Tatsuya Sugawara

Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light-scattering detection

Glycolipids from edible plant sources were accurately quantified by silica-based, normal-phase high-performance liquid chromatography using an evaporative light-scattering detector. Five major glycolipid classes (acylated steryl glucoside, steryl glucoside, ceramide monohexoside, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol) were separated and determined with a binary gradient system consisting of chloroform and methanol/water (95∶5, vol/vol) without any interference from other lipid classes and pigments. The described method was applied to 48 edible plants available in Japan including cereals, legumes, vegetables, and fruits. Examined plant species contained glycolipids in wide concentration ranges, such as 5–645 mg/100 g tissue.

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.

Isolation of Sphingoid Bases of Sea Cucumber Cerebrosides and Their Cytotoxicity against Human Colon Cancer Cells

Sea cucumber is a health-beneficial food, and contains a variety of physiologically active substances including glycosphingolipids. We show here the sphingoid base composition of cerebrosides prepared from sea cucumber and the cytotoxicity against human colon cancer cell lines. The composition of sphingoid bases prepared from sea cucumber was different from that of mammals, and the major constituents estimated from mass spectra had a branched C17–19 alkyl chain with 1–3 double bonds. The viability of DLD-1, WiDr and Caco-2 cells treated with sea cucumber sphingoid bases was reduced in a dose-dependent manner and was similar to that of cells treated with sphingosine. The sphingoid bases induced such a morphological change as condensed chromatin fragments and increased the caspase-3 activity, indicating that the sphingoid bases reduced the cell viability by causing apoptosis in these cells. Sphingolipids of sea cucumber might therefore serve as bioactive dietary components to suppress colon cancer.

Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and β-carotene significantly inhibited the antigen-induced release of β-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (FcϵRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited FcϵRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of FcϵRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of FcϵRI to lipid rafts, which are known as platforms of the aggregation of FcϵRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of FcϵRI to lipid rafts. This is the first report that focused on the aggregation of FcϵRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.

Siphonaxanthin, a marine carotenoid from green algae, effectively induces apoptosis in human leukemia (HL-60) cells.

BACKGROUND Bioactive marine molecules have recently received considerable attention for their nutraceutical characteristics. Considering the ever-increasing demand of nutraceuticals for anti-cancer therapy, we investigated the apoptosis-inducing effects of marine carotenoids, including siphonaxanthin, on human leukemia (HL-60) cells. METHODS Apoptotic effects were evaluated by cell viability assay, TUNEL assay, and caspase-3 activity. The expression of apoptosis-inducing death receptor-5 (DR5), Bcl-2 and Bax were assayed by Western blot analysis, and mRNA expression of GADD45α was assayed by quantitative RT-PCR analysis. RESULTS Siphonaxanthin potently inhibited the viability of HL-60 cells compared with the other carotenoids evaluated. In comparison with fucoxanthin, siphonaxanthin at a concentration of 20μM markedly reduced cell viability (p<0.05) as early as within 6h of treatment. The effective apoptotic activity of siphonaxanthin was observed by increases in TUNEL-positive cells, and by increased chromatin condensation in HL-60 cells. This induction of apoptosis was associated with the decreased expression of Bcl-2, and the subsequently increased activation of caspase-3. In addition, siphonaxanthin up-regulated the expression of GADD45α and DR5. CONCLUSIONS These data suggest that the dietary carotenoid siphonaxanthin could be potentially useful as a chemo-preventive and/or chemotherapeutic agent. GENERAL SIGNIFICANCE Our findings demonstrate for the first time the novel functional property of siphonaxanthin as a potent inducer of apoptosis in HL-60 cells.

Protective Effect of Fucoxanthin against UVB-Induced Skin Photoaging in Hairless Mice

Fucoxanthin, a major carotenoid in brown algae, has various beneficial effects. In this study, we evaluated the effect of topical fucoxanthin on UVB-induced skin photoaging in hairless mice. The dorsal skins were treated topically with a 0.001% fucoxanthin solution 2 h each time before UVB irradiation (5 times a week) for 10 weeks. The formation of wrinkles in UVB-irradiated skin treated with vehicle alone significantly increased, as compared with the non-irradiated control. Treatment with fucoxanthin tended to suppress UVB-induced wrinkle formation, but there was no significant difference between wrinkle formation in the control group and the fucoxanthin treatment group. However, topical treatment with fucoxanthin significantly lessened UVB-induced epidermal hypertrophy, VEGF, and MMP-13 expression in the epidermis and thiobarbituric acid reactive substances (TBARS) in the skin. These results indicate that topical treatment with fucoxanthin prevents skin photoaging in UVB-irradiated hairless mice, possibly via antioxidant and antiangiogenic effects.

Anti-angiogenic effect of siphonaxanthin from green alga, Codium fragile.

Since anti-angiogenic therapy has becoming a promising approach in the prevention of cancer and related diseases, the present study was aimed to examine the anti-angiogenic effect of siphonaxanthin from green alga (Codium fragile) in cell culture model systems and ex vivo approaches using human umbilical vein endothelial cells (HUVECs) and rat aortic ring, respectively. Siphonaxanthin significantly suppressed HUVEC proliferation (p<0.05) at the concentration of 2.5 μM (50% as compared with control) and above, while the effect on chemotaxis was not significant. Siphonaxanthin exhibited strong inhibitory effect on HUVEC tube formation. It suppressed the formation of tube length by 44% at the concentration of 10 μM, while no tube formation was observed at 25 μM, suggesting that it could be due to the suppression of angiogenic mediators. The ex vivo angiogenesis assay exhibited reduced microvessel outgrowth in a dose dependent manner and the reduction was significant at more than 2.5 μM. Our results imply a new insight on the novel function of siphonaxanthin in preventing angiogenesis related diseases.

Trans geometric isomers of EPA decrease LXRα-induced cellular triacylglycerol via suppression of SREBP-1c and PGC-1β

Dietary mono- or di-trans fatty acids with chain lengths of 18–22 increase the risk of cardiovascular diseases because they increase LDL cholesterol and decrease HDL cholesterol in the plasma. However, the effects of trans isomers of PUFAs on lipid metabolism remain unknown. Dietary PUFAs, especially eicosapentaenoic acid (EPA) in marine oils, improve serum lipid profiles by suppressing liver X receptor α (LXRα) activity in the liver. In this study, we compared the effects of trans geometric isomers of eicosapentaenoic acid (TEPA) on triacylglycerol synthesis induced by a synthetic LXRα agonist (T0901317) with the effects of EPA in HepG2 cells. TEPA significantly decreased the amount of cellular triacylglycerol and the expression of mRNAs encoding fatty acid synthase, stearoyl-CoA desaturase-1, and glycerol-3-phosphate acyltransferase induced by T0901317 compared with EPA. However, there was no significant difference between the suppressive effect of TEPA or EPA on the expression of sterol-regulatory element binding protein-1c (SREBP-1c) induced by T0901317. We found that TEPA, but not EPA, decreased the mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β), which is a coactivator of both LXRα and SREBP-1. These results suggest that the hypolipidemic effect of TEPA can be attributed to a decrease not only in SREBP-1 but also in PGC-1β expression.

Intestinal absorption of dietary maize glucosylceramide in lymphatic duct cannulated rats

Sphingolipids are ubiquitous in all eukaryotic organisms. Various physiological functions of dietary sphingolipids, such as preventing colon cancer and improving the skin barrier function, have been recently reported. One of the common sphingolipids used as a foodstuff is glucosylceramide from plant sources, which is composed of sphingoid bases distinct from those of mammals. However, the fate of dietary sphingolipids derived from plants is still not understood. In this study, we investigated the absorption of maize glucosylceramide in the rat intestine using a lipid absorption assay of lymph from the thoracic duct. The free and complex forms of trans-4,cis-8-sphingadienine, the predominant sphingoid base of maize glucosylceramide, were found in the lymph after administration of maize glucosylceramide. This plant type of sphingoid base was detected in the ceramide fraction and N-palmitoyl-4,8-sphingadienine (C16:0-d18:2) and N-tricosanoyl-4,8-sphingadienine (C23:0-d18:2) were identified by LC-MS/MS. The cumulative recovery of 4t,8c-sphingadienine in the lymph was very low. These results indicate that dietary glucosylceramide originating from higher plants is slightly absorbed in the intestine and is incorporated into ceramide structures in the intestinal cells. However, it appears that the intact form of sphingoid bases is not reutilized well in the tissues.

Efflux of Sphingoid Bases by P-Glycoprotein in Human Intestinal Caco-2 Cells

The aim of this study was to determine whether sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 μM digoxin for 48 h caused up-regulation of murtidrug-resistance (MDR)1 mRNA and decreased the accumulation of sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive tract.