Organic Synthesis of New Secosteroids from Fucosterol, Its Intestinal Absorption by Caco-2 Cells, and Simulation of the Biological Activities of Vitamin D.

We previously examined the cellular uptake of six types of vitamin D in human intestinal Caco-2 cells. Since vitamins D5-D7 were commercially unavailable, we synthesized these compounds organically before studying them. This process led us to understand that new secosteroids could be generated as vitamin D candidates, depending on the sterol used as the starting material. We obtained two new secosteroids-compounds 3 and 4-from fucosterol in the current study. We investigated the intestinal absorption of these compounds using Caco-2 cells cultured in Transwells and compared the results with vitamin D3, a representative secosteroid. The intestinal absorption of compound 4 was comparable to that of vitamin D3. Compound 3 showed similar uptake levels but transported about half as much as vitamin D3. These compounds demonstrated intestinal absorption at the cellular level. Vitamin D is known for its diverse biological activities manifest after intestinal absorption. Using PASS online simulation, we estimated the biological activity of compound 3's activated form. In several items indicated by PASS, compound 3 exhibited stronger biological activity than vitamins D2-D7 and was also predicted to have unique biological activities.

Effect of an Emulsified Formulation on Vegetable Carotenoid Bioaccessibility.

We investigated the effect of neutral lipids, polar lipids, and an emulsified formulation (EMF) on carotenoid bioaccessibility in an in vitro digestion assay of vegetables. These reagents enhanced carotenoid bioaccessibility. Contrary to our previous report, they also exhibited effects on lutein. Bile extracts/pancreatin concentrations also participated in the bioaccessibility. The EMF, which consisted of lower amounts of oil, had the same effect on lutein as rapeseed oil. These reagents also showed effects in the aging model, with more reduced bile extract/pancreatin concentrations, suggesting that lipids and EMF contributed to carotenoid bioaccessibility in bile/pancreatic juice secretions due to aging and disease.

Uptake of Vitamins D2, D3, D4, D5, D6, and D7 Solubilized in Mixed Micelles by Human Intestinal Cells, Caco-2, an Enhancing Effect of Lysophosphatidylcholine on the Cellular Uptake, and Estimation of Vitamins D' Biological Activities.

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.

Semi-dominant effects of a novel ripening inhibitor (rin) locus allele on tomato fruit ripening.

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

Effect of dispersed form on the bioavailability of ß-carotene from daily intake in humans.

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

Allelic Mutations in the Ripening -Inhibitor Locus Generate Extensive Variation in Tomato Ripening.

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.

Reduction in the Radiocesium in Meats of the Sika Deer and Wild Boar by Cooking.

ABSTRACT: 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.

Diversity in γ-oryzanol profiles of Japanese black-purple rice varieties.

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.

Simultaneous Synthesis of Vitamins D2, D4, D5, D6, and D7 from Commercially Available Phytosterol, ß-Sitosterol, and Identification of Each Vitamin D by HSQC NMR.

We succeeded in simultaneously synthesizing the vitamin D family, vitamins D2, D4, D5, D6, and D7, from ß-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 {ß-sitosterol (40-45%), campesterol (20-30%), 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.

Degradation of Fucoxanthin to Elucidate the Relationship between the Fucoxanthin Molecular Structure and Its Antiproliferative Effect on Caco-2 Cells.

Fucoxanthin has an antiproliferative effect on cancer cells, but its detailed structure⁻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 ß,γ-epoxy ketone group and the other with an allenic bond. In order to elucidate the structure⁻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.

Re-evaluation of the rin mutation and the role of RIN in the induction of tomato ripening.

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.

Egg white hydrolysate inhibits oxidation in mayonnaise and a model system.

The flavor deterioration of mayonnaise is induced by iron, which is released from egg yolk phosvitin under acidic conditions and promotes lipid oxidation. To prevent oxidative deterioration, natural components, rather than synthetic chemicals such as ethylenediaminetetraacetic acid have been required by consumers. In the present study, we evaluated the inhibitory effects of three egg white components with the same amino acid composition, namely egg white protein, hydrolysate, and the amino acid mixture, on lipid oxidation in mayonnaise and an acidic egg yolk solution as a model system. We found that the hydrolysate had the strongest inhibitory effect on lipid oxidation among the three components. The mechanism underlying the antioxidant effect was associated with Fe2+-chelating activity. Thus, egg white hydrolysate may have the potential as natural inhibitors of lipid oxidation in mayonnaise.

The content and distribution of steryl ferulates in wheat produced in Japan.

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.

3'-hydroxy-ε,ε-caroten-3-one inhibits the differentiation of 3T3-L1 cells to adipocytes.

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,ß-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.

Lysoglyceroglycolipids Improve the Intestinal Absorption of Micellar Fucoxanthin by Caco-2 Cells.

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.

Fucoxanthin Derivatives: Synthesis and their Chemical Properties.

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.

Glyceroglycolipids Affect Uptake of Carotenoids Solubilized in Mixed Micelles by Human Intestinal Caco-2 Cells.

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 ß-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.

A 3-hydroxy ß-end group in xanthophylls is preferentially oxidized to a 3-oxo ε-end group in mammals.

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-ß,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 ß-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo ß-end group. Other xanthophylls such as ß-cryptoxanthin and zeaxanthin, which have a 3-hydroxy ß-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 ß,ε-caroten-3'-one was significantly increased by the ingestion of ß-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy ß-end group of xanthophylls to a 3-oxo ε-end group.

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 ß-carotene present in spinach, but not of lutein and α-tocopherol, which are less hydrophobic than ß-carotene. Free fatty acid, monoacylglycerol, and diacylglycerol also enhanced the bioaccessibility of ß-carotene present in spinach. In addition to the long-chain triacylglycerols, their hydrolyzates formed during digestion would facilitate the dispersion and solubilization of ß-carotene into mixed micelles. Dietary fats and oils would therefore enhance the bioaccessibility of hydrophobic carotenes present in vegetables.

Effects of mixed micellar lipids on carotenoid uptake by human intestinal Caco-2 cells.

We reported previously that lysophosphatidylcholine remarkably enhanced ß-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 ß-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.