Administration timing and duration-dependent effects of sesamin isomers on lipid metabolism in rats.

Metabolism of lipids such as cholesterol and triglycerides has daily variations and is controlled by a circadian clock. Sesamin isomers, a mixture of sesamin and episesamin (SE), are types of lignans in sesame seed that have shown the improvement of lipid metabolism with various diseases in an animal model. We therefore tested whether the effects of SE on lipid metabolism are influenced by timing of administration. High-fat diet (HFD)-loaded rat was administered SE in the ZT13 or 14 (at the beginning of the active phase) or ZT23 or 22 (at the end of the active phase) every day for 7 or 28 days, and the effects on lipid metabolism were evaluated. The effects of SE were enhanced by duration-dependency: 28-day administration of SE strongly affected some parameters related to lipid metabolism, particularly cholesterol metabolism, as compared to 7-day administration. In particular, in 28-day administration, the analysis of serum and liver cholesterol levels revealed that SE administration decreases more effectively at the beginning of the active phase when compared to at the end of that. Furthermore, quantitative real-time polymerase chain reaction (QRT-PCR) and functional analysis indicated that suppression of cholesterol synthesis in the liver and promotion of cholesterol excretion from the liver, as well as inhibition of the functional activity and gene expression of sterol response element-binding protein 2 (Srebp2), which is a transcriptional factor and controls the gene expression involved in cholesterol-metabolism enzymes, contribute to enhancement of SE's effects at this administration timing. No significant differences were observed in triglyceride metabolism with regard to timing of SE administration. After 28-day administration of SE, administration at the beginning of the active phase only affected the expression of clock genes in the liver with phase-advance. In the pharmacokinetic study, administration time had no effect on the level of sesamin, episesamin or their metabolites in the liver after administration of SE for 28 days. The present results suggest that continuous long administration of SE at the beginning of the active phase is preferable for obtaining beneficial effects on lipid metabolism.

Mechanisms of chromosomal aberrations induced by sesamin metabolites in Chinese hamster lung cells.

Sesamin is a major lignan in sesame seeds and oil. We previously demonstrated that sesamin induces chromosomal aberrations (CA) in Chinese hamster lung (CHL/IU) cells in the presence of a metabolic activation system (S9 mix), although no genotoxicity was detected in vivo. To clarify the mechanism of CA induction by sesamin, we identified its principal active metabolite. A mono-catechol derivative, [2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabi-cyclo[3.3.0]octane (SC-1)], was previously identified in culture medium when sesamin was incubated with S9 mix. In the present study, we show that SC-1 induces CA in CHL/IU cells but not in human hepatoblastoma (HepG2) cells. SC-1 was unstable in culture medium. Addition of glutathione (GSH) to the incubation mixture decreased the rate of decomposition and also suppressed induction of CA in CHL/IU cells. These results indicate that SC-1 itself may not contribute to the induction of CA. Two GSH adducts of SC-1 were identified when SC-1 was incubated with GSH, suggesting that SC-1 was converted to the semiquinone/quinone form and then conjugated with GSH in the culture medium. Sodium sulfite (a quinone-responsive compound) also suppressed CA induction by SC-1. These findings strongly suggest that SC-1 is oxidized to semiquinone/quinone derivatives extracellularly in culture medium, that these derivatives are responsible for the induction of CA in CHL/IU cells, and therefore that the positive results obtained with sesamin in in vitro CA tests using CHL/IU cells may not be relevant to the assessment of in vivo activity.

Absorption, distribution, metabolism, and excretion of [14 C]sesamin in rats.

SCOPE: Sesamin is a major lignan in sesame seeds and has various physiological effects. Although metabolism of sesamin by cytochrome P450 or intestinal microflora has been reported, little is known concerning the mass balance, pharmacokinetics, and tissue distribution of sesamin. METHODS AND RESULTS: Absorption, distribution, metabolism, and excretion of [14 C]sesamin were investigated after a single oral dose of 5 mg/kg in rats. Sesamin was absorbed with peak plasma radioactivity at 1.0 h and declined with a terminal half-life 4.7 h. The cumulative excretion of radioactivity was 37.5 ± 3.1% in urine and 58.7 ± 4.8% in feces. In bile duct-cannulated rats, the cumulative excretion of radioactivity was 66.3 ± 8.4% in bile and 27.8 ± 10.2% in urine. Tissue distribution was investigated using quantitative whole-body autoradiography. Radioactivity was widely distributed over the whole body and was highly detected in the liver and kidney. The metabolites profile was examined using radiochromatography. Sesamin was mainly distributed in the form of conjugate metabolites. CONCLUSIONS: Sesamin was absorbed efficiently and distributed over the whole body. In particular, sesamin was highly distributed in the form of the metabolites in the liver and kidney. The results of this study are useful in elucidating the action mechanism of sesamin.

Sesame Lignans and Vitamin E Supplementation Improve Subjective Statuses and Anti-Oxidative Capacity in Healthy Humans With Feelings of Daily Fatigue.

Sesamin has anti-oxidative functions in vivo. Fatigue is caused in part by oxidative stress. We evaluated whether sesame lignans (sesamin/episesamin=1/1, 10 mg) with vitamin E (55 mg of alpha-tocopherol) (SVE) could improve subjective statuses and anti-oxidative capacity in humans using questionnaires on fatigue, sleep and physical appearance, as well as low-density lipoprotein oxidation lag time. A placebo-controlled, double-blind, parallel-group study was conducted with subjects experiencing daily fatigue. After a run-in period, subjects were administered oral SVE or a placebo (P) for 8 weeks. A questionnaire regarding fatigue, sleep and physical appearance was conducted at 0, 4, and 8 weeks. Plasma low-density lipoprotein oxidation lag time was measured as an indicator of anti-oxidative capacity. The per-protocol analysis revealed significant improvements in fatigue status at 4 and 8 weeks compared to 0 weeks in both groups (p<0.01), and sleep and physical appearance at 8 weeks compared to 0 weeks only in the SVE group (p<0.01). There were no significant differences observed between the groups. According to the 72-subject subgroup analysis (aged 40 and over), the sleep and physical appearance significantly improved compared to the P group (p<0.05), and fatigue status showed a tendency for improvement compared to the P group. Anti-oxidative capacity in the SVE group significantly increased compared to the P group (p<0.01). No adverse events relating to SVE supplementation were confirmed. These results suggest SVE supplementation could safely alleviate daily fatigue and oxidative stress.

Pharmacokinetics and safety of the sesame lignans, sesamin and episesamin, in healthy subjects.

A single-blind, placebo-controlled, parallel-group and multiple oral dose study was conducted in 48 healthy subjects to investigate the pharmacokinetics and safety of multiple oral doses of sesame lignans (sesamin and episesamin). Subjects were randomly divided into two groups. Each subject was administered 50 mg of sesame lignans (sesamin/episesamin=1/1) or placebo once daily for 28 days. The pharmacokinetics of the sesame lignans were investigated using 10 of the 24 subjects in the sesame lignans group. No serious adverse events were observed in this study. Sesamin was absorbed with a peak plasma concentration at 5.0 h. The plasma concentration of the main metabolite, SC-1, reached a peak at 5.0 h and decreased rapidly with a terminal half-life of 2.4 h. Episesamin was also absorbed with a peak plasma concentration at 5.0 h and decreased with a terminal half-life of 7.1 h. The plasma concentration of the main metabolite, EC-1, reached a peak at 5.0 h and decreased rapidly with a terminal half-life of 3.4 h. The plasma concentrations of sesamin and episesamin reached a steady state by day 7. Sesame lignans were confirmed to be safe and tolerable in healthy subjects. The results of the pharmacokinetic study demonstrate that no accumulation was observed following multiple 50 mg doses of sesame lignans.

Identification of the metabolites of episesamin in rat bile and human liver microsomes.

Episesamin is an isomer of sesamin, resulting from the refining process of non-roasted sesame seed oil. Episesamin has two methylendioxyphenyl groups on exo and endo faces of the bicyclic skeleton. The side methylendioxyphenyl group was metabolized by cytochrome-P450. Seven metabolites of episesamin were found in rat bile after treatment with glucuronidase/arylsulfatase and were identified using NMR and MS. The seven metabolites were (7α,7'ß,8α,8'α)-3,4-dihydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane (EC-1-1), (7α,7'ß,8α,8'α)-3,4-methylenedioxy-3',4'-dihydroxy-7,9':7',9-diepoxylignane (EC-1-2) and (7α,7'ß,8α,8'α)-3,4:3',4'-bis(dihydroxy)-7,9':7',9-diepoxylignane (EC-2), (7α,7'ß,8α,8'α)-3-methoxy-4-hydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane (EC-1m-1), (7α,7'ß,8α,8'α)-3,4-methylenedioxy-3'-methoxy-4'-hydroxy-7,9':7',9-diepoxylignane (EC-1m-2), (7α,7'ß,8α,8'α)-3-methoxy-4-hydroxy-3',4'-dihydroxy-7,9':7',9-diepoxylignane (EC-2m-1) and (7α,7'ß,8α,8'α)-3,4-dihydroxy-3'-methoxy-4'-hydroxy-7,9':7',9-diepoxylignane (EC-2m-2). EC-1-1, EC-1-2 and EC-2 were also identified as metabolites of episesamin in human liver microsomes. These results suggested that similar metabolic pathways of episesamin could be proposed in rats and humans.

The mechanism underlying the synergetic hypocholesterolemic effect of sesamin and α-tocopherol in rats fed a high-cholesterol diet.

Sesamin is a major lignan in sesame seed. We confirmed that ingestion of sesamin and α-tocopherol synergistically reduced the concentration of blood cholesterol in rats given a high-cholesterol diet. To elucidate the molecular mechanism behind this effect, we analyzed the gene-expression profiles in rat liver after co-ingestion of sesamin and α-tocopherol. Six-week-old male Sprague-Dawley rats were fed a 1% cholesterol diet (HC) or HC containing 0.2% sesamin, 1% α-tocopherol or sesamin + α-tocopherol for 10 days. Blood samples were collected on days 1, 3, 7, and 10 and livers were excised on day 10. The gene expressions of ATP-binding cassette, sub-family G (WHITE), members 5 (ABCG5) and 8 (ABCG8) were significantly increased, while the gene expression of apolipoprotein (Apo) A4 was significantly decreased. ABCG5 and ABCG8 form a functional heterodimer that acts as a cholesterol efflux transporter, which contributes to the excretion of cholesterol from the liver. ApoA4 controls the secretion of ApoB, which is a component of low-density-lipoprotein cholesterol. These studies indicate that the cholesterol-lowering mechanism underlying the effects of co-ingestion of sesamin and α-tocopherol might be attributable to increased biliary excretion of cholesterol and reduced ApoB secretion into the bloodstream.

Involvement of heme oxygenase-1 induction via Nrf2/ARE activation in protection against H2O2-induced PC12 cell death by a metabolite of sesamin contained in sesame seeds.

Induction of phase II antioxidant enzymes by activation of Nrf2/ARE (antioxidant response element) signaling has been considered as a promising strategy to combat with oxidative stress-related diseases. In the present study, we tested for potential effects of sesamin, a major lignan contained in sesame seeds, its stereoisomer episesamin, and their metabolites on Nrf2/ARE activation in rat pheochromocytoma PC12 cells. Luciferase reporter assays showed that primary metabolites of sesamin and episesamin, SC-1 and EC-1 were the most potent ARE activators among all tested compounds. SC-1 {(1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane} enhanced nuclear translocation of Nrf2 and up-regulated expression of phase II antioxidant enzymes including heme oxygenase-1 (HO-1). Treatment with SC-1 resulted in increased phosphorylation of p38 MAP kinase and transient increase in intracellular ROS levels. N-acetylcysteine (NAC) treatment abolished p38 phosphorylation as well as HO-1 induction caused by SC-1, indicating that ROS are upstream signals of p38 in Nrf2/ARE activation by SC-1. Furthermore, preconditioning with SC-1 attenuated H(2)O(2)-induced cell death in a dose-dependent manner. Finally, treatment with a HO-1 inhibitor, Zn-protoporphyrin (ZnPP), and overexpression of a dominant-negative mutant of Nrf2 diminished SC-1-mediated neuroprotection. Our results demonstrate that SC-1 is capable of protecting against oxidative stress-induced neuronal cell death in part through induction of HO-1 via Nrf2/ARE activation, suggesting its potential to reduce oxidative stress and ameliorate oxidative stress-related neurodegenerative diseases.

Metabolites of sesamin, a major lignan in sesame seeds, induce neuronal differentiation in PC12 cells through activation of ERK1/2 signaling pathway.

Sesamin, a major lignan in sesame seeds, exhibits various health benefits. Here, we investigated effects of sesamin, its stereoisomer episesamin, and their metabolites on neuronal differentiation in rat pheochromocytoma PC12 cells. Among all compounds tested, primary metabolites of sesamin and episesamin, SC-1 and EC-1 {S- and R-epimer of 2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo [3.3.0]octane}, were the most potent to induce neuronal differentiation. SC-1 alone induced neuronal differentiation through extracellular signal-regulated kinase (ERK) 1/2 activation that is essential for nerve growth factor (NGF)-induced neuronal differentiation, as shown by the suppression with MEK1/2 inhibitors, PD98059 and U0126. However, SC-1 did not increase phosphorylation of TrkA, a high-affinity NGF receptor, and a TrkA inhibitor, K252a, did not affect SC-1-induced neuronal differentiation. Furthermore, SC-1 potentiated neuronal differentiation in cells co-treated with NGF, which was associated with enhanced ERK1/2 activation and increased expression of neuronal differentiation markers. Interestingly, when treated with SC-1 and a high dose of NGF, formation of synaptic connections and synaptophysin accumulation at the neurite terminals were markedly enhanced. These results indicate that (1) SC-1 alone induces neuronal differentiation, (2) SC-1 potentiates neuronal differentiation in NGF-treated cells, (3) SC-1 enhances formation of synaptic connections in cells treated with a high dose of NGF, all of which are associated with ERK1/2 activation. It is therefore concluded that SC-1 may promote neuronal differentiation by tapping into the ERK1/2-MAPK (mitogen-activated protein kinase) signaling pathway downstream from the TrkA receptor in PC12 cells.

First chemical synthesis of antioxidative metabolites of sesamin.

The first chemical synthesis of two metabolites ((1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3,3,0]octane (SC-1) and (1R,2S,5R,6S)-2,6-bis(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3,3,0]octane (SC-2)) of sesamin was achieved by a simple two-step approach from sesamin. The approach consists of acetoxylation of the methylenedioxy moiety(ies) with lead(IV) tetraacetate and acid hydrolysis of the resulting hemiorthoester to SC-1 and SC-2.

Formation of two methylenedioxy bridges by a Sesamum CYP81Q protein yielding a furofuran lignan, (+)-sesamin.

(+)-Sesamin, a furofuran class lignan, is widespread in vascular plants and represented by Sesamum spp. (+)-Sesamin has been of rapidly growing interest because of its beneficial biological effects in mammals, but its biosynthesis and physiological roles in plants remain to be clarified. It is speculated to be synthesized from (+)-pinoresinol by means of (+)-piperitol by formation of two methylenedioxy bridges mediated by two distinct Sesamum indicum cytochrome P450 (SiP450) proteins. Here, we report an SiP450, CYP81Q1, that alone catalyzes (+)-sesamin biosynthesis from (+)-pinoresinol by means of (+)-piperitol by forming two methylenedioxy bridges. The CYP81Q1 gene expression profile was temporally consistent with the accumulation pattern of (+)-sesamin during seed development. The CYP81Q1-GFP chimera protein was colocalized with an endoplasmic reticulum (ER)-targeting chimera protein, indicating that (+)-sesamin biosynthesis occurs on the ER cytoplasmic surface. Moreover, we isolated two CYP81Q1 homologs from other Sesamum spp. Sesamum radiatum CYP81Q2 showed dual (+)-piperitol/(+)-sesamin synthetic activity. CYP81Q2, as well as CYP81Q1, therefore, corresponds to a (+)-piperitol/(+)-sesamin synthase in lignan biosynthesis. In contrast, Sesamum alatum CYP81Q3 showed no activity, in accord with (+)-sesamin being deficient in S. alatum. Our findings not only provide insight into lignan biosynthesis but also unravel a unique mode of cytochrome P450 action.

Sesamin metabolites induce an endothelial nitric oxide-dependent vasorelaxation through their antioxidative property-independent mechanisms: possible involvement of the metabolites in the antihypertensive effect of sesamin.

Sesamin, a major lignan in sesame seeds and oil, has been known to lower blood pressure in several types of experimental hypertensive animals. A recent study demonstrated that sesamin metabolites had in vitro radical-scavenging activities. Thus, we determined whether the antioxidative effect of sesamin metabolites modulate the vascular tone and contribute to the in vivo antihypertensive effect of sesamin. We used four demethylated sesamin metabolites: SC-1m (piperitol), SC-1 (demethylpiperitol), SC-2m [(1R,2S,5R,6S)-6-(4-hydroxy-3-methoxyphenyl)-2-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3,3,0]octane], and SC-2 [(1R,2S,5R, 6S)-2,6-bis(3,4-dihydroxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane]. SC-1, SC-2m, and SC-2, but not SC-1m, exhibited potent radical-scavenging activities against the xanthine/xanthine oxidase-induced superoxide production. On the other hand, SC-1m, SC-1, and SC-2m produced endothelium-dependent vasorelaxation in phenylephrine-precontracted rat aortic rings, whereas SC-2 had no effect. The SC-1m- and SC-1-induced vasorelaxations were markedly attenuated by pretreatment with a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (NOARG), or a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Neither SC-1m nor SC-1 changed the expression level of endothelial NOS protein in aortic tissues. The antihypertensive effects of sesamin feeding were not observed in chronically NOARG-treated rats or in deoxycorticosterone acetate-salt-treated endothelial NOS-deficient mice. These findings suggest that the enhancement of endothelium-dependent vasorelaxation induced by sesamin metabolites is one of the important mechanisms of the in vivo antihypertensive effect of sesamin.

A novel vitamin C analog, 2-O-(beta-D-Glucopyranosyl)ascorbic acid: examination of enzymatic synthesis and biological activity.

2-O-(beta-D-Glucopyranosyl)ascorbic acid (AA 2 beta G) isolated from a popular traditional Chinese food (Lycium fruit) was synthesized using cellulase derived from Trichoderma sp. with cellobiose as a glucose donor. 6-O-(beta-D-Glucopyranosyl)ascorbic acid as well as AA 2 beta G was also synthesized in this reaction. The vitamin C activity of AA 2 beta G was also evaluated using inherently scorbutic (osteogenic disorder Shionogi [ODS]) rats. The rats were fed vitamin C-deficient food and water containing AA 2 beta G for 21. AA 2 beta G supported their growth and the level of vitamin C in tissues was moderately maintained. The vitamin C level in some tissues depended on the hydrolytic activity of AA 2 beta G (beta-glucosidase activity) although the correlation was not statistically significant (P=0.08). The results indicate that AA 2 beta G has pro-vitamin C activity.

2-O-(beta-D-Glucopyranosyl)ascorbic acid, a novel ascorbic acid analogue isolated from Lycium fruit.

A novel stable precursor of ascorbic acid (vitamin C), 2-O-(beta-D-glucopyranosyl)ascorbic acid, was isolated from both the ripe fresh fruit and dried fruit of Lycium barbarum L., a plant of the Solanaceae family. The chemical structure was inferred by instrumental analyses and confirmed by chemical synthesis. The dried fruit of Lycium barbarum L. contained ca. 0.5% of it, which is comparable to the ascorbic acid content of fresh lemons. It increased the blood ascorbic acid by oral administration to rats, and it was also detected in blood from the portal vein.