaxanthin isomers of the present study showed varying degrees of cellular antioxidant and anti-inflammatory effects as seen in elevated SOD and CAT activities and decreased IL-8 expression in Caco-2 cells stimulated by H2O2. H2O2 stimulation did not significantly affect the activity of GPx (Figure S4) and GR (data not shown) in Caco-2 cells (p > 0.05). None of the three astaxanthin isomers exhibited significant effect on these enzymes either (p > 0.05). In terms of GSH, H2O2 stimulation significantly increased its concentration by 32.7% over that in the NC (Figure S5); however, all three astaxanthin isomers were able to restore the redox state by significantly modulating the concentration of GSH in Caco-2 cells stimulated by H2O2, although at varied degrees (Figure S5). All-E-astaxanthin and 9Z-astaxanthin

or systemic antioxidative processes. IL-8 is a pro-inflammatory mediator that is secreted abundantly in intestinal epithelial cells. It helps mediate the inflammatory response by activating and recruiting neutrophils to the site of infection.44 IL-8 secretion promoted by H2O2-induced oxidative stress in Caco-2 cells can thus be used as an indicator of oxidative stress.45 As shown in Figure 4A, 2 mM H2O2 treatment (positive control, PC) indeed induced IL-8 secretion in Caco-2 cells by

Figure 4. Effect of astaxanthin isomers on IL-8 secretion (A) and IL-8 gene expression in H2O2-induced Caco-2 cells. Cells were pretreated with 0.6 μM astaxanthin isomers for 1 h, treated with 2 mM H2O2 for 1 h, and then post-treated with astaxanthin isomers for another 22 h at 37 °C. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to analyze data. Values with different letters are significantly different from each other (p < 0.05). NC means negative control; PC represents positive control, which is treated with H2O2, and 13Z-AST, all-E-AST, and 9Z-AST represent 13Z-astaxanthin, all-E-astaxanthin, and 9Z-astaxanthin, respectively.

almost 7-fold compared to that in nontreated cells (negative control, NC), a result similar to what was reported by Zhao et al.24 All three astaxanthin isomers showed significant inhibition of H2O2-induced IL-8 secretion (p < 0.05) against PC at 0.6 μM after a total of 24 h exposure (Figure 4A), although the difference in the inhibitory effects among the three isomers was 10229


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In conclusion, this study demonstrated that the trans−cis isomerization of all-E-astaxanthin and the cis−trans isomerization of Z-astaxanthins could happen during both in vitro GI digestion and cellular uptake processes. 13Z-Astaxanthin showed higher bioaccessibility than 9Z- and all-E-astaxanthins during in vitro digestion, and 9Z-astaxanthin exhibited higher transport efficiency than all-E- and 13Z-astaxanthins during cellular transport in Caco-2 cell monolayers. These might explain why 13Z- and 9Z-astaxanthins are found at higher concentrations in human plasma than that of all-E astaxanthin in the reported study. More interestingly, all three astaxanthin isomers were involved in maintaining cellular redox homeostasis in H2O2-stimulated Caco-2 cells as seen in the antioxidant enzyme activities study, and the two cis isomers, i.e., 9Z- and 13Z- astaxanthins, seemed to exhibit higher protective effects than that of all-E-astaxanthin against oxidative stress considering the relatively lower cellular uptake of Z-astaxanthins and the lower secretion and gene expression of the pro-inflammatory cytokine IL-8 in Caco-2 cells treated with H2O2. By carefully examining the digestive stability, changes in isomeric profile, cellular absorption, and transport efficiency of individual isomers of all-E-, 9Z-, and 13Z-astaxanathins and their antioxidant and anti-inflammatory effects together with existing knowledge in the literature, we can reasonably conclude, for the first time, that Z-astaxanthin isomers may play a more important role in preventing oxidative stress-induced intestinal diseases. Z-Astaxanthin isomers such as 9Z- and 13Zastaxanthins should be further explored as superior forms of astaxanthin as ingredients for functional foods and nutraceuticals.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04254. Table S1, Primers used for RT-PCR for IL-8 gene expression; Figure S1, changes in the isomeric profile of all-E-, 13Z-, and 9Z-astaxanthin during in vitro digestion; Figure S2, effect of astaxanthin isomers on IL-8 gene expression in H2O2-induced Caco-2 cells; Figure S3, effect of astaxanthin isomers on cell viability in H2O2induced Caco-2 cells; Figure S4, effects of astaxanthin isomers on glutathione peroxidase activity in H2O2treated Caco-2 cells; Figure S5, effects of astaxanthin isomers on the concentration of intracellular total glutathione in H2O2-treated Caco-2 cells (PDF)

Figure 5. Effects of astaxanthin isomers (0.6 μM) on catalase (CAT) (A) and superoxide dismutase (SOD) (B) activities in H2O2 -treated Caco-2 cells. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to analyze data. Values with different letters are significantly different from each other (p < 0.05).

completely diminished the effect of H2O2 on GSH. In addition, Chew et al. found that pretreatment of human umbilical vein endothelial cells with astaxanthin for 48 h followed by treatment of 100 μM H2O2 overnight showed no effect on GPx activity, although there was a significant difference between the NC and PC groups.46 The glutathione system (GPx and GR activities) is highly complex and many factors, including the innate ability to maintain redox balance by GPx and GR, the concentration of H2O2 used, treatment time, and concentration of astaxanthin isomers, may all be attributed to the lack of significant activity on these two enzymes by astaxanthin isomers in the cell model of the present study. Nevertheless, results of this study suggest that astaxanthin isomers are not only strong antioxidants via direct reaction with ROS as shown in chemical-based assays but can also act by stimulating the expression of antioxidant enzymes, particularly CAT and SOD, as found in the present study.6 The overall antioxidant effect in the cell can be a combination of both direct and indirect actions by astaxanthin isomers.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-510-85917025. Fax: +86-510-85917025. E-mail: [email protected]. *Tel: +1 226 217 8180. Fax: +1 226 217 8183. E-mail: Rong. [email protected]. ORCID

Rong Tsao: 0000-0001-6537-1820 Funding

This work was funded by grants from the Natural Sciences Foundation of China (31171724) and the Fundamental Research Funds for the Central Universities (JUSRP51501) and the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP 20130093110008). Cheng Yang is a visiting Ph. D. student funded by China Scholarship Council 10230


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through the Agriculture & Agri-Food Canada-Ministry of Education of China (AAFC-MOE Ph.D. Research Program. This study was also partially supported by the Abase fund of AAFC (Project # J-000283.001.01). Notes

The authors declare no competing financial interest.

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Bioaccessibility, Cellular Uptake, and Transport of Astaxanthin

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