Yukari Egashira

Purification and characterization of a feruloyl esterase from the intestinal bacterium Lactobacillus acidophilus

ABSTRACT Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37°C, respectively). The metal ions Cu2+ and Fe3+ (at a concentration of 5 mmol liter−1) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-l-arabinofuranose (FAA) as a substrate, the enzyme exhibited a Km of 0.0953 mmol liter−1 and a Vmax of 86.27 mmol liter−1 min−1 mg−1 of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-α-l-arabinofuranosyl)-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.

Identification of a Hepatoprotective Peptide in Wheat Gluten Hydrolysate against d-Galactosamine-Induced Acute Hepatitis in Rats

A hepatoprotective peptide, pyroglutamyl leucine (pyroGlu-Leu), was identified in wheat gluten hydrolysate through an in vivo activity-guided fractionation approach based on D-galactosamine-induced acute hepatitis in rats and fractionation of peptides with large-scale preparative ampholine-free isoelectric focusing. The active acidic fraction predominantly consisted of pyroglutamyl peptides and free pyroglutamic acid. Pyroglutamyl peptides were derivatized with phenyl isothiocyanate after removal of a pyroglutamyl residue by pyroglutamate aminopeptidase. The derivatives were purified by reversed-phase HPLC and subjected to sequence analysis. The active fraction contained pyroGlu-Ile, pyroGlu-Leu, pyroGlu-Gln, pyroGlu-Gln-Gln, and free pyroGlu. Ingestion of pyroGlu-Leu at 20 mg/kg body weight significantly decreased serum aspartate and alanine aminotransferases to approximately 30% and 20% of those values of the vehicle group, respectively, which were near the normal levels. Thirty minutes after ingestion of pyroGlu-Leu at 20 mg/kg, the concentration of pyroGlu-Leu in portal blood plasma increased to approximately 2 μM.

Hypocholesterolemic Mechanism of Chlorella: Chlorella and Its Indigestible Fraction Enhance Hepatic Cholesterol Catabolism through Up-Regulation of Cholesterol 7α-Hydroxylase in Rats

Chlorella powder (CP) has a hypocholesterolemic effect and high bile acid-binding capacity; however, its effects on hepatic cholesterol metabolism are still unclear. In the present study, male Wistar rats were divided into four groups and fed a high sucrose + 10% lard diet (H), an H + 10% CP diet (H+CP), an H + 0.5% cholesterol + 0.25% sodium cholate diet (C), or a C + 10% CP diet (C+CP) for 2 weeks. CP decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but did not affect these parameters in rats fed H-based diets. CP increased the hepatic mRNA level and activity of cholesterol 7α-hydroxylase (CYP7A1). CP increased hepatic HMG-CoA reductase (HMGR) activity in the rats fed H-based diets, but not in rats fed C-based diets. CP did not affect hepatic mRNA levels of sterol 27-hydroxylase, HMGR, low-density lipoprotein (LDL) receptor, scavenger receptor class B1, ATP-binding cassette (ABC) A1, ABCG5, or ABCB11. Furthermore, the effect of a 3.08% Chlorella indigestible fraction (CIF, corresponding to 10% CP) on hepatic cholesterol metabolism was determined using the same animal models. CIF also decreased serum and liver cholesterol levels significantly in rats fed C-based diets. CIF increased hepatic CYP7A1 mRNA levels. These results suggest that the hypocholesterolemic effect of CP involves enhancement of cholesterol catabolism through up-regulation of hepatic CYP7A1 expression and that CIF contributes to the hypocholesterolemic effect.

Protection by dietary Spirulina platensis against D-galactosamine- and acetaminophen-induced liver injuries.

Increasing attention has been paid to Spirulina for its potential clinical uses. The present study investigated the protection by dietary Spirulina platensis against d-galactosamine (d-GalN)- and acetaminophen (APAP)-induced hepatitis in ICR mice. Mice in each group (n 6) were fed with a standard diet (American Institute of Nutrition (AIN)-93G), a positive control diet containing 0.5 % butylated hydroxytoluene (BHT), or a diet containing 3, 6 or 9 % S. platensis for 1 week. On the last day the mice were treated with d-GalN (300 mg/kg body weight, intraperitoneally) or APAP (150 mg/kg body weight, intraperitoneally) and 24 h later the mice were killed. The doses of both 6 and 9 % S. platensis were found to significantly alleviate the increase of serum glutamate oxaloacetoacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) activities in d-GalN- or APAP-intoxicated mice. The observation was very similar to that of the positive control groups. Two more experiments were carried out to investigate the involvement of thiobarbituric acid-reactive substances (TBARS) and IL-18 in the suppression of 6 % S. platensis on d-GalN- and APAP-induced hepatitis. The significant increase of GOT and GPT activities was found to be accompanied with the elevation of hepatic TBARS level, IL-18 mRNA expression and serum IL-18 concentration, and was significantly alleviated by supplementation with 6 % S. platensis in diets. These results showed that dietary S. platensis could provide a significant protection against d-GalN- and APAP-induced liver injuries, and IL-18 and lipid peroxidation might be involved in the protective influence of S. platensis.

One-Week Antihypertensive Effect of Ile-Gln-Pro in Spontaneously Hypertensive Rats

The antihypertensive effect of an angiotensin I-converting enzyme (ACE) inhibitory peptide Ile-Gln-Pro (IQP), whose sequence was derived from Spirulina platensis , was investigated in spontaneously hypertensive rats (SHRs) for 1 week. The weighted systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the peptide IQP-treated group were significantly lower than those of the negative control group from the third and fourth days, respectively. Accompanying the blood pressure reduction, a significant regulation of the expression of major components of the renin-angiotensin system (RAS) was found in the treatment group, including downregulation of the mRNA levels of renin, ACE, and the angiotensin II type 1 (AT1) receptor in the kidney, as well as serum angiotensinogen (Ang), ACE, and angiotensin II (Ang II) concentrations. The treatment group also showed upregulation of mRNA expression of the angiotensin II type 2 (AT2) receptor in the kidney. Our findings suggested that IQP might be of potential use in the treatment of hypertension.

Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages.

AIMS Food-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages. MAIN METHODS RAW 264.7 macrophages were treated with LPS and various concentrations of pyroglutamyl-leucine (pyroGlu-Leu), -valine (pyroGlu-Val), -methionine (pyroGlu-Met), and -phenylalanine (pyroGlu-Phe). Cell viability/proliferation and various inflammatory parameters were measured by the established methods including ELISA and western blotting. The binding of fluorescein isothiocyanate-labeled LPS to RAW 264.7 cells was also measured fluorescently. KEY FINDINGS All the tested dipeptides significantly inhibited the secretion of nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 from LPS-stimulated RAW 264.7 macrophages. Above all, pyroGlu-Leu inhibited the secretion of all these inflammatory mediators even at the lowest dose (200μg/ml). PyroGlu-Leu dose-dependently suppressed IκBα degradation and MAPK (JNK, ERK, and p38) phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, it did not affect the binding of LPS to the cell surface. SIGNIFICANCE Our results indicated that pyroGlu-Leu inhibits LPS-induced inflammatory response via the blocking of NF-κB and MAPK pathways in RAW 264.7 macrophages.

L-Gln and L-Ser suppress the D-galactosamine-induced IL-18 expression and hepatitis

D-Galactosamine (GalN) induces acute hepatitis in experimental animals and this hepatitis has been shown to be suppressed by preceding ingestion of amino acids such as Gly, L-Ser, and L-Gln. However, little is known about the mechanism of its action. The present study shows for the first time that IL-18 reduction is involved in the suppressive actions of L-Gln and L-Ser on GalN-induced hepatitis. Elevation of IL-18 mRNA expression in liver and its concentration in serum in GalN-treated rats were found to be suppressed by preceding ingestion of 10% L-Gln- or 10% L-Ser diets, and resulted in the attenuation of the increase in serum transaminase (ALT and AST) activities, indexes of hepatic injury. These results suggest that suppressive effects of some dietary amino acids on the GalN-induced hepatitis are mediated by IL-18 reduction.

Effect of dietary linoleic acid on the tryptophan-niacin metabolism in streptozotocin-diabetic rats

To make clear the mechanism of change of tryptophan-niacin metabolism in diabetic rats, we investigated the effect of dietary linoleic acid on the tryptophan-niacin metabolites and the activity of liver, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), a key enzyme of tryptophan-niacin metabolism, in streptozotocin diabetic rats. Moreover, we investigated the involvement of linoleic acid in the induction of hepatic ACMSD activity by streptozotocin diabetes. In diabetic rats, the sum of urinary excretion of nicotinamide, N1-methylnicotinamide (MNA), N1-methyl-2-pyridone-5-carboxamide (2-Py) and N1-methyl-4-pyridone-3-carboxamide (4-Py) was higher in the fat free diet group than in the linoleic acid group, that was accompanied by the increase of tryptophan intake and reduction of body weight in the fat free diet group. In diabetic rats, hepatic ACMSD activity was higher in the fat free diet group than in the linoleic acid group. The results indicated that the induction of hepatic ACMSD activity by diabetes was not due to removal of the suppressive effect of the linoleic acid on the enzyme. In the diabetic+insulin group, hepatic ACMSD activity was significantly lower than in the diabetic group.

Tryptophan-Niacin Metabolism in Rat with Puromycin Aminonucleoside-Induced Nephrosis

We investigated the change of tryptophan-niacin metabolism in rats with puromycin aminonucleoside PAN-induced nephrosis, the mechanisms responsible for their change of urinary excretion of nicotinamide and its metabolites, and the role of the kidney in tryptophan-niacin conversion. PAN-treated rats were intraperitoneally injected once with a 1.0% (w/v) solution of PAN at a dose of 100 mg/kg body weight. The collection of 24-hour urine was conducted 8 days after PAN injection. Daily urinary excretion of nicotinamide and its metabolites, liver and blood NAD, and key enzyme activities of tryptophan-niacin metabolism were determined. In PAN-treated rats, the sum of urinary excretion of nicotinamide and its metabolites was significantly lower compared with controls. The kidney alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) activity in the PAN-treated group was significantly decreased by 50%, compared with the control group. Although kidney ACMSD activity was reduced, the conversion of tryptophan to niacin tended to be lower in the PAN-treated rats. A decrease in urinary excretion of niacin and the conversion of tryptophan to niacin in nephrotic rats may contribute to a low level of blood tryptophan. The role of kidney ACMSD activity may be minimal concerning tryptophan-niacin conversion under this experimental condition.

Gene expression analysis of the liver and skeletal muscle of psyllium-treated mice

Psyllium, a dietary fibre rich in soluble components, has both cholesterol- and TAG-lowering effects. Many studies have verified these actions using liver samples, whereas little information is available on the effects of psyllium treatment on other organs. The purpose of the present study was to evaluate the possible beneficial effects of psyllium. We investigated the gene expression profiles of both liver and skeletal muscle using DNA microarrays. C57BL/6J mice were fed a low-fat diet (LFD; 7 % fat), a high-fat diet (HFD; 40 % fat) or a HFD with psyllium (40 % fat+5 % psyllium; HFD+Psy) for 10 weeks. Body weights and food intake were measured weekly. After 10 weeks, the mice were killed and tissues were collected. Adipose tissues were weighed, and plasma total cholesterol and TAG blood glucose levels were measured. The expression levels of genes involved in glycolysis, gluconeogenesis, glucose transport and fatty acid metabolism were measured by DNA microarray in the liver and skeletal muscle. In the HFD+Psy group, plasma total cholesterol, TAG and blood glucose levels significantly decreased. There was a significant reduction in the relative weight of the epididymal and retroperitoneal fat tissue depots in mice fed the HFD+Psy. The expression levels of genes involved in fatty acid oxidation and lipid transport were significantly up-regulated in the skeletal muscle of the HFD+Psy group. This result suggests that psyllium stimulates lipid transport and fatty acid oxidation in the muscle. In conclusion, the present study demonstrates that psyllium can promote lipid consumption in the skeletal muscle; and this effect would create a slightly insufficient glucose state in the liver.