Akihiko Nagao

Antioxidant activity of xanthophylls on peroxyl radical-mediated phospholipid peroxidation

The ability of xanthophylls (canthaxanthin, zeaxanthin, and astaxanthin) as chain-breaking antioxidants was investigated in peroxyl radical-mediated peroxidation of phosphatidylcholine (PC) liposomes under atmospheric conditions using lipid-soluble and water-soluble radical generators. These xanthophylls retarded the chain propagation reaction of phosphatidylcholine hydroperoxides (PC-OOH) formation, although their activities to trap chain-carrying peroxyl radical were much less than that of alpha-tocopherol. In chick plasma studies, it was observed that endogenious xanthophylls participated in the antioxidant defenses against the attack of aqueous peroxyl radical. It was concluded that xanthophylls possess the ability to act as chain-breaking antioxidants in the peroxidation of membraneous phospholipids. Dietary xanthophylls may, therefore, be helpful in resisting membraneous phospholipids against oxidative damage in vivo.

Acyclic Carotenoids and Their Oxidation Mixtures Inhibit the Growth of HL-60 Human Promyelocytic Leukemia Cells

Lycopene has been known as a potential food component for cancer prevention, since tomato consumption was shown to be associated with reduced risk of certain cancers. We used HL-60 cells as a model of cancer cells to investigate whether acyclic carotenoids, such as phytoene, phytofluene, and ζ-carotene present in tomatoes, other than lycopene, as well as oxidation mixtures of these carotenoids, are potentially involved in the cancer-preventive action of tomatoes. When HL-60 cells were grown in the carotenoid-supplemented medium for 120 hours, ζ-carotene and phytofluene at 10 μM inhibited cell growth to 3.7% and 22.6% of the growth in control culture, respectively, although they were extremely unstable in the culture medium. The oxidation mixture of each carotenoid, which was prepared by incubation in toluene at 37°C for 24 hours, more strongly inhibited cell growth than each intact carotenoid. The growth inhibition by lycopene was remarkably enhanced by its oxidation before supplementation to the medium. Phytofluene, ζ-carotene, and the oxidation mixture of lycopene induced apoptosis in HL-60 cells during incubation for 24 hours. The addition of α-tocopherol to the medium did not eliminate growth inhibition by the oxidation mixture of lycopene. These results suggest that the acyclic carotenoids inhibit cell growth through apoptosis induction and that oxidation products of the carotenoids participate in the growth inhibition.

Phospholipids Affect the Intestinal Absorption of Carotenoids in Mice

Previously, we have shown that uptake of carotenoids solubilized with mixed micelles by human intestinal Caco-2 cells is enhanced by lysophosphatidylcholine (lysoPC) and suppressed by PC. This study determined the effect of PC and lysoPC in mixed micelles on the accumulation of β-carotene and lutein in mice in order to elucidate the roles of micellar phospholipid in the intestinal uptake of carotenoids in vivo. Mixed micelles were composed of 2.5 mM monooleoylglycerol, 7.5 mM oleic acid, 12 mM sodium taurocholate, 200 μM carotenoid, and 3 mM phospholipid in PBS. The mice were fed single doses of β-carotene or lutein solubilized in PC (PC group), lysoPC (LPC group), and no phospholipid (NoPL group) micelles. The β-carotene responses in the plasma and liver of the PC group were markedly lower than those of the other two groups, whereas no differences were noticed between the LPC and NoPL groups. The average level of lutein in the plasma of the PC group after administration was significantly (P<0.05) lower than those of the other groups. Moreover, the average level of lutein in the liver was significantly (P<0.05) different among the groups in the order of LPC>NoPL>PC. Thus, the results clearly indicate that PC suppressed the accumulation of β-carotene and lutein in plasma and liver and that lysoPC enhanced the accumulation of lutein in liver. These results suggest that the hydrolysis of PC to lysoPC plays an important role in the intestinal uptake of carotenoids solubilized in mixed micelles.

Formation of cleavage products by autoxidation of lycopene

The cleavage products formed by autoxidation of lycopene were evaluated in order to elucidate possible oxidation products of lycopene in biological tissues. Lycopene solubilized at 50 μM in toluene, aqueous Tween 40, or liposomal suspension was oxidized by incubating at 37°C for 72 h. Among a number of oxidation products formed, eight products in the carbonyl compound fraction were identified as 3,7,11-trimethyl-2,4,6,10-dodecatetraen-1-al, 6,10,14-trimethyl-3,5,7,9,13-pentadecapentaen-2-one, acycloretinal, apo-14′-lycopenal, apo-12′-lycopenal, apo-10′-lycopenal, apo-8′-lycopenal, and apo-6′-lycopenal. These correspond to a series of products formed by cleavage in the respective 11 conjugated double bonds of lycopene. The maximal formation of acycloretinal was 135 nM in toluene, 49 nM in aqueous Tween 40, and 64 nM in liposomal suspension. Acycloretinoic acid was also formed by autoxidation of lycopene, although its formation was lower in the aqueous media than in toluene. The pig liver homogenate had the ability to convert acycloretinal to acycloretinoic acid, comparable to the conversion of all-trans-retinal to all-trans-retinoic acid. These results suggest that lycopene might be cleaved to a series of apolycopenals and short-chain carbonyl compounds under the oxidative conditions in biological tissues and that acycloretinal is further enzymatically converted to acycloretinoic acid.

Low bioavailability of dietary epoxyxanthophylls in humans

Epoxyxanthophylls (epoxide-containing xanthophylls), a group of carotenoids, are ubiquitously distributed in edible plants. Among them, neoxanthin in green leafy vegetables and fucoxanthin in brown algae have been reported to exhibit an antiproliferative effect on several human cancer cells in vitro. However, there is little information about the intestinal absorption and metabolic fate of dietary epoxyxanthophylls in humans. To estimate the intestinal absorption of neoxanthin and fucoxanthin in humans, we evaluated the plasma epoxyxanthophyll concentrations before and after 1-week dietary interventions with spinach (Spinacia oleracea) and wakame (Undaria pinnatifida). The epoxyxanthophylls and their metabolites in the plasma extracts were determined by HPLC after partial purification and concentration with solid-phase extraction cartridges. Even after 1 week of spinach intake (3.0 mg neoxanthin/d), the plasma concentrations of neoxanthin and its metabolites (neochrome stereoisomers) remained very low (about 1 nmol/l), whereas those of beta-carotene and lutein were markedly increased. Similarly, the plasma concentration of fucoxanthinol, a gastrointestinal metabolite of fucoxanthin, was < 1 nmol/l after 1 week of wakame intake (6.1 mg fucoxanthin/d). These results indicated that the plasma response to dietary epoxyxanthophylls was very low in humans even after 1-week intake of epoxyxanthophyll-rich diets.

Absorption and Metabolism of Xanthophylls

Dietary carotenoids, especially xanthophylls, have attracted significant attention because of their characteristic biological activities, including anti-allergic, anti-cancer, and anti-obese actions. Although no less than forty carotenoids are ingested under usual dietary habits, only six carotenoids and their metabolites have been found in human tissues, suggesting selectivity in the intestinal absorption of carotenoids. Recently, facilitated diffusion in addition to simple diffusion has been reported to mediate the intestinal absorption of carotenoids in mammals. The selective absorption of carotenoids may be caused by uptake to the intestinal epithelia by the facilitated diffusion and an unknown excretion to intestinal lumen. It is well known that β-carotene can be metabolized to vitamin A after intestinal absorption of carotenoids, but little is known about the metabolic transformation of non provitamin A xanthophylls. The enzymatic oxidation of the secondary hydroxyl group leading to keto-carotenoids would occur as a common pathway of xanthophyll metabolism in mammals. This paper reviews the absorption and metabolism of xanthophylls by introducing recent advances in this field.

Characterization of Apoptosis Induced by Fucoxanthin in Human Promyelocytic Leukemia Cells

Apoptosis induced by fucoxanthin in HL-60 cells was associated with a loss of mitochondrial membrane potential at an early stage, but not with an increase in reactive oxygen species. Fucoxanthin treatment caused cleavages of procaspase-3 and poly (ADP-ribose) polymerase without any effect on the protein level of Bcl-2, Bcl-XL, or Bax. Apoptosis induction by fucoxanthin may be mediated via mitochondrial membrane permeabilization and caspase-3 activation.

Inhibition of myeloperoxidase-catalyzed tyrosylation by phenolic antioxidants in vitro.

We have developed an in vitro assay system for the evaluation of the inhibitory effects of phenolic antioxidants on myeloperoxidase (MPO) activity. The formation of dityrosine from the MPO/H2O2/L-tyrosine system was used as an indicator of the MPO activity. Because the buffer system used does not include chloride ion, this assay has the advantage of exclusion of direct reaction between an antioxidant and HOCl. In this assay, ferulic acid, gallic acid, and quercetin strongly inhibited the dityrosine formation, and curcumin and caffeic acid were also effective.

Soluble Fibers Inhibit Carotenoid Micellization in Vitro and Uptake by Caco-2 Cells

We evaluated the effects of soluble fibers on β-carotene and lutein micellization during simulated digestion in vitro, and on carotenoid uptake from mixed micelles by Caco-2 cells. Medium- and high-viscosity alginates and pectins inhibited carotenoid micellization and cellular uptake relative to the fiber-free control. Alginates, carboxy-methylcelluloses, and methylcelluloses inhibited β-carotene uptake mainly by increasing medium viscosity, but pectins might inhibit carotenoid uptake by additional mechanisms.

Effects of fats and oils on the bioaccessibility of carotenoids and vitamin E in vegetables.

The low bioavailability of lipophilic micronutrients is mainly caused by their limited solubilization to an aqueous micelle, which hinders their ability to be taken up by the intestines. Bioaccessibility is the ratio of the solubilized portion to the whole amount ingested. We evaluated in this study the effects of individual fats and oils and their constituents on the bioaccessibility of carotenoids and vitamin E in vegetables by simulated digestion. Various fats and oils and long-chain triacylglycerols enhanced the bioaccessibility of β-carotene present in spinach, but not of lutein and α-tocopherol, which are less hydrophobic than β-carotene. Free fatty acid, monoacylglycerol, and diacylglycerol also enhanced the bioaccessibility of β-carotene present in spinach. In addition to the long-chain triacylglycerols, their hydrolyzates formed during digestion would facilitate the dispersion and solubilization of β-carotene into mixed micelles. Dietary fats and oils would therefore enhance the bioaccessibility of hydrophobic carotenes present in vegetables.