Black Soybean Seed Coat Extract Improves Endothelial Function and Upregulates Oxidative Stress Marker Expression in Healthy Volunteers by Stimulating Nitric Oxide Production in Endothelial Cells.

Black soybean seed coat extract (BE) contains multiple bioactive polyphenols, including flavan-3-ols and anthocyanins. BE improves endothelial function; however, it is unclear whether BE protects endothelial cells from senescence. In this study, we examined the effects of BE on endothelial cell senescence and vascular function in healthy individuals. High concentrations of glucose were used to induce senescence in bovine aortic endothelial cells incubated with BE. Senescence, vascular function, and oxidative stress markers were measured. Incubation with BE remarkably inhibited senescence-associated ß-galactosidase and lactate dehydrogenase activities and dose dependently reduced intracellular reactive oxygen species levels in bovine aortic endothelial cells. BE treatment increased the levels of endothelial nitric oxide synthase (eNOS) mRNA and endothelial nitric oxide (NO) metabolites and increased the mRNA expression of klotho, a gene associated with an antiaging phenotype. To examine the effects of BE in humans, we conducted a clinical study using the second derivative of the fingertip photoplethysmogram to investigate vascular function and aging in 24 healthy volunteers. The participants consumed BE supplements (100 mg/day) or a placebo for 2 weeks. When compared with the placebo group, the BE group showed considerably improved vascular function, NO metabolite levels, and oxidative stress. These results suggest that BE supplementation improves endothelial function, possibly through antioxidant activity and NO production, and may consequently reduce the cardiovascular risk associated with aging. BE supplementation may be an effective and safe approach to reduce the risk of atherosclerosis and cardiovascular disease; however, additional studies investigating chronic vascular inflammation are needed.

Black Soybean Improves Vascular Function and Blood Pressure: A Randomized, Placebo Controlled, Crossover Trial in Humans.

Vascular dysfunction and injurious stimuli such as oxidative stress are closely related to the risk of cardiovascular diseases (CVD). Dietary polyphenols are reported to exert beneficial effects in reducing the risk of CVD. Black soybean has been used as a nutritionally rich food and contains abundant polyphenols in its seed coat and grain. Black soybean has many beneficial physiological activities, and its prevention effects on CVD risk were reported mainly in animal experiments. In this study, we performed a randomized, single blind, placebo controlled, crossover trial to investigate the effect of black soybean consumption on the vascular function in healthy humans. Twenty-two healthy adults aged from 30 to 60 completed the four week trial with daily consumption of about a 40 g test material cookie containing 20 g roasted black soybean powder. Body composition, vascular function, biomarkers for oxidative stress, and polyphenol contents in the urine and the plasma were measured. After ingestion of the black soybean cookie, vascular function, which was evaluated by plethysmogram using a Pulse Analyzer®, was improved and systolic blood pressure was decreased. Moreover, nitric oxide levels in plasma and urine were increased, while an oxidative stress biomarker, 8-hydroxy-2'-deoxyguanosine level, in the plasma was decreased accompanied by an increase in the concentration of polyphenols derived from black soybean in plasma and urine. These results suggest that the antioxidant activity of black soybean polyphenols and an increase in the nitric oxide level may contribute to the improvement of vascular function. Thus, black soybean is an attractive food material for improvement of vascular function through decreasing oxidative stress by its potent antioxidant activity and increasing the nitric oxide level in healthy humans.

Black soybean improves the vascular function through an increase in nitric oxide and a decrease in oxidative stress in healthy women.

Vascular dysfunction and injurious stimuli such as oxidative stress is closely related to the risk of cardiovascular diseases (CVD). Dietary polyphenols is reported to exert the beneficial effects on reducing the risk of CVD. Black soybean is rich in polyphenols, including isoflavones, anthocyanidins and flavan-3-ols, and its prevention effects on CVD risk were reported in the animal experiments. In this study, we investigated the effect of black soybean consumption on the vascular function and oxidative stress associating with the polyphenol concentrations in healthy women. Lowered vascular age was observed in 33 out of 44 volunteers who completed the 8-week trial. It was observed that improvement of the vascular stiffness, increasing in the urinary NO2 and NO3 level, and decreasing in the oxidative stress markers, 8-hydroxy-2'-deoxyguanosine, hexanoyl-lysine and myeloperoxidase. In addition, concentration of 12 polyphenols in black soybean increased in the plasma and urine. Increased concentration of polyphenols would be involved in the decreased oxidative stress. Thus, black soybean consumption improved the vascular function through an increase in nitric oxide and a decrease in oxidative stress accompanied by increasing the polyphenol concentrations in healthy women.

NRF2 and HSF1 coordinately regulate heme oxygenase-1 expression.

Heme oxygenase-1 (HO-1) is an inducible enzyme responding to various stresses and has cytoprotective activities. Although HO-1 has been referred to as heat shock protein (HSP) 32, the heat-mediated induction of HO-1 varies among different species and cell lines. We examined the effects of heat shock on HO-1 expression in mouse embryonic fibroblast (MEF) cells deficient in heat shock factor 1 (HSF1) or nuclear factor-erythroid-2-related factor 2 (NRF2). Heme-induced expression of HO-1 was 2-fold higher in Hsf1-/- cells than in the wild-type cells at both mRNA and protein levels. In Nrf2-/- cells, heme-induced expression of HO-1 was not detected. In contrast, HO-1 expression was markedly induced by heat shock at 40-42 °C in Nrf2-/- cells while the wild-type cells were not responsive. The heat-induced expression of HO-1 in Nrf2-/- cells were almost completely diminished by transfection of siRNA against Hsf1 gene. These results suggest that HSF1 and NRF2 suppress heme-induced and heat-induced HO-1 expression, respectively.

Multi-residue analysis of pesticides in agricultural products by liquid chromatography time-of-flight mass spectrometry.

The applicability of liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) for determining pesticide residues in agricultural products was investigated. TOF-MS conditions for monitoring target ions, together with their fragment ions, were carefully optimized. The developed LC-TOF-MS method was evaluated for 154 pesticides in soybean and spinach by using matrix-matched standards. No significant matrix effect was observed for most of the tested pesticides at a concentration level of 0.01 mg/kg, where the limits of quantification were less than 0.01 mg/kg for 145 of the 154 pesticides (S/N>10). In addition, no significant interference was observed in the chromatograms of the blank extracts. These results indicate that LC-TOF-MS determination may become a powerful tool for multi-residue analysis of pesticides in agricultural products.

[Determination of pindone in animal products, fishery products, and honey by LC-MS/MS].

A sensitive and selective analytical method for the determination of the rodenticide pindone in animal products, fishery products, and honey by LC-MS/MS was developed. Pindone was extracted with acidified acetone, and the crude extract was purified by liquid-liquid partitioning, followed by silica gel and ODS column chromatography. LC separation was performed on an ODS column with methanol/water containing ammonium acetate as the mobile phase, and detection was carried out using tandem mass spectrometry (MS/MS) with electrospray ionization (ESI) in the negative mode. The average recoveries from fortified bovine muscle, bovine liver, bovine fat, chicken muscle, salmon, eel, freshwater clam, egg, milk, and honey spiked at 0.001 mg/kg were in the range of 76-92%, and the relative standard deviations were 4-8%. The limit of quantitation (S/N≥10) of the developed method was 0.001 mg/kg for all the tested foods.

[Determination of pindone in agricultural products by LC-MS/MS].

A sensitive and selective analytical method for the determination of pindone in agricultural products by LC-MS/MS was developed. Pindone was extracted with acetone, and an aliquot of the crude extract was re-extracted with hexane. For lipid-rich samples, the crude extract was further cleaned up by acetonitrile-hexane partitioning. The extract was cleaned up on a tandem graphitized carbon-silica gel column. For brown rice, soybean, and tea, PSA column cleanup was added prior to LC-MS/MS determination. Average recoveries of pindone from brown rice, soybean, potato, spinach, cabbage, apple, orange, tomato, cucumber, and tea fortified at 0.001 mg/kg were 81-93%, and the relative standard deviations were 2-7%. The limit of quantitation (S/N ≥ 10) of the developed method was 0.001 mg/kg for all the tested agricultural products.

[Determination of 4-hydroxycoumarin rodenticides in animal products, fishery products, and honey by liquid chromatography-tandem mass spectrometry].

A sensitive and selective method for the determination of 4-hydroxycoumarin-type rodenticides (warfarin, coumatetralyl, bromadiolone, and brodifacoum) in animal products, fishery products, and honey was developed. 4-Hydroxycoumarin rodenticides were extracted with acidified acetone, and the crude extract was purified by liquid-liquid partitioning followed by PSA column cleanup. Gradient liquid chromatographic separation was performed by using an Inertsil ODS-4 column, with methanol and water containing ammonium acetate as the mobile phase. Detection was carried out on a tandem mass spectrometer with electrospray ionization in the negative mode. Average recoveries from bovine muscle, bovine liver, bovine fat, swine muscle, salmon, eel, freshwater clam, egg, milk, and honey spiked at 0.0005-0.001 mg/kg were in the range of 79-108%, and the relative standard deviations were 2-8%. The limits of quantitations of the developed method were 0.0005 mg/kg for brodifacoum, 0.001 mg/kg for warfarin, coumatetralyl, and bromadiolone.

Clarifying springtime temperature reconstructions of the medieval period by gap-filling the cherry blossom phenological data series at Kyoto, Japan.

We investigated documents and diaries from the ninth to the fourteenth centuries to supplement the phenological data series of the flowering of Japanese cherry (Prunus jamasakura) in Kyoto, Japan, to improve and fill gaps in temperature estimates based on previously reported phenological data. We then reconstructed a nearly continuous series of March mean temperatures based on 224 years of cherry flowering data, including 51 years of previously unused data, to clarify springtime climate changes. We also attempted to estimate cherry full-flowering dates from phenological records of other deciduous species, adding further data for 6 years in the tenth and eleventh centuries by using the flowering phenology of Japanese wisteria (Wisteria floribunda). The reconstructed tenth century March mean temperatures were around 7 degrees C, indicating warmer conditions than at present. Temperatures then fell until the 1180s, recovered gradually until the 1310s, and then declined again in the mid-fourteenth century.

A novel oxidative dimer from protocatechuic esters: contribution to the total radical scavenging ability of protocatechuic esters.

A novel oxidative dimer was isolated as a major product from a reaction mixture of methyl protocatechuate and DPPH radical in methanol. Its unusual benzobicyclo[3.2.1]octane structure was elucidated by extensive spectral analysis. This result suggests that the regeneration of catechol structures by the nucleophilic addition of an alcohol molecule on o-quinones and subsequent dimerization is one of the key reactions in the high radical-scavenging activity of protocatechuic esters in an alcoholic solvent.

Inhibitory effects of 5,6,7-trihydroxyflavones on tyrosinase.

Baicalein (1), 6-hydroxyapigenin (6), 6-hydroxygalangin (13) and 6-hydroxy-kaempferol (14), which are naturally occurring flavonoids from a set of 14 hydroxy-flavones tested, exhibited high inhibitory effects on tyrosinase with respect to L-DOPA, while each of the 5,6,7-trihydroxyflavones 1, 6, 13 or 14 acted as a cofactor to monophenolase. Moreover, 6-hydroxykaempferol (14) showed the highest activity and was a competitive inhibitor of tyrosinase compared to L-DOPA. 5,6,7-Trihydroxyflavones 1, 6, 13 or 14 showed also high antioxidant activities. Hence, we conclude that the 5,6,7-trihydroxy-flavones are useful as good depigmentation agents with inhibitory effects in addition to their antioxidant properties.

Calyciphylline D, a novel alkaloid with an unprecedented fused-pentacyclic skeleton from Daphniphyllum calycinum.

[structure: see text]. Calyciphylline D (1), a novel Daphniphyllum alkaloid with an unprecedented fused-pentacyclic skeleton containing a 8-azatricyclo[4.2.1.0.4,8]nonane ring system, has been isolated from the leaves of Daphniphyllum calycinum (Daphniphyllaceae), and the structure and relative stereochemistry were elucidated on the basis of spectroscopic data.

Paratunamides A-D, oxindole alkaloids from Cinnamodendron axillare.

Four new oxindole alkaloids, paratunamides A-D (1-4), containing a secologanin unit, were isolated from the bark of Cinnamodendron axillare, and their structures and relative configurations were elucidated by spectroscopic data. The absolute configuration at C-7 in 1-4 was assigned as S, S, R, and S, respectively, on the basis of the CD spectra.

Radical scavenging activity of dicaffeoyloxycyclohexanes: contribution of an intramolecular interaction of two caffeoyl residues.

Six regio- and stereoisomers of dicaffeoyloxycyclohexanes and 2,4-di-O-caffeoyl-1,6-anhydro-beta-D-glucose were synthesized as model compounds of dicaffeoylquinic acids, and their radical scavenging activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical scavenging tests. Both DPPH and ABTS radical scavenging reactions of these compounds consisted of two different steps. In the first step, catechol moieties of the caffeoyl residues were rapidly converted to o-quinone structures and no significant difference in the reactivity was observed among the tested compounds. In the second step, however, the rate of the reaction increased as the intramolecular distance of the two caffeoyl residues decreased. A novel intramolecular coupling product, which could scavenge additional radicals, was isolated from the reaction mixture of trans-1,2-dicaffeoyloxycyclohexane and DPPH radical. The result suggests that the second step of the radical scavenging reaction is arising from an intramolecular interaction between the two caffeoquinone residues to regenerate catechol structures, and that the closer their distance is, the more rapidly they react. The radical scavenging activity of natural dicaffeoylquinic acids in a biological aqueous system might also depend on the positions of caffeoyl ester groups.

Synergistic effects of thiols and amines on antiradical efficiency of protocatechuic acid.

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of protocatechuic acid and its structural analogues (methyl protocatechuate, 3',4'-dihydroxyacetophenone, 3,4-dihydroxybenzaldehyde, and 3,4-dihydroxybenzonitrile) were examined in aprotic and protic solvents. In aprotic acetonitrile, all test compounds scavenged two radicals. In protic methanol, however, these compounds rapidly scavenged five radicals except for protocatechuic acid, which consumed only two radicals. The result indicated that higher radical scavenging activity in methanol than in acetonitrile was due to a nucleophilic addition of the methanol molecule on the oxidized quinones, which led to a regeneration of catechol structures. To investigate the importance of the nucleophilic addition on the quinones for the high radical scavenging activity, DPPH radical scavenging activity of protocatechuic acid and its analogues was examined in the presence of a variety of nucleophiles. The addition of a strong nucleophile such as a cysteine derivative significantly increased the radical scavenging equivalence. Furthermore, thiol adducts at C-2 and C-2,5 of protocatechuic acid and its analogues were isolated from the reaction mixtures. These results strongly suggest that the quinone of protocatechuic acid and its analogues undergo a nucleophilic attack at C-2 to yield 2-substituted-3,4-diols. Then, a regenerated catechol moiety of adducts scavenge two additional radicals by reoxidation into quinones, which undergo the second nucleophilic attack at the C-5. This mechanism demonstrates a possibility of synergistic effects of various nucleophiles on the radical scavenging ability of plant polyphenols containing a 3,4-dihydroxy substructure like protocatechuic acid and its analogues.

Effects of alcoholic solvents on antiradical abilities of protocatechuic acid and its alkyl esters.

DPPH radical scavenging reactions of protocatechuic acid and its methyl ester were investigated in various solvents. In alcoholic solvents, methyl protocatechuate rapidly scavenged more than four equivalents of the radical, whereas approximately two equivalents were consumed in aprotic solvents. Methyl, ethyl, butyl, isopropyl, and tert-butyl protocatechuates were examined for their DPPH radical scavenging abilities in methanol or ethanol. As a result, the radical scavenging equivalence of sterically bulky esters tended to decrease compared to that of methyl or ethyl ester. The ABTS radical scavenging ability of those esters in water also showed the same tendency. Since 2-methoxy derivatives were detected in the reaction mixture of methyl protocatechuate and DPPH radical in methanol, a nucleophilic attack of an alcoholic molecule on the o-quinone intermediate, which is sensitive to steric hindrance from alkyl groups of both esters and alcoholic solvents, must be crucial for total radical scavenging abilities.

Radical scavenging mechanism of phenol carboxylic acids: reaction of protocatechuic esters.

Solvent-dependency of the antiradical reaction of protocatechuic acid (3,4-dihydroxybenzoic acid, PA) and its esters has been investigated. In aprotic solvents, methyl protocatechuate (PAMe) was readily oxidized by two molar equivalents of DPPH radical and converted to protocatechuquinone methyl ester (PQMe). On the other hand, in alcoholic solvents such as methanol, PAMe rapidly consumed five radicals in 30 min and changed to complex oxidized mixtures. A 1H-NMR analysis of the reaction mixture of PAMe and DPPH radical in methanol showed that PAMe was rapidly converted to PQMe and its 3-hemiacetal. In addition, a signal of 2-methoxy-PQMe 3-hemiacetal was also detected in the reaction mixture. The results suggested that PQMe undergoes a nucleophilic attack by the solvent alcohol molecule at the C-2 of the ring in methanol, leading to a regeneration of catechol structure, which accounts well for the higher DPPH radical scavenging activity of PAMe in alcohols than in aprotic solvents.

Quinone hemiacetal formation from protocatechuic acid during the DPPH radical scavenging reaction.

Protocatechuic acid was rapidly converted to protocatechuquinone 3-methyl hemiacetal and protocatechuquinone during the reaction with DPPH radical in methanol. The structure of the acetal was determined by comparing the NMR data with those of an authentic compound prepared by (diacetoxy)iodobenzene oxidation of protocatechuic acid.