Atsushi Sano

Procyanidin B1 is detected in human serum after intake of proanthocyanidin-rich grape seed extract.

To confirm the absorption of proanthocyanidin (PA) into the human body, four healthy adults were administered 2.0 g of PA-rich grape seed extract (GSE). Blood were drawn before intake and 2 h after intake. Through the enzymatic treatment of sulfatase and β-glucuronidase, blood samples were analyzed by HPLC coupled with mass-spectrometry (LC/MS). Procyanidin B1 [epicatechin-(4β→8)-catechin] was detected in human serum 2 h after intake. Its concentration was 10.6±2.5 nmol/l.

Antihypertensive Effect of Peptide-Enriched Soy Sauce-Like Seasoning and Identification of Its Angiotensin I-Converting Enzyme Inhibitory Substances

We have developed a peptide-enriched soy sauce-like seasoning termed Fermented Soybean Seasoning (FSS), by modifying the process of soy sauce brewing. The FSS has a 2.7-fold higher concentration of total peptides than regular soy sauce. The angiotensin I-converting enzyme (ACE) inhibitory activity of FSS (IC(50) = 454 microg/mL) was greater than that of regular soy sauce (IC(50) = 1620 microg/mL). The FSS demonstrated antihypertensive effects both in spontaneously hypertensive rats and in Dahl salt-sensitive rats during continuous feeding. The ACE inhibitory substances were purified from FSS by reversed-phase chromatography. Ala-Trp IC(50) = 10 microM; Gly-Trp IC(50) = 30 microM; Ala-Tyr IC(50) = 48 microM; Ser-Tyr, IC(50) = (67 microM; Gly-Tyr, IC(50) = 97 microM; Ala-Phe, IC(50) = 190 microM; Val-Pro, IC(50) = (480 microM; Ala-Ile, IC(50) = 690 microM; Val-Gly, IC(50) = 1100 microM; and a nicotianamine, IC(50) = 0.26 microM. [corrected] The concentrations of these substances in the FSS were revealed to be higher than that of regular soy sauce through quantitative LC-MS/MS analysis.

Antihypertensive Effect of a γ-Aminobutyric Acid Rich Tomato Cultivar ‘DG03-9’ in Spontaneously Hypertensive Rats

This study aimed to investigate the effects of a gamma-aminobutyric acid (GABA) rich tomato (Solanum lycopersicum L.) cultivar DG03-9 in comparison with Momotaro, a commonly consumed tomato cultivar in Japan, on systolic blood pressure (SBP) in spontaneously hypertensive rats (SHR). In a single administration study, treatment with the GABA-rich cultivar elicited a significant decrease in SBP compared to the control group. In a chronic administration study, SHR were fed diets containing one of the tomato cultivars for 4 weeks. Both cultivars significantly reduced the increase in SBP compared to the control. The antihypertensive effect of the GABA-rich cultivar was higher than that of the commonly consumed cultivar in both the single- and chronic-administration studies. Treatment with a comparable amount of GABA elicited a similar response to treatment with the GABA-rich cultivar. These results suggest that the GABA-rich cultivar DG03-9 is a potent antihypertensive food and may be useful for treating hypertension effectively.

Procyanidin B3 Prevents Articular Cartilage Degeneration and Heterotopic Cartilage Formation in a Mouse Surgical Osteoarthritis Model

Osteoarthritis (OA) is a common disease in the elderly due to an imbalance in cartilage degradation and synthesis. Heterotopic ossification (HO) occurs when ectopic masses of endochondral bone form within the soft tissues around the joints and is triggered by inflammation of the soft tissues. Procyanidin B3 (B3) is a procyanidin dimer that is widely studied due to its high abundance in the human diet and antioxidant activity. Here, we evaluated the role of B3 isolated from grape seeds in the maintenance of chondrocytes in vitro and in vivo. We observed that B3 inhibited H2O2-induced apoptosis in primary chondrocytes, suppressed H2O2- or IL-1ß−induced nitric oxide synthase (iNOS) production, and prevented IL-1ß−induced suppression of chondrocyte differentiation marker gene expression in primary chondrocytes. Moreover, B3 treatment enhanced the early differentiation of ATDC5 cells. To examine whether B3 prevents cartilage destruction in vivo, OA was surgically induced in C57BL/6J mice followed by oral administration of B3 or vehicle control. Daily oral B3 administration protected articular cartilage from OA and prevented chondrocyte apoptosis in surgically-induced OA joints. Furthermore, B3 administration prevented heterotopic cartilage formation near the surgical region. iNOS protein expression was enhanced in the synovial tissues and the pseudocapsule around the surgical region in OA mice fed a control diet, but was reduced in mice that received B3. Together, these data indicated that in the OA model, B3 prevented OA progression and heterotopic cartilage formation, at least in a part through the suppression of iNOS. These results support the potential therapeutic benefits of B3 for treatment of human OA and heterotopic ossification.

Development of a rapid positive/absent test for coliforms using sensitive bioluminescence assay

I. MASUDA‐NISHIMURA, S. FUKUDA, A. SANO, K. KASAI and H. TATSUMI.2000.We have developed a sensitive bioluminescence assay for β‐galactosidase using a luminescent substrate, d‐luciferin‐O‐β‐galactopyranoside (LuGal). The detection limit for β‐galactosidase was 3u2003×u200310−20 mol per assay, which was approximately 50‐fold more sensitive than the test using a fluorescent substrate. This assay was applied to a positive/absent (P/A) test for coliforms. Observations made after 7 h of culture followed by a 10‐min enzyme assay using LuGal were comparable to those made after a 22–24‐h culture by the current method. Therefore, the LuGal method allows a rapid P/A test for coliforms.

Metabolite profiling of soy sauce using gas chromatography with time-of-flight mass spectrometry and analysis of correlation with quantitative descriptive analysis

Soy sauces, produced from different ingredients and brewing processes, have variations in components and quality. Therefore, it is extremely important to comprehend the relationship between components and the sensory attributes of soy sauces. The current study sought to perform metabolite profiling in order to devise a method of assessing the attributes of soy sauces. Quantitative descriptive analysis (QDA) data for 24 soy sauce samples were obtained from well selected sensory panelists. Metabolite profiles primarily concerning low-molecular-weight hydrophilic components were based on gas chromatography with time-of-flightmass spectrometry (GC/TOFMS). QDA data for soy sauces were accurately predicted by projection to latent structure (PLS), with metabolite profiles serving as explanatory variables and QDA data set serving as a response variable. Moreover, analysis of correlation between matrices of metabolite profiles and QDA data indicated contributing compounds that were highly correlated with QDA data. Especially, it was indicated that sugars are important components of the tastes of soy sauces. This new approach which combines metabolite profiling with QDA is applicable to analysis of sensory attributes of food as a result of the complex interaction between its components. This approach is effective to search important compounds that contribute to the attributes.

Identification and Quantification of Metabolites of Orally Administered Naringenin Chalcone in Rats

Naringenin chalcone is the main active component of tomato skin extract, which has an antiallergic activity. In this study, naringenin chalcone was orally administered to rats, and the chemical structures and levels of the major metabolites in the plasma and urine of rats were determined. HPLC analysis indicated the presence of three major metabolites in the urine. LC-MS and NMR analyses tentatively identified these as naringenin chalcone-2-O-beta-D-glucuronide, naringenin-7-O-beta-D-glucuronide, and naringenin-4-O-beta-D-glucuronide. Naringenin chalcone-2-O-beta-D-glucuronide was the only metabolite detected in the plasma, and its peak plasma level was observed 1 h after naringenin chalcone administration. Naringenin chalcone-2-O-beta-D-glucuronide also inhibited histamine release from rat peritoneal mast cells stimulated with compound 48/80. This activity might contribute to the antiallergic activity of naringenin chalcone in vivo. To the best of the authors knowledge, this study is the first to report determination of naringenin chalcone metabolites in rat plasma and urine.

Antihypertensive Mechanism of a Peptide‐Enriched Soy Sauce‐Like Seasoning: The Active Constituents and Its Suppressive Effect on Renin–Angiotensin–Aldosterone System

We previously reported that a peptide-enriched soy sauce-like seasoning called fermented soybean seasoning (FSS) demonstrated antihypertensive effects both in spontaneously hypertensive rats (SHR) and Dahl salt-sensitive rats. Angiotensin I-converting enzyme (ACE) inhibitory substances (9 kinds of dipeptides and a nicotianamine) were identified from FSS. In the present study, we clarified the mechanisms underlying the antihypertensive effects of FSS in SHR. FSS was divided into the nicotianamine fraction and the peptide fraction. The peptide fraction was found to exert a more prevalent antihypertensive effect than the nicotianamine fraction in SHR. Among the peptides, we identified Gly-Tyr and Ser-Tyr as the 2 primary substances in FSS that contributed to the antihypertensive effect in SHR. These peptides were neither degraded by acid nor gastrointestinal proteases, and were absorbed into the circulating blood. FSS (2000 mg/kg) exerted ACE inhibitory activity in the lung of rats and provided a decrease (P= 0.0067) in the level of serum aldosterone after a single oral administration in SHR, resulting in the antihypertensive effect. The antihypertensive mechanism was found to be similar to therapeutic ACE inhibitors and other food-derived ACE inhibitory peptides, which are in wide use and are recognized as safe.

Safety assessment of 4-week oral intake of proanthocyanidin-rich grape seed extract in healthy subjects.

A clinical study was conducted to assess the safety and tolerability of oral doses of proanthocyanidin-rich grape seed extract (GSE) in healthy Japanese adult volunteers. In an open-label, 4-week toxicity test, 29 subjects daily received 1000, 1500, or 2500 mg GSE orally. Serum Fe levels of two subjects in the 2500 mg GSE group decreased to 61 and 60 μg/100 mL from 205 and 182 μg/100 mL at baseline respectively, at second week of GSE consumption; these values are low but within the normal range for the Japanese population. Two weeks after completing the 4-week course of GSE ingestion, the serum Fe levels of both subjects returned to near baseline levels (210 and 189 μg/100 mL). No subject discontinued the study. Oral intake of GSE up to 2500 mg for 4 weeks was found to be generally safe and well tolerated in humans. Research with a larger number of subjects is required to confirm these findings.

Proanthocyanidin-rich grape seed extract reduces leg swelling in healthy women during prolonged sitting.

BACKGROUNDnLeg swelling is a modern-day affliction of sedentary working women. The aim of this study was to evaluate the effectiveness of the intake of grape seed extract (GSE) on leg swelling in healthy Japanese women while sitting.nnnRESULTSnSingle intake trials and 14 day intake trials were held in a double-blind, placebo-controlled, crossover clinical study. A prolonged sedentary position was maintained for 6 h after GSE or placebo administration. Leg volume distension, increase in body extracellular fluid, and leg water were significantly suppressed in the GSE groups.nnnCONCLUSIONnThe intake of GSE is a contributing factor in the inhibition of leg swelling in healthy women during prolonged sitting.