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National Food Research Institute, 2–1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2To whom correspondence should be addressed. E-mail: nagao{at}nfri.affrc.go.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • absorption • Caco-2 cells • fucoxanthin • fucoxanthinol • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). Furthermore, some epidemiologic studies have indicated that the ubiquitous consumption of seaweeds in Japan is a protective factor against some types of cancers (5
). Thus, seaweeds seem to be useful foodstuffs for human health.
Fucoxanthin (Fig. 1
) is a major carotenoid of edible brown seaweeds (6
). There have recently been several reports that fucoxanthin has beneficial effects on chemoprevention of cancer (7
–11
). Fucoxanthin inhibited the growth of human neuroblastoma GOTO cells (7
) and intestinal carcinogenesis in animal experiments (8
,9
), and induced apoptosis of human leukemia HL-60 cells (10
). Previously, we had found that fucoxanthin reduced the viability of prostate cancer cells by inducing apoptosis to a greater extent than the other carotenoids present in foodstuffs (11
). Although the potential biological activities of fucoxanthin have been demonstrated in vitro, the digestion, absorption and metabolism of dietary fucoxanthin are still not well understood.
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). The Caco-2 cells derived from human colonic carcinoma have been a useful model for studying the metabolism and transport of drugs and dietary compounds by intestinal absorptive cells (13
,14
). The object of the present study was to characterize the absorption and metabolism of fucoxanthin using Caco-2 cells. Furthermore, we present herein evidence of the absorption of orally administered fucoxanthin by mice. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Lysophosphatidylcholine (1-palmitoyl-sn-glycero-3-phosphocholine), monoolein, sodium taurocholate and sulfatase from Helix pomatia (type H-5), possessing ß-glucuronidase and ß-glucosidase activity, were purchased from Sigma Chemical (St. Louis, MO). Lipase from Candida cylindracea was purchased from Fluka (Buchs, Switzerland). Brown algae (Undaria pinnatifida) were purchased from a local market in Tsukuba, Japan. Fucoxanthin was extracted and refined from the brown algae as described previously (12
). Other chemicals and solvents were of reagent grade.
Caco-2 cells (American Type Culture Collection, Rockville, MD) were maintained in 10-cm dishes (Corning Glassworks, Corning, NY) containing Dulbecco’s modified Eagle’s medium (DMEM)3
supplemented with 100 mL/L fetal bovine serum(FBS), 4 mmol/L L-glutamine, 40,000 U/L penicillin, 40 mg/L streptomycin and 0.1 mmol/L nonessential amino acids. Cells were kept at 37°C in a humidified atmosphere of 95% air and 5% CO2. The growth medium was replenished every 2–3 d. Cells were reseeded when the cell monolayers became semiconfluent. For the experiments, cells at passages 30–60 were seeded on 12-well plates at 1.2 x 105 cells/well or on Transwells (polycarbonate membrane, 24-mm diameter, 0.4-µm pore size; Coster, Cambridge, MA) at 7.5 x 105 cells/filter and grown under the same conditions as those described above. The experiments were performed at 20–22 d postseeding on 12-well plates. The cell cultures on Transwell were used for the experiments in which the transepithelial electrical resistance exceeded 500 
· cm2 at 25–30 d postseeding.
Preparation of fucoxanthin solubilized in micelles.
Fucoxanthin was delivered to the cells as mixed micelles prepared as previously described (12
). Briefly, appropriate volumes of stock solutions were transferred to glass tubes, and the organic solvent was removed under a stream of argon. The residue was dissolved in serum-free DMEM with a Vortex mixer. The final concentrations of each component in the medium were as follows: 2 mmol/L sodium taurocholate, 100 µmol/L monoolein, 33.3 µmol/L oleic acid, 50 µmol/L lysophosphatidylcholine and 1.0 µmol/L fucoxanthin. Lysophosphatidylcholine was used for micellarization of fucoxanthin because lysophosphatidylcholine enhances the uptake of carotenoids by Caco-2 cells (12
). The resultant solutions were optically clear. The medium was sterilized by passage through a presterilized 0.22-µm filter and was used as a fucoxanthin-supplement medium. The concentration of micellar fucoxanthin after filtration was confirmed to be 1.00 ± 0.05 µmol/L by HPLC before the fucoxanthin was used in the following experiments.
Incubation of Caco-2 cells with micellar fucoxanthin.
The differentiated monolayers of Caco-2 cells on 12-well plates were washed twice with 0.5 mL of serum-free DMEM and then supplemented with 1 mL of DMEM containing micellar fucoxanthin. After incubation, as described in the cell-culture section, for the indicated time, the cell-culture plates were placed on ice, the media were removed and monolayers were washed twice with PBS containing 10 mmol/L sodium taurocholate to remove surface-bonded carotenoids, followed by two additional washings with PBS. The washed cells were harvested in 1 mL of PBS and pelleted by centrifugation at 1000 x g for 5 min at 4°C. The supernatants were discarded, and the cell pellets were homogenized with a microtube homogenizer in 0.5 mL of ice-cold PBS. To extract the carotenoids, 1.5 mL of dichloromethane/methanol (1:2, v/v) containing 70 µmol/L 
-tocopherol was added to 0.4 mL of the cell homogenate and mixed well with a Vortex mixer. Hexane (0.75 mL) was mixed with the solution, and the resultant upper layer of hexane/dichloromethane was withdrawn. The lower layer was similarly extracted with 0.5 mL of dichloromethane, then with 0.75 mL of hexane. The hexane/dichloromethane layer was combined with the initial extract. The combined extract was dried under a stream of argon gas, dissolved in 200 µL of methanol and subjected to HPLC analysis as described below. We also analyzed the concentrations of carotenoids in DMEM before and after incubation. An aliquot of medium (100 µL) was mixed with 400 µL of dichloromethane/methanol (1:4, v/v) and subjected to HPLC analysis.
In the case of the culture on Transwells, the inserts were washed twice with 1 mL of serum-free medium. Unless otherwise stated, cells received 1.5 mL of the serum-free DMEM containing micellar fucoxanthin on the apical side and 2 mL of DMEM containing 100 mL/L FBS on the basolateral side. After incubation, the apical and basolateral media were collected. An aliquot of the apical medium (100 µL) was mixed with 400 µL of dichloromethane/methanol (1:4, v/v) and subjected to HPLC analysis. Fucoxanthin was extracted from the basolateral medium and cells as described above and subjected to HPLC analysis.
For enzymatic hydrolysis of a possible conjugate of fucoxanthin or its metabolites, an aliquot of apical and basolateral media, cell homogenate, and their extracts were mixed with 50 U of a sulfatase type H-5 solution in 0.2 mL of 0.1 mol/L acetate buffer, pH 5.0, and incubated at 37°C for 45 min. Carotenoids were extracted from the reaction mixture as described above and subjected to HPLC analysis.
Fucoxanthin hydrolysis by lipase.
An acetone solution (10 µL) of fucoxanthin (29 pmol) was added to 1 mL of Britton-Robinson buffer (26 mmol/L phosphoric acid, acetic acid, boric acid and 70 mmol/L sodium hydroxide, pH 7.0) containing 5 mg lipase from Candida cylindracea, and the mixture was then incubated at 37°C for 6 h. Then carotenoids were extracted from the reaction mixture as described above and subjected to HPLC analysis.
Fucoxanthin hydrolytic activities in Caco-2 cells and medium.
The differentiated Caco-2 cell monolayers cultured on 12-well plates as described above were washed twice and replaced with 1.0 mL of serum-free DMEM. The medium and cells were collected after a 24-h incubation, and the cells were then homogenized in 1.0 mL of serum-free DMEM. The cell homogenate (0.5 mL) or the medium (0.5 mL) was added to 0.5 mL of 1 µmol/L fucoxanthin solubilized with micelles in serum-free DMEM. After the reaction mixture was incubated at 37°C for 6 h, an aliquot of solution (100 µL) was mixed well with 400 µL of dichloromethane/methanol (1:4, v/v) and then centrifuged at 1000 x g for 5 min. The supernatant was subjected to HPLC analysis as described below.
Male ICR mice (7 wk old; CLEA Japan, Tokyo) were housed at 25°C with a 12-h light:dark cycle and acclimated with free access to tap water and a commercial diet (MF, Oriental Yeast, Tokyo, Japan). After 7 d of feeding, mice (30–35 g body weight, n = 8) were deprived of diet for 14–15 h before fucoxanthin administration. Four mice were orally administered 40 nmol fucoxanthin solubilized in 0.2 mL of PBS containing 12 mmol/L sodium taurocholate, 2.5 mmol/L monoolein, 7.5 mmol/L oleic acid and 1.25 mmol/L lysophosphatidylcholine by direct intubation to the stomach; the other four mice were untreated (control). One hour after administration, all mice were anesthetized with ether, and blood was collected from the caudal vena cava with a heparinized syringe. Plasma was immediately prepared by centrifugation at 1000 x g for 15 min at 4°C. Carotenoids were then extracted from the plasma as described above and subjected to HPLC analysis. The experimental procedures used in this study met the guidelines for experimental animals of the National Food Research Institute, Japan.
The HPLC system consisted of an LC-10AD pump (Shimadzu, Kyoto, Japan), an SPD-10A UV-visible (UV-VIS) absorbance detector (Shimadzu), an AS-8020 autosampler (Tosoh, Tokyo, Japan), and a personal computer with EZChrome Chromatography Data System software (Scientific Software, Pleasanton CA). Carotenoids were separated on a TSK gel ODS-80Ts (Tosoh), 4.6 x 250 mm, attached to a precolumn (2 x 20 mm) of Pelliguard LC-18 (Supelco, Bellefonte, PA). The solvent used was acetonitrile/methanol/water (75:15:10, v/v/v) containing 1 g/L ammonium acetate. Isocratic analyses were performed at 1.0 mL/min, and fucoxanthin was detected at 450 nm. Fucoxanthin and fucoxanthinol were quantified from their peak area by use of a standard curve with purified fucoxanthin because a fucoxanthinol standard was not available. The peak identities for fucoxanthin and fucoxanthinol were further confirmed from their characteristic UV-VIS spectra recorded with a model 1100 HPLC system equipped with a photodiode array detector (Hewlett-Packard, Palo Alto, CA) and from their positive ions recorded with the HPLC system connected to a LCQ mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an interface of atmospheric pressure chemical ionization. All procedures were carried out under dim yellow light to minimize degradation and isomerization of fucoxanthin by light irradiation.
Data are means ± SD. Statistical analyses were carried out using Student’s t test or one-way ANOVA with Scheffé’s F-test to identify significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Figure 2A
shows the HPLC chromatogram of the extract from the cells after 6 h of incubation with 1 µmol/L fucoxanthin solubilized in micelles. The peak corresponding to fucoxanthin was detected at 12.5 min. The other peaks ascribed to metabolites of fucoxanthin were detected at 6.8 min and 8.8 min. The retention time and UV-VIS spectrum of the main metabolite peak detected at 6.8 min were consistent with those of the fucoxanthin hydrolysate by lipase (Figs. 2
and 3
). Thus, the metabolite eluting at 6.8 min was assumed to be the deacetylation product of fucoxanthin (Fig. 1)
. The mass spectra of the peak detected at 6.8 min in both the cell extract and the hydrolysate by lipase gave positive ions at m/z 617 and 599, which corresponded to [M + H]+ and [M + H - 18]+, of fucoxanthinol, respectively. Thus, fucoxanthinol was identified as a metabolite of fucoxanthin. The mass spectrum of the minor peak detected at 8.8 min in the cell extract showed a molecular ion identical to that of fucoxanthinol. In addition, we found that the UV-VIS spectrum of this minor metabolite peak showed a hypochromic shift and a cis-peak at 335 nm (Fig. 3
C). This peak component was assumed to be a cis-isomer of fucoxanthinol. In Caco-2 cells not incubated with fucoxanthin, no peaks corresponding to fucoxanthin and fucoxanthinol were detected.
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shows the time-course changes of fucoxanthin and fucoxanthinol levels in Caco-2 cells incubated with 1 µmol/L fucoxanthin solubilized in micelles. The cellular fucoxanthin level reached a maximum after 6 h and subsequently declined. In contrast, the fucoxanthinol level increased steadily throughout the 24-h period. In the medium, fucoxanthinol also appeared during incubation (Fig. 4
B). Fucoxanthinol was not formed, however, when fucoxanthin was incubated in the medium without cells. The levels of total carotenoids (fucoxanthin and fucoxanthinol) from the culture (cells and medium) were lower than those in medium incubated without cells (Fig. 4
C).
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We examined the secretion of fucoxanthin and fucoxanthinol into the basolateral side from Caco-2 cells cultured on Transwells (Fig. 5
). The levels of fucoxanthin and fucoxanthinol in the apical medium and the cells cultured on Transwells showed a similar trend to those in the culture on 12-well plates. On the basolateral side, both fucoxanthin and fucoxanthinol accumulated to the same level after 6 h of incubation. Thereafter, the fucoxanthin level did not increase, whereas after 24 h of incubation, the fucoxanthinol level had increased to eight times that after 6 h of incubation.
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. In the chromatogram of cellular extracts after enzyme treatment, some peaks ascribed to the enzyme reagent appeared, but no increases in fucoxanthin and fucoxanthinol were observed. Similar results were shown when the cell homogenate and both apical and basolateral media were subjected directly to the enzymatic reaction. In addition, no peaks corresponding to conjugated forms such as glucuronide and sulfate were detected by HPLC with the photodiode array detector in either the cells or the media after incubation with micellar fucoxanthin. These results suggest that the conjugation reaction did not occur during uptake of fucoxanthin by Caco-2 cells.
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shows the HPLC chromatogram of the plasma extract of a mouse fed 40 nmol fucoxanthin. In the plasma of a control mouse, no fucoxanthin or fucoxanthinol was found. One hour after ingestion of fucoxanthin, peaks at 6.8 min and 8.8 min of retention time appeared in the HPLC chromatogram of the plasma extract. Peak A (6.8 min) of plasma extract produced the same UV-VIS spectrum as fucoxanthinol (Fig. 8
) and gave the characteristic ions of fucoxanthinol: [M + H]+ and [M + H - 18]+ ions at m/z 617 and 599, respectively, in the mass spectrum. Plasma fucoxanthinol levels 1 h after ingestion of fucoxanthin were 10.4 ± 5.3 nmol fucoxanthinol/L plasma (n = 4). The UV-VIS spectrum of peak B (8.8 min) differed from those of fucoxanthinol and fucoxanthin (Fig. 8)
. Because relatively large amounts of biological compounds without visible absorption coeluted with peak B under the present HPLC conditions, it was difficult to evaluate the molecular ion by liquid chromatography-mass spectrometry.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The aim of the present study was to further characterize the absorption and metabolism of dietary fucoxanthin. We found that fucoxanthin is hydrolyzed to fucoxanthinol during the uptake by Caco-2 cells, and that fucoxanthinol but not fucoxanthin appears in mouse plasma after ingestion of fucoxanthin.
It has been suggested that fatty acid esters of carotenoids are hydrolyzed in the small intestine in humans because no esters have been detected in chylomicrons or serum (15
,16
). Strand et al. (17
) reported that fucoxanthinol but not fucoxanthin was transferred to the egg yolks of laying hens fed a diet with 15% seaweed meal. This hydrolysis of fucoxanthin is consistent with the general phenomenon that animals absorb the free carotenoid form. Our results suggest that fucoxanthin is hydrolyzed mainly by an extracelluar enzyme secreted from Caco-2 cells and that some part of fucoxanthin is hydrolyzed after cellular uptake. As reported previously, the enzymes for hydrolysis might be lipase and carboxylesterase. Spalinger et al. (18
) observed endogenous lipase activity distributed on the apical membrane as well as in the apical medium in Caco-2 cell culture, suggesting the secretion of cellular lipase. Carboxylesterase activity in Caco-2 cells has also been reported (19
,20
). In the present study, we found that the secretion of fucoxanthinol into the basolateral medium by Caco-2 cells was much higher than that of fucoxanthin, and that fucoxanthinol but not fucoxanthin appeared in plasma when the mice ingested fucoxanthin. The more rapid appearance of fucoxanthinol in mouse plasma compared with Caco-2 cell cultures likely is due to the presence of pancreatic juice containing lipase, phospholipase and cholesterol esterase. Thus, dietary fucoxanthin is certainly hydrolyzed to fucoxanthinol in the intestinal tract by lipase and esterase from the pancreas or in intestinal cells, and this hydrolyzed product is taken up by the intestinal cells and secreted into the lymph in vivo.
Human intestinal mucosa has UDP-glucuronosyltransferase (UGP) and sulfotransferase activities, both of which are drug-metabolizing enzymes that conjugate hydrophobic drugs. UGP activity has been reported in Caco-2 cells (21
), and the presence of sulfotransferase activity in Caco-2 cells has also been mentioned (22
). Recently, several researchers found that dietary phenolic compounds such as flavonoids were metabolized to their conjugates by Caco-2 cells (23
,24
). Strand et al. (17
) reported that fucoxanthinol sulfate was present in the egg yolks of laying hens fed a fucoxanthin-rich diet. However, we could not confirm the presence of conjugates of fucoxanthin and fucoxanthinol when Caco-2 cells were exposed to micellar fucoxanthin. Their metabolic conjugation could occur, however, in the liver and kidney after intestinal absorption.
In addition, other metabolic or oxidative degradations of fucoxanthin in the cells might occur because the combined recovery of fucoxanthin and fucoxanthinol from the culture (cells plus medium) decreased in a time-dependent manner. Furthermore, we observed an unknown peak that could correspond to a metabolite of fucoxanthin in the HPLC chromatogram of mouse plasma extract 1 h after intubation of fucoxanthin. The UV-VIS spectrum of this metabolite of fucoxanthin was different from that of cis-fucoxanthinol or halocynthiaxanthin, which has been suggested to be a metabolite of fucoxanthin in marine animals (25
,26
). In mammals, provitamin A carotenoids are converted to vitamin A through the cleavage by ß-carotene-15,15'-dioxygenase. However, fucoxanthin cannot be cleaved by the enzyme because of its high substrate specificity for carotenoids with the ß-ionone ring (27
). Eccentric cleavage was also proposed as an additional pathway of vitamin A formation (28
), but its involvement in the metabolism of nonprovitamin A carotenoids has not been shown so far. Further in vivo studies are required to clarify the metabolic fates of dietary fucoxanthin in mammals.
Epoxyxanthophylls such as fucoxanthin, neoxanthin, and violaxanthin are widely distributed in nature and represent a major portion of dietary carotenoids in a number of fruits, vegetables and seaweeds. Recently, we found that neoxanthin as well as fucoxanthin remarkably reduced the viability of prostate cancer cells by inducing apoptosis (11
). Chang et al. (29
) reported that neoxanthin inhibited chemically induced carcinogenesis in the hamster buccal pouch. Despite their abundance in foodstuffs and their potential health benefits, there has been no evidence that dietary epoxyxanthophylls are absorbed by mammals. In the present study, we demonstrated for the first time that fucoxanthin is converted to fucoxanthinol and one unknown metabolite in mice. Fucoxanthinol rather than fucoxanthin itself should therefore be considered in mechanistic studies of the biological actions of fucoxanthin. The absorption and metabolism of other epoxyxanthophylls such as neoxanthin and violaxanthin, which are major carotenoids in green vegetables, warrant future study.
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FOOTNOTES |
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3 Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; UGP, UDP-glucuronosyltransferase; UV-VIS, UV-visible.
Manuscript received 26 October 2001. Initial review completed 4 December 2001. Revision accepted 29 January 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T. Sugawara, M. Kinoshita, M. Ohnishi, J. Nagata, and M. Saito Digestion of Maize Sphingolipids in Rats and Uptake of Sphingadienine by Caco-2 Cells J. Nutr., September 1, 2003; 133(9): 2777 - 2782. [Abstract] [Full Text] [PDF]
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