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In a randomized double-blind crossover study, a canned beverage was prepared using an emulsion dispersion formulation (EM) of β-carotene and a crystal dispersion formulation (CR) of β-carotene; the beverages were ingested by human subjects daily for 2 weeks to compare the β-carotene bioavailability. EM-β-carotene enhanced the β-carotene concentrations in human plasma approximately 4-fold, but CR-β-carotene showed no statistically significant enhancement. Bioaccessibility is the ratio of the solubilized fraction to the whole amount ingested. Bioaccessibility of β-carotene from EM-β-carotene was higher than that from CR-β-carotene in an in vitro digestion test. Contrarily, β-carotene from CR-β-carotene, consists of all-trans-β-carotene, was higher than that from EM-β-carotene, consists of a mixture of cis and all-trans-β-carotene, on the uptake by intestinal Caco-2 cells, suggesting that bioaccessibility was a critical factor in β-carotene bioavailability in this study. EM-β-carotene thus has potential as a food coloring agent with value added because it enhances β-carotene bioavailability.

RIPENING INHIBITOR (RIN) is a transcription factor with transcriptional activator activity that plays a major role in regulating fruit ripening in tomato (Solanum lycopersicum). Recent studies have revealed that (1) RIN is indispensable for full ripening but not for the induction of ripening; and (2) the rin mutation, which produces nonripening fruits that never turn red or soften, is not a null mutation but instead converts the encoded transcriptional activator into a repressor. Here, we have uncovered aspects of RIN function by characterizing a series of allelic mutations within this locus that were produced by CRISPR/Cas9. Fruits of RIN-knockout plants, which are characterized by partial ripening and low levels of lycopene but never turn fully red, showed excess flesh softening compared to the wild type. The knockout mutant fruits also showed accelerated cell wall degradation, suggesting that, contrary to the conventional view, RIN represses over-ripening in addition to facilitating ripening. A C-terminal domain-truncated RIN protein, encoded by another allele of the RIN locus (rinG2), did not activate transcription but formed transcription factor complexes that bound to target genomic regions in a manner similar to that observed for wild-type RIN protein. Fruits expressing this truncated RIN protein exhibited extended shelf life, but unlike rin fruits, they accumulated lycopene and appeared orange. The diverse ripening properties of the RIN allelic mutants suggest that substantial phenotypic variation can be produced by tuning the activity of a transcription factor.

The γ-oryzanol contents and the composition of steryl ferulates distributed in Japanese pigmented rice varieties were investigated using the high-performance liquid chromatography–ultraviolet detection method for the purpose of expanding their utilisation. The average γ-oryzanol content in nine black-purple, four red, four green and three brown rice varieties was 54.2, 47.3, 44.3 and 43.3 mg γ-oryzanol equivalent/100 g dried weight, respectively. Among the nine varieties of black-purple rice, five varieties showed steryl ferulate composition similar to that of brown, red and green varieties. In contrast, the composition of steryl ferulates in other four black-purple rice varieties was partially specific and was characterised by a low amount of campesteryl ferulate and high of campestanyl ferulate and stigmastanyl ferulate. The latter two steryl ferulates have been recognised as minor components of γ-oryzanol in rice and as major components in wheat and corn. These results indicate that the compositions of steryl ferulates vary among Japanese black-purple rice varieties. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13197-019-03767-w) contains supplementary material, which is available to authorized users.

Vitamins D have various biological activities, as well as intestinal calcium absorption. There has been recent concern about insufficient vitamin D intake. In addition to vitamins D2 and D3, there are lesser-known vitamins D4–D7. We synthesized vitamins D5–D7, which are not commercially available, and then evaluated and compared the mixed micelles-solubilized vitamins D uptake by Caco-2 cells. Except for vitamin D5, the uptake amounts of vitamins D4–D7 by differentiated Caco-2 cells were similar to those of vitamins D2 and D3. The facilitative diffusion rate in the ezetimibe inhibited pathway was approximately 20% for each vitamin D type, suggesting that they would pass through the pathway at a similar rate. Lysophosphatidylcholine enhanced each vitamin D uptake by approximately 2.5-fold. Lysophosphatidylcholine showed an enhancing effect on vitamin D uptake by reducing the intercellular barrier formation of Caco-2 cells by reducing cellular cholesterol, suggesting that increasing the uptakes of vitamins D and/or co-ingesting them with lysophosphatidylcholine, would improve vitamin D insufficiency. The various biological activities in the activated form of vitamins D4–D7 were estimated by Prediction of Activity Spectra for Substances (PASS) online simulation. These may have some biological activities, supporting the potential as nutritional components.

The tomato (Solanum lycopersicum)ripening inhibitor(rin) mutation completely represses fruit ripening, asrinfruits fail to express ripening-associated genes and remain green and firm. Moreover, heterozygousrinfruits (rin/+) ripen normally but have extended shelf life, an important consideration for this perishable fruit crop; therefore, heterozygousrinhas been widely used to breed varieties that produce red tomatoes with improved shelf life. We previously used CRISPR/Cas9 to produce novel alleles at therinlocus. The wild-type alleleRINencodes a MADS-box transcription factor and the novel allele, named asrinG2, generates an early stop codon, resulting in C-terminal truncation of the transcription factor. Likerinfruits,rinG2fruits exhibit extended shelf life, but unlikerinfruits, which remain yellow-green even after long-term storage,rinG2fruits turn orange due to ripening-associated carotenoid production. Here, to explore the potential of therinG2mutation for breeding, we characterized the effects ofrinG2in the heterozygous state (rinG2/+) compared to the effects ofrin/+. The softening ofrinG2/+fruits was delayed compared to the wild type but to a lesser degree thanrin/+fruits. Lycopene and β-carotene levels inrinG2/+fruits were similar to those of the wild type, whereasrin/+fruits accumulated half the amount of β-carotene compared to the wild type. TherinG2/+fruits produced lower levels of ethylene than wild-type andrin/+fruits. Expression analysis revealed that inrinG2/+fruits, therinG2mutation (likerin) partially inhibited the expression of ripening-associated genes. The small differences in the inhibitory effects ofrinG2vs.rincoincided with small differences in phenotypes, such as ethylene production, softening, and carotenoid accumulation. Therefore,rinG2represents a promising genetic resource for developing tomato cultivars with extended shelf life.

Although oryzanol in rice bran is one of the most utilised steryl ferulates (SFs, ferulic acid/coumaric acid esters of phytosterol/triterpene alcohol), SFs are also present in other cereals. Thus, we herein report the SF profiles present in various cereals, pseudo-cereals and millets using a high performance liquid chromatography (HPLC) method. The SF contents of whole foxtail millet, whole proso millet, and whole barnyard millet were 28.9, 0.2, and 4.8 mg-oryzanol equivalent/100 g, respectively, indicating that the SF content in whole foxtail millet was about 80% of oryzanol in brown rice. Using an atmospheric pressure chemical ionisation method combined with mass spectrometry (APCI-MS), the SF components from several millets and cereals were identified. It revealed that SFs of whole foxtail millet was composed of stigmastanyl ferulate (73%), stigmastanyl coumarate (10%), and others (17%). Furthermore, SF compositions of whole barnyard millet and naked barley were firstly determined and they were considerably different from those of other cereals.

We previously showed that Caco-2 cell absorption of β-carotene from taurocholic acid (TA)-based mixed micelles differed depending on the composition of the micelles. In this study, the shapes and sizes of TA-based mixed micelles, that is, mixed micelles of TA, 1-oleoyl-rac-glycerol (MG), oleic acid (OLA), and either 1-palmitoyl-sn-glycero-3-phosphocholine (MPPC; i.e., a lysophospholipid) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; i.e., a phospholipid) (60:3:1:0.75–12) were determined by using small-angle X-ray scattering (SAXS). We found that increasing the ratio of MPPC in mixed micelles of TA, MG, OLA, and MPPC was responsible for the previously observed enhanced β-carotene absorption by Caco-2 cells and changed the micelle shape from core–shell spherical to core–shell ellipsoidal. In contrast, increasing the ratio of POPC in mixed micelles of TA, MG, OLA, and POPC was responsible for the suppressed β-carotene absorption by the cells, changed the micelle shape from core–shell spherical to core–shell ellipsoidal to core–shell cylindrical, and caused a rapid increase in micelle volume. These results will be useful for understanding the mechanisms that mediate β-carotene absorption by cells and for developing technologies to improve the intestinal absorption of lipophilic components of drugs and nutrients.

Tomato (Solanum lycopersicum) rin mutants completely fail to ripen: they do not produce red pigmentation, soften or induce an ethylene burst. Therefore, RIN has long been believed to function as a major regulator that is essential for the induction of ripening. Here, we provide evidence contradicting this concept of RIN function, showing induction of fruit ripening in the absence of RIN. A CRISPR/Cas9-mediated RIN-knockout mutation did not repress initiation of ripening and the mutant fruits showed moderate red colouring. Moreover, inactivation of the rin mutant allele partially restored the induction of ripening. Therefore, RIN is not required for the initiation of ripening and rin is not a null mutation, but rather is a gain-of-function mutation that produces a protein that actively represses ripening. Since the discovery of the rin mutant a half-century ago, many models have depicted RIN as indispensable for the induction of ripening; these models should be reconsidered in light of these results.

Oryzanol contained in rice bran is a complex mixture of steryl ferulates (SFs) with many identified health benefits. Recently, SF has been shown to exist in other cereals such as wheat, rye, and corn. In this study, SFs in several wheats produced in Japan were analyzed. For instance, SF content of whole wheat grain, Yumekaori (Japan) was 15.2 ± 1.4 mg-oryzanol-equivalent/100 g grain, while that of the imported one, 1CW (Canada) was 11.4 ± 1.3 mg-oryzanol-equivalent/100 g grain. The main SF components in the examined wheats were campesteryl ferulate, campestanyl ferulate, and sitostanyl ferulate. SF distribution in whole wheat grain was investigated using 14 fractions produced by a conventional test milling machine. SF was intensively accumulated in the four bran fractions (24 − 95 mg-oryzanol-equivalent/100 g bran fraction). These results suggest that the wheat bran would be an important source of SF.

The behavior of radiocesium in wild animal meats upon cooking was investigated. The ratio of the concentration change (processing factor, Pf), remaining ratio (food processing retention factor, Fr), and removal ratio of radiocesium in the meats by grilling, boiling, and steaming were determined. Differences in cooking methods, rather than differences in meat parts or animal species, clearly influenced the Pf, Fr, and removal ratios. The mean Fr values were 0.9 (range, 0.7 to 1.0) for grilling, 0.6 (range, 0.4 to 0.7) for boiling, and 0.5 (range, 0.4 to 0.7) for steaming. The removal effect of grilling (11%) was lower than that of boiling (41%) or steaming (47%). The mean value of Pf was 1.2 (range, 1.1 to 1.6) for grilling, 0.8 (range, 0.6 to 0.9) for boiling, and 0.8 (range, 0.7 to 1.0) for steaming. The radiocesium concentration in the meats increased only upon grilling, but not by boiling or steaming. This difference is due to the lower removal effect of grilling than that of boiling and steaming. Therefore, boiling and steaming were more effective than grilling for removing radiocesium and reducing its concentration in wild animal meats. Furthermore, the ratio of water content fluctuations due to boiling was negatively correlated with Pf and Fr. It was evident that greater reductions in water content resulted in lower concentrations and improved radiocesium removal in the meats. These results suggest that some of the radiocesium naturally present in the meats is soluble in water and that the radiocesium dissolved in water can be removed from the meat with the release of water from the tissue. HIGHLIGHTS

We succeeded in simultaneously synthesizing the vitamin D family, vitamins D2, D4, D5, D6, and D7, from &beta, sitosterol, which is sold as a commercially available reagent from Tokyo Chemical Industry Co., Ltd. It is officially sold as a mixture of four phytosterols {&beta, sitosterol (40&ndash, 45%), campesterol (20&ndash, 30%), stigmasterol, and brassicasterol}. Owing to this, we anticipated that, using this reagent, various vitamin D analogs could be synthesized simultaneously. We also synthesized vitamin D3 from pure cholesterol and analyzed and compared all vitamin D analogs (D2, D3, D4, D5, D6, and D7) by HSQC NMR. We succeeded in clearly demonstrating the difference in the NMR chemical shifts for each vitamin D analog.

We previously found that mice fed lutein accumulated its oxidative metabolites (3'-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3'-dione) as major carotenoids, suggesting that mammals can convert xanthophylls to keto-carotenoids by the oxidation of hydroxyl groups. Here we elucidated the metabolic activities of mouse liver for several xanthophylls. When lutein was incubated with liver postmitochondrial fraction in the presence of NAD(+), (3'R,6'R)-3'-hydroxy-β,ε-caroten-3-one and (6RS,3'R,6'R)-3'-hydroxy-ε,ε-caroten-3-one were produced as major oxidation products. The former accumulated only at the early stage and was assumed to be an intermediate, followed by isomerization to the latter. The configuration at the C3' and C6' of the ε-end group in lutein was retained in the two oxidation products. These results indicate that the 3-hydroxy β-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo β-end group. Other xanthophylls such as β-cryptoxanthin and zeaxanthin, which have a 3-hydroxy β-end group, were also oxidized in the same manner as lutein. These keto-carotenoids, derived from dietary xanthophylls, were confirmed to be present in plasma of normal human subjects, and β,ε-caroten-3'-one was significantly increased by the ingestion of β-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy β-end group of xanthophylls to a 3-oxo ε-end group.

Novel fucoxanthin derivatives that could change the size of mixed micelles were synthesized. The mixed micelles under consideration consist of a bile acid and some additives. To change the affinity against a bile acid, we designed the synthesis of a fucoxanthin-lithocholic acid complex. Lithocholic acid is one of the bile acids. The 3-OH on lithocholic acid was protected by a levulinyl group, and the protected lithocholic acid was selectively coupled via an ester linkage to the 3-OH on fucoxanthin to obtain levulinyl-protected lithocholyl fucoxanthin (LevLF). The levulinyl group was then selectively deprotected using hydrazine to obtain a lithocholyl fucoxanthin (LF). The average sizes of the micelles that contained these compounds (fucoxanthin, LevLF, and LF) with a bile acid (sodium taurocholate) were measured. The LevLF induced larger micelles than fucoxanthin or LF. Interestingly, the addition of 1-oleoyl-rac-glycerol induced a more efficient change in the micelle size. The large micelles grew larger, and the small micelles became smaller. Triple-mixed micelles with LevLF, sodium taurocholate, and 1-oleoyl-rac-glycerol formed the largest micelle with a diameter of 68 nm. On the other hand, triple-mixed micelles using LF, sodium taurocholate, and 1-oleoyl-rac-glycerol made the smallest micelles with diameters as low as 12 nm. We also investigated the hydrolysis of these compounds with enzymes (esterase from porcine liver, lipase from porcine pancreas, and cholesterol esterase from Pseudomonas sp.). The ester linkage between the lithocholic acid and fucoxanthin of LevLF was hydrolyzed with cholesterol esterase. In addition, the intestinal absorption was examined with Caco-2 cells, and no advantageous change in absorption efficiency was observed by chemically modifying the fucoxanthin unless different micelles sizes and increasing hydrophobicity are induced.

The antioxidant behaviour of major dietary carotenoids accumulated at high concentrations in human hepatoma HepG2 cells was evaluated, in comparison with α-tocopherol. The cells that accumulated carotenoids and α-tocopherol at levels higher than the values reported in the human liver were exposed to mild oxidative stress with tert -butylhydroperoxide. β-Carotene (>2.6 nmol/mg protein) and astaxanthin (>1.8 nmol/mg protein) significantly suppressed lipid peroxidation, while β-cryptoxanthin and lutein did not. α-Tocopherol remarkably suppressed lipid peroxidation with an IC 50 value of 0.16 nmol/mg protein. Neither α-tocopherol nor any of the carotenoids except for lycopene showed pro-oxidant action even at high cellular concentrations. The antioxidant behaviours of carotenoids in a cellular milieu were quite different from those previously found in liposomes and homogeneous solutions. Further studies are required to assess the implications of the antioxidant behaviours found in the cultured cells on human health.

Fucoxanthin has an antiproliferative effect on cancer cells, but its detailed structure&ndash, activity correlation has not yet been elucidated. To elucidate this correlation, fucoxanthin was degraded by ozonolysis. The degraded compounds of fucoxanthin obtained by ozonolysis were purified by HPLC and analyzed by NMR. The polyene chain of fucoxanthin was cleaved by ozonolysis, and the fucoxanthin was divided into two types of cyclohexyl derivatives, one with a &beta, &gamma, epoxy ketone group and the other with an allenic bond. In order to elucidate the structure&ndash, activity correlation, Caco-2 cells (human colorectal carcinoma) were treated with fucoxanthin degradation compounds. It was found that the entire structure of fucoxanthin is not essential for its antiproliferative effect and that even a partial structure exerts this effect.

We previously found that endothelin-2/vasoactive intestinal contractor (ET-2/VIC) greatly increased in mouse epidermis after birth. In the present study, we evaluated whether ET-2/VIC expression was associated with the calcium-induced differentiation of cultured mouse keratinocytes. The differentiation induction was revealed by morphological change, cornified envelope (CE) formation, and involucrin and transglutaminase 1 (TG 1) expressions. ET-2/VIC gene expression and peptide production subsequently increased in the induction of the differentiation. We also found that Y-27632, a Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) inhibitor, suppressed up-regulation of ET-2/VIC gene expression, the induction of morphological change, the CE formation, and TG 1 expression, but not involucrin expression. These results indicate new three findings, (1) ET-2/VIC expression increases and has potential as a differentiation marker, (2) ET-2/VIC expression is mediated by ROCK, and (3) the ROCK regulated TG 1 expression, on the calcium-induced differentiation of mouse keratinocytes.

An oxidative metabolite of lutein, 3′-hydroxy-ε,ε-caroten-3-one, inhibited the differentiation of 3T3-L1 cells to adipocytes and the subsequent triacylglycerol production, but lutein did not. The α,β-unsaturated carbonyl structure of 3′-hydroxy-ε,ε-caroten-3-one was considered to participate in the inhibitory effect, suggesting that this lutein metabolite has the potential to prevent metabolic syndrome.

To improve the intestinal absorption of fucoxanthin, we evaluated the effects of dietary glyceroglycolipids on the uptake and secretion of fucoxanthin solubilized in mixed micelles by human intestinal Caco-2 cells. Although digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol suppressed fucoxanthin uptake and secretion, their lyso-types, digalactosylmonoacylglycerol and sulfoquinovosylmonoa cylglycerol, remarkably enhanced them. Thus, some dietary glyceroglycolipids may be potential enhancers of fucoxanthin bioavailability in humans.

This paper reviews the local hormone endothelin-2 (ET-2), or vasoactive intestinal contractor (VIC), a member of the vasoconstrictor ET peptide family, where ET-2 is the human orthologous peptide of the murine VIC. While ET-2/VIC gene expression has been observed in some normal tissues, ET-2 recently has been reported to act as a tumor marker and as a hypoxia-induced autocrine survival factor in tumor cells. A recently published study reported that the hypoxic mimetic agent CoCl2at 200 µM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced intracellular reactive oxygen species (ROS) increase and neurite outgrowth in neuronal model PC12 cells. The ROS was generated by addition of CoCl2to the culture medium, and the CoCl2-induced effects were completely inhibited by the antioxidantN-acetyl cysteine. Furthermore, interleukin-6 (IL-6) gene expression was up-regulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by CoCl2-induced ROS may be associated with neuronal differentiation through the regulation of IL-6 expression. CoCl2acts as a pro-oxidant, as do Fe(II, III) and Cu(II). However, some biological activities have been reported for CoCl2that have not been observed for other metal salts such as FeCl3, CuSO4, and NiCl2. The characteristic actions of CoCl2may be associated with the differentiation of PC12 cells. Further elucidation of the mechanism of neurite outgrowth and regulation of ET-2/VIC expression by CoCl2may lead to the development of treatments for neuronal disorders.

Endothelin (ET)-2, an ET family peptide, is highly expressed in intestine. However, the specific distribution and function of ET-2 remain unknown. We elucidated the expression profile and localization of ET-2 in mouse gastrointestinal tract. Real-time PCR analysis revealed that ET-2 gene expression in the gastrointestinal tract of healthy animals was relatively high in the colon. Immunohistochemical analysis revealed ET-2-like immunoreactivity mainly in epithelial cells of the mucosa throughout the intestinal tract of healthy animals. Intracellularly, ET-2 was concentrated close to the basement membrane of intestinal epithelial cells. A weak ET-2-like immunoreactivity was also localized to some neurofibers and the myenteric plexus of the muscle layer, coexpressing with vasoactive intestinal peptide. ET-2-like immunoreactivity was also detected at Brunner’s glands of the duodenum and follicle-associated epithelium of Peyer’s patch. In contrast, ET-1-like immunoreactivity was uniformly distributed in epithelial cells. In dextran sulfate sodium (DSS)-induced colitis, colonic ET-2 was upregulated during the late stage of DSS treatment. These results suggest that in intestinal epithelial cells ET-2 could be secreted into the lamina propria and the dome region in Peyer’s patch, and that it might modulate immune cells in these sites for mucosal defense.

We investigated whether endothelin-2/vasoactive intestinal contractor (ET-2/VIC) gene expression, upregulated by hypoxia in cancer cells, was associated with differentiation in neuronal cells. RT-PCR analysis, morphological observations, and immunostaining revealed that CoCl2, a hypoxic mimetic agent, at 200 μM increased expression of the ET-2/VIC gene, decreased expression of the ET-1 gene, and induced neurite outgrowth in PC-12 rat pheochromocytoma cells. These effects induced by 200 μM CoCl2 were completely inhibited by the antioxidant N-acetyl cysteine at 20 mM. In addition, CoCl2 increased the level of intracellular reactive oxygen species (ROS) at an early stage. Furthermore, interleukin (IL)-6 gene expression was upregulated upon the differentiation induced by CoCl2. These results suggest that expression of ET-2/VIC and ET-1 mediated by ROS may be associated with neuronal differentiation through the regulation of IL-6. When the cells were treated with 500 μM CoCl2 for 24 hr, however, ET-2/VIC gene expression disappeared, IL-6 gene expression was downregulated, and necrosis was subsequently induced in the PC-12 cells. © 2005 Wiley-Liss, Inc.

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-X(L), or Bax. Apoptosis induction by fucoxanthin may be mediated via mitochondrial membrane permeabilization and caspase-3 activation.

Dietary carotenoids have been thought to have beneficial effects on human health through their antioxidant activity, provitamin A activity, and effects on cancer cell propagation. Recent studies suggest that oxidation products or metabolites are involved in biological activities of carotenoids. We previously reported that an autoxidation mixture of lycopene induced apoptosis in HL-60 human promyelocytic leukemia cells, but lycopene alone did not. In the present study, bioassay-directed fractionations of autoxidized lycopene led to isolation of a novel cleavage product of lycopene. Spectral analyses elucidated its structure as (E,E,E)-4-methyl-8-oxo-2,4,6-nonatrienal (MON), suggesting the formation through the oxidative cleavages at the 5, 6- and 13, 14-double bonds of lycopene. MON was proved to cause a dose-dependent reduction of viability in HL-60 cells with morphological changes such as chromatin condensation and nuclear fragmentation. Treatment of HL-60 cells with MON could induce DNA fragmentation and increase apoptotic cells in a time- and dose-dependent manner. The MON treatment could enhance both caspase 8 and caspase 9 activities. Moreover, it reduced the expression of Bcl-2 and Bcl-XL proteins, whereas it had no effect on the level of Bax protein. These results clearly indicated that MON induced apoptosis in HL-60 cells, associated with the down regulation of Bcl-2 and Bcl-XL and the activation of caspase cascades. The concentration of MON attained by treatment of the autoxidized lycopene preparation was far less than the IC50 (10 microM) value of MON alone in reducing the viability of HL-60 cells. The fractionation of the oxidized lycopene indicated the presence of other active oxidation products. Thus, unidentified products as well as MON would be responsible for the apoptosis-inducing activity of the autoxidized lycopene.

The oil content of the silkworm, Bombyx mori, in the pupal stage was 4.8% for the male and 9.0% for the female (wet basis), respectively. Total lipid (TL) extracted from silkworm pupae mainly consisted of triacylglycerol (TG), phosphatidylethanolamine, and phosphatidylcholine. TL and TG contained approximately 40% α-linolenic acid as the predominant fatty acid. 20:3n-3 was also present in the pupa, but the quantities were very small. Total tocopherol in TL was 125.2 μg/g lipid for the male and 224.1 μg/g lipid for the female, respectively. The oxidative stability of TL was confirmed by measuring the decrease in oxygen in the headspace. The oxidative stability of TL from silkworm pupae was very high and no difference between sexes was appeared. It has been suggested that a synergistic effect between phospholipids and tocopherol in silkworm pupa plays an important role in protecting the lipids against oxidation. Furthermore, the silkworm pupa contained carotenoids such as lutein and neoxanthin. These carotenoids may act as antioxidants in TL. The silkworm pupa would be a good source of the functional fatty acid, α-linolenic acid, and of the functional pigments, lutein and neoxanthin.

We previously reported that phospholipids markedly affected the uptake of carotenoids solubilized in mixed micelles by human intestinal Caco-2 cells. In the present study, we found that two classes of dietary glyceroglycolipids and the corresponding lysoglyceroglycolipids affected uptake of β-carotene and lutein by differentiated Caco-2 cells. The levels of carotenoid uptake from micelles containing digalactosyldiacylglycerol or sulfoquinovosyldiacylglycerol were significantly lower than that from control micelles. On the other hand, the uptakes from micelles containing digalactosylmonoacylglycerol or sulfoquinovosylmonoacylglycerol were significantly higher than that from control micelles. In dispersed cells and Caco-2 cells with poor cell-to-cell adhesion, however, the levels of uptake from micelles containing these lyso-lipids were much lower than that from control micelles. The uptake levels from control micelles were markedly decreased depending on the development of cell-to-cell/cell-matrix adhesion in Caco-2 cells, but the uptake levels from the micelles containing these lyso-lipids were not substantially changed, suggesting that the intercellular barrier formed by cell-to-cell/cell-matrix adhesion inhibited the uptake from control micelles, but not from the lyso-lipid-containing micelles. The lyso-lipids appeared to enhance carotenoid uptake by decreasing the intercellular barrier integrity. The results showed that some types of glyceroglycolipids have the potential to modify the intestinal uptake of carotenoids.

The effects were evaluated of various glycerophospholipids on the uptake of beta-carotene solubilized in mixed micelles by human intestinal Caco-2 cells. Phosphatidylethanolamine markedly enhanced the transfer of beta-carotene from the micelles to the cells, whereas phosphatidylcholine suppressed it. All the lysoglycerophospholipids enhanced the transfer, irrespective of the polar head group. Glycerophospholipids therefore have the potential to modify the intestinal absorption of carotenoids.

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.

We reported previously that lysophosphatidylcholine remarkably enhanced β-carotene uptake from bile acid-mixed micelles by human intestinal Caco-2 cells. In the present study, we evaluated how mixed micelle components other than phospholipids, viz., fatty acids, monoolein, and cholesterol, affect carotenoid uptake by Caco-2 cells. Each component influenced the β-carotene uptake in a different way depending on micellar composition. Oleic acid at 200 µM significantly enhanced uptake in the absence of lysophosphatidylcholine. Cholesterol at 40 µM significantly reduced uptake in the presence of lysophosphatidylcholine, while no reduction was found in the presence of 200 µM oleic acid. Facilitated diffusion was suggested partly to mediate uptake in mixed micelles, except for mixed micelles containing 200 µM oleic acid. Uptake mediated by facilitated diffusion was approximately 20% of total uptake. Mixed micellar lipids have the potential to modify intestinal uptake.

CoCl(2) and MnCl(2) are hypoxic mimetic agents. We previously found that expression of ET-2/VIC, one of hypoxia-related factors, and the induction of neurite outgrowth in PC12 cells through ROS induced by CoCl(2). MnCl(2) also are known to induce neurite outgrowth in PC12 cells. However, it is unclear whether the mechanism of the effect induced by these metals is same. In the present study, we evaluated biological effects induced by MnCl(2) and compared with those induced by CoCl(2). Furthermore, we analyzed sources of CoCl(2)-induced ROS generation. MnCl(2) up-regulated ET-2/VIC gene expression and ET-2/VIC peptide production as CoCl(2) did, but not affect ET-1 gene expression, in the neurite outgrowth of PC12 cells. NAC did not at all inhibit the effects induced by MnCl(2). Furthermore, addition of MnCl(2) to the culture medium did not generate ROS as CoCl(2) did. These results indicate that ET-2/VIC expression is a common pathway in neurite outgrowth induced by CoCl(2) and MnCl(2), but the effects induced by CoCl(2) are ROS dependent, whereas the effects induced by MnCl(2) are ROS independent. Taken together, the mechanism for the effects by CoCl(2) was different from that by MnCl(2). The ROS, were not decomposed by catalase or SOD, were rapidly generated by reaction of CoCl(2) mainly with components of HS rather than with FBS or DMEM. Some ROS generated by reaction of CoCl(2) with components of HS may participate in the observed neurite outgrowth of PC12 cells.

Acyclo-retinoic acid, a novel acyclic analogue of all-trans-retinoic acid, has been previously isolated as one of the in vitro oxidation products of lycopene. The effect of acyclo-retinoic acid on the growth of human prostate cancer cells was compared with those of the four retinoids: all-trans-retinoic acid, geranylgeranoic acid, 9-cis-retinoic acid and N-(4-hydroxyphenyl)retinamide. When prostate cancer cells, PC-3, DU 145 and LNCaP, were cultured in a retinoid-supplemented medium, acyclo-retinoic acid remarkably reduced the viability of the cells except for LNCaP. This effect was significantly higher than that of geranylgeranoic acid, all-trans-retinoic acid and 9-cis-retinoic acid, but was comparable to that of N-(4-hydroxyphenyl)retinamide. DNA fragmentations of nuclei in PC-3 and DU 145 cells treated with acyclo-retinoic acid were detected by in situ TUNEL assay. Furthermore, an apoptotic DNA ladder was observed in PC-3 cells. These results showed that acyclo-retinoic acid reduced cell viability by inducing apoptosis in human prostate cancer cells.

We investigated whether various carotenoids present in foodstuffs were potentially involved in cancer-preventing action on human prostate cancer. The effects of 15 kinds of carotenoids on the viability of three lines of human prostate cancer cells, PC-3, DU 145 and LNCaP, were evaluated. When the prostate cancer cells were cultured in a carotenoid-supplemented medium for 72 h at 20 micromol/L, 5,6-monoepoxy carotenoids, namely, neoxanthin from spinach and fucoxanthin from brown algae, significantly reduced cell viability to 10.9 and 14.9% for PC-3, 15.0 and 5.0% for DU 145, and nearly zero and 9.8% for LNCaP, respectively. Acyclic carotenoids such as phytofluene, zeta-carotene and lycopene, all of which are present in tomato, also significantly reduced cell viability. On the other hand, phytoene, canthaxanthin, beta-cryptoxanthin and zeaxanthin did not affect the growth of the prostate cancer cells. DNA fragmentation of nuclei in neoxanthin- and fucoxanthin-treated cells was detected by in situ TdT-mediated dUTP nick end labeling (TUNEL) assay. Neoxanthin and fucoxanthin were found to reduce cell viability through apoptosis induction in the human prostate cancer cells. These results suggest that ingestion of leafy green vegetables and edible brown algae rich in neoxanthin and fucoxanthin might have the potential to reduce the risk of prostate cancer.