Effects of fermented soymilk with Lactobacillus casei Shirota on skin condition and the gut microbiota: a randomised clinical pilot trial.

Several clinical studies have shown that isoflavones and Lactobacillus casei Shirota (LcS) have beneficial effects on skin condition and the gut microbiota, respectively. Thus, we investigated the effects of consecutive intake of fermented soymilk (FSM) with LcS on skin condition and the gut microbiota, as well as isoflavone bioavailability, in a randomised, double-blind, placebo-controlled trial as a pilot study. Sixty healthy premenopausal Japanese women received FSM containing a moderate level of isoflavone aglycones and a probiotic LcS, or soymilk (SM) containing neither of them, twice a day for 8 weeks. Skin condition was assessed by a subjective questionnaire for face and morphological analysis of the stratum corneum on the inner forearm. Faecal microbiota and urinary isoflavone were analysed by 16S rRNA gene amplicon sequencing and high-performance liquid chromatography tandem mass spectrometry, respectively. Both the FSM and SM groups had improved skin condition as assessed from scores of overall satisfaction, dryness, moisture, elasticity, coarseness, pigmentation and/or stratum corneum morphology, as well as significantly increased levels of urinary isoflavones during the intake period compared with the pre-intake period, although there were no significant differences between the two groups. There was a significant positive correlation between urinary isoflavone levels and skin questionnaire scores. In contrast, the relative abundance levels of Lactobacillaceae significantly increased and those of Bifidobacteriaceae tended to increase during the intake period compared with the pre-intake period. For the after-intake period they only decreased significantly in the FSM group. The levels of Enterobacteriaceae and Porphyromonadaceae significantly decreased during the intake period in the FSM group. These findings suggest that daily intake of FSM, as well as SM, provides health benefits that improve skin condition via increased levels of isoflavone absorption in the body, and that only FSM beneficially modifies the gut microbiota in premenopausal healthy women.

Bifidobacterium fermented milk and galacto-oligosaccharides lead to improved skin health by decreasing phenols production by gut microbiota.

A questionnaire survey found that women suffering from abnormal bowel movements have many skin problems such as a high frequency of dry skin. Although there are similarities between the structure and barrier function mechanism of the gut and skin, experimental data are insufficient to show an association between the intestinal environment and skin conditions. Phenols, for example phenol and p-cresol, as metabolites of aromatic amino acids produced by gut bacteria, are regarded as bioactive toxins and serum biomarkers of a disturbed gut environment. Recent studies have demonstrated that phenols disturb the differentiation of monolayer-cultured keratinocytes in vitro, and that phenols produced by gut bacteria accumulate in the skin via the circulation and disrupt keratinocyte differentiation in hairless mice. Human studies have demonstrated that restriction of probiotics elevated serum free p-cresol levels and harmed skin conditions (reduced skin hydration, disrupted keratinisation). In contrast, daily intake of the prebiotic galacto-oligosaccharides (GOS) restored serum free p-cresol levels and skin conditions in adult women. Moreover, a double-blind placebo-controlled trial demonstrated that the daily intake of fermented milk containing the probiotic Bifidobacterium breve strain Yakult and prebiotic GOS reduced serum total phenol levels and prevented skin dryness and disruption of keratinisation in healthy adult women. It is concluded that phenols produced by gut bacteria are one of the causes of skin problems. Probiotics and/or prebiotics, such as B. breve strain Yakult and/or GOS, are expected to help maintain a healthy skin by decreasing phenols production by gut microbiota. These findings support the hypothesis that probiotics and prebiotics provide health benefits to the skin as well as the gut.

High production of beta-thujaplicin glycosides by immobilized plant cells of Nicotiana tabacum.

beta-Thujaplicin (hinokitiol) is a tropolone derivative present in the heartwood of cupressaceous plants and is used as a medicine, a food additive, and a preservative, and in cosmetics as hair tonic. The cultured plant cells of Nicotiana tabacum glycosylated beta-thujaplicin to two glucosides, 4-isopropyltropolone 2-O-beta-D-glucoside (6%) and 6-isopropyltropolone 2-O-beta-D-glucoside (12%), and two gentiobiosides, 4-isopropyltropolone 2-O-beta-D-gentiobioside (2%) and 6-isopropyltropolone 2-O-beta-D-gentiobioside (5%) after 48 h incubation. The use of immobilized cells of N. tabacum in sodium alginate gel much improved the yield of the products; the glycosylation of beta-thujaplicin with immobilized N. tabacum gave the glycoside products, 4-isopropyltropolone 2-O-beta-D-glucoside (11%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (6%), 6-isopropyltropolone 2-O-beta-D-glucoside (20%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (10%). On the other hand, 4-isopropyltropolone 2-O-beta-D-glucoside (14%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (7%), 6-isopropyltropolone 2-O-beta-D-glucoside (33%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (13%) were obtained through the biotransformation with immobilized cells in the medium without iron ions. In comparison with the case of bioconversion in the normal medium containing iron ions, removal of iron ions improved the yields of products.

Effect of three flavonoids, 5,7,3',4'-tetrahydroxy-3-methoxy flavone, luteolin, and quercetin, on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophil.

The effect of three flavonoids, 5,7,3',4'-tetrahydoxy-3-methoxy flavone (THMF), luteolin, and quercetin, on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils were investigated. When the cells were preincubated with these flavonoids, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed, showing a dependence on amounts of the flavonoid. The suppressing effect of the flavonoid was THMF > luteolin > quercetin. These flavonoids also suppressed the superoxide generation induced by phorbol 12-myristate 13-acetate. In this case also, THMF was more effective than luteolin and quercetin. On the other hand, the superoxide generation induced by arachidonic acid was markedly suppressed by quercetin. The suppressing effect was quercetin >> THMF > luteolin. THMF, luteolin, and quercetin significantly suppressed tyrosyl phosphorylation of 80.1-, 58.0-, and 45.0-kDa proteins in fMLP-treated human neutrophils. The suppression depended on the concentration of the flavonoids, and the inhibition of tyrosyl phosphorylation was in parallel to that of the fMLP-induced superoxide generation, respectively. While luteolin and quercetin showed a weak hemolytic activity at 2.5 mM, THMF showed almost no hemolytic activity even at 5 mM, suggesting an advantage of THMF for its clinical use.

cDNA cloning and expression of mutant catalase from the hypocatalasemic mouse: comparison with the acatalasemic mutant.

Mutant catalase cDNAs from the hypocatalasemic and acatalasemic mice were cloned and expressed in bacteria. A novel missense mutation, Asp (AAT) to Ser (AGT), was identified at amino acid position 439 of the hypocatalasemic catalase. Analysis of recombinant catalase mutants revealed that the mutation is responsible for the reduced activity of hypocatalasemic catalase and the unstable tetrameric structure of acatalasemic catalase was also suggested.

Hypocholesterolemic effect of indigestible fraction of Chlorella regularis in cholesterol-fed rats.

The effects of Chlorella regularis powder (CP) and Chlorella regularis indigestible fraction (CIF) on serum and liver lipid concentrations and on fecal steroid excretion were estimated in rats fed diets containing 5 g/kg cholesterol and 2.5 g/kg sodium cholate. The ingestion of 12.7% CP or 5.3% CIF did not influence food intake or growth. CP and CIF decreased the levels of serum cholesterol, but had no effect on the levels of serum triacylglycerol and phospholipid. Liver cholesterol contents were lower in the CP and CIF groups than in the control group, but CP and CIF did not affect liver triacylglycerol content. CP and CIF increased the total amount of fecal neutral steroids excreted, but did not modify the total bile acid excretion. However, the soluble bile acid concentrations of reconstituted fecal water in the rats fed CP and CIF diets were lower than the control value. Moreover, CP and CIF had a high bile acid binding capacity in vitro. These results indicated that CIF had a hypocholesterolemic effect and enhanced fecal neutral steroid excretion while decreasing the soluble fecal bile acid concentration.

Effect of soy milk and bifidobacterium-fermented soy milk on plasma and liver lipids in ovariectomized Syrian hamsters.

The effects of soy milk and fermented soy milk on lipid metabolism were studied in ovariectomized Syrian hamsters. Five mo-old Syrian hamsters were randomly assigned to four treatment groups: ovariectomized (OVX)+control diet (OVX-C); OVX+soy milk diet (OVX-SM); OVX+fermented soy milk diet (OVX-FSM); and sham-operated+control diet (Sham-C). The hamsters were fed on these diets for 4 wk. The atherogenic index value of the OVX-FSM group was lower than that of the OVX-C group. The plasma triglyceride level of the OVX-FSM group was significantly lower than that of the OVX-C group. The liver total cholesterol contents in the OVX-SM and OVX-FSM groups were significantly lower than that in the OVX-C group. Thus, these results demonstrate that bifidobacterium-fermented soy milk had a hypolipidemic effect in ovariectomized hamsters.

Lower plasma triglyceride level in Syrian hamsters fed on skim milk fermented with Lactobacillus casei strain Shirota.

The effect of fermented skim milk (FSM) by Lactobacillus casei strain Shirota on plasma lipids in hamsters was examined. Hamsters fed on cholesterol-free and -enriched diets containing 30% FSM had lower levels of plasma triglyceride than those fed on the control diet. In the experiment with the cholesterol-enriched diet-fed hamsters, the plasma triglyceride level was suppressed by FSM at concentrations of 10% to 30%. Unfermented milk tended to lower the level of triglyceride, but not significantly. The plasma cholesterol concentration was not affected by an FSM and unfermented skim milk supplement to the diet. L. casei strain Shirota grew well in the presence of mixed lipid micelles containing bile acid, but did not have the ability to remove cholesterol from the culture broth. These results indicate that FSM lowered the plasma triglyceride level in hamsters.

Formation of gamma-glutamylpropargylglycylglycine from propargylglycine in human blood and erythrocytes.

Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase) from human blood incubated with D,L-propargylglycine in the presence of L-glutamate and glycine, and identified by fast-atom-bombardment mass spectrometry, indicating that human blood can metabolize propargylglycine to gamma-Glu-PPG-Gly. When whole blood was incubated with 2 mM D,L-propargylglycine in the presence of 10 mM L-glutamate and 10 mM glycine at 37 degrees C for 16h, 0.094+/-0.013 micromol of gamma-Glu-PPG-Gly was formed per ml of whole blood. When erythrocytes were incubated under the same conditions for 16h, 0.323+/-0.060 micromol of gamma-Glu-PPG-Gly was formed per ml of erythrocytes, suggesting a large contribution of erythrocytes to gamma-Glu-PPG-Gly formation in whole blood. The apparent Km value of gamma-Glu-PPG-Gly formation in human erythrocytes for D,L-propargylglycine was 0.32 mM. The observed rate of gamma-Glu-PPG-Gly formation and the Km value for D,L-propargylglycine suggest that metabolism of propargylglycine to gamma-Glu-PPG-Gly can play a definite biological role in human subjects who are loaded with propargylglycine.

Determination of glutathione peroxidase activity and its contribution to hydrogen peroxide removal in erythrocytes.

A new method for the determination of glutathione peroxidase activity in erythrocytes was developed. The present method was applied to the measurement of hydrogen peroxide removal rates by glutathione peroxidase in erythrocytes at 70 microM hydrogen peroxide under simulated in vivo conditions. The removal rates by glutathione peroxidase in mouse erythrocytes were twenty-times faster than those in human ones and were 5.2 mumol/sec/g of Hb. The removal rates in acatalasemic mouse erythrocytes indicate that glutathione peroxidase is the main means of hydrogen peroxide removal in acatalasemic mouse erythrocytes. Based on these results, we concluded that glutathione peroxidase in mouse erythrocytes had sufficient ability to remove hydrogen peroxide at even relatively high concentrations. This may be one of the reasons why acatalasemic mice suffer no health problems while Japanese acatalasemic patients suffer from Takahara disease when infected with hydrogen peroxide-generating bacteria.

Different effect of diastereoisomers of L-cystathionine sulfoxide on the stimulus coupled responses of human neutrophils.

The priming effect of L-cystathionine sulfoxide, which is one of the unusual cystathionine metabolites found in the urine of patients with cystathioninuria, on the stimulus-induced superoxide generation by human neutrophils was examined. The synthetic L-cystathionine sulfoxide significantly enhanced the superoxide generations induced by N-formyl-methionyl-leucyl-phenylalanine [fMLP], opsonized zymosan [OZ], arachidonic acid [AA], and phorbol 12-myristate 13-acetate [PMA]. Then the synthetic L-cystathionine sulfoxide was separated into two diastereoisomers, CS-I and CS-II, which showed a peak at 76 and 83 min on chromatogram by amino acid analyzer, respectively. CS-I enhanced the superoxide generations induced by AA and PMA but not those induced by fMLP and OZ. On the contrary, CS-II enhanced the superoxide generations induced by fMLP and OZ but not those induced by AA and PMA. The superoxide generation induced by PMA with CS-I was suppressed by H-7 and was enhanced by genistein, while that by fMLP with CS-II was suppressed by genistein and was enhanced by H-7.

Characterization of hydrogen peroxide removal activities in mouse hemolysates: catalase activity and hydrogen peroxide removal activity by hemoglobin.

Hydrogen peroxide removal activities in normal and acatalasemic mouse hemolysates were examined to determine the optimal temperature of catalase. From thermal stability of the removal activities in hemolysates, the removal activities were divided into two activities. The removal activity deactivated at lower temperature was catalase, and the 50% inactivation was observed after 10 min incubation at 47.2 +/- 0.5 degrees C for normal hemolysates and 34.0 +/- 0.8 degrees C for acatalasemic ones. The removal activity deactivated at a higher temperature remained after the addition of sodium azide, and the 50% inactivation was observed at 63.5 +/- 1.4 degrees C. After separation of the removal activities by carboxymethyl-cellulose column chromatography, the removal activity deactivated at higher temperature was attributed to the activity by hemoglobin. From Lineweaver-Burk plot analysis of the removal rates by hemoglobin at 37 degrees C, the Michaelis constant for hydrogen peroxide and the maximum velocity were 201 +/- 53 microM and 5.37 +/- 1.39 micromol/s per g of Hb, respectively. Removal rates by hemoglobin in mouse hemolysates at 37 degrees C in 70 microM hydrogen peroxide were 1.32 +/- 0.12 micromol/s per g of Hb. Catalase activity (k/g Hb: rate constant related to the hemoglobin content) in normal mouse hemolysates was 104 +/- 12 at 25 degrees C and 117 +/- 10 at 37 degrees C, and that in acatalasemic hemolysates was 10.5 +/- 1.7 at 25 degrees C. These results indicate that activity of hydrogen peroxide removal by hemoglobin is substantial and the activity in acatalasemic hemolysates is predominant at low concentration of hydrogen peroxide.

Priming effect of N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine in human neutrophils and tyrosyl phosphorylation of 45 kDa protein.

Human peripheral blood polymorphonuclear leukocytes were preincubated with N-acetylcystathionine and N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine (NAc-OCPC) found in the urine of a patient with cystathioninuria. NAc-OCPC significantly enhanced the N-formyl-methionyl-leucyl-phenylalanine-induced superoxide generation, whereas N-acetylcystathionine did not enhance the superoxide generation. When the cells were incubated with NAc-OCPC, the tyrosyl phosphorylation of 45 kDa protein of the cell was markedly increased with time. The phosphorylation process was dependent on the concentration of NAc-OCPC. Both the superoxide generation and the tyrosyl phosphorylation of 45 kDa protein increased by NAc-OCPC were inhibited by genistein and herbimycin A, the inhibitors of protein tyrosine kinase, but were rather enhanced by staurosporine, an inhibitor of protein kinase C.

Spectrophotometric determination of hydrogen peroxide: catalase activity and rates of hydrogen peroxide removal by erythrocytes.

A new method of hydrogen peroxide determination for the measurement of catalase activity and rates of hydrogen peroxide removal by erythrocytes was described. Hydrogen peroxide was determined by converting it to the indamine dye with a water-soluble ironporphyrin and measuring the absorbance at 590 nm. This method was applied to the assay of catalase in hemolysates from human, rat and mouse blood. The activities obtained were in agreement with those obtained by other methods including UV method. The present method was also applied to the determination of rates of hydrogen peroxide removal by intact erythrocytes from human subjects, rats and mice. Data suggested that normal erythrocytes have substantial capacity to remove extracellular hydrogen peroxide. From the measurement of catalase activity in erythrocytes treated with 3-amino-1,2,4-triazole and rates of hydrogen peroxide removal by the erythrocytes, it is deduced that rate constants related to the hemoglobin content (k/g Hb) for hydrogen peroxide removal by catalase in normal and acatalasemic erythrocytes are 42.0 +/- 6.0 and 8.0 +/- 3.0, respectively.

Identification of cyclic cystathionine sulfoxide and N-acetylcyclic cystathionine in the urine of a patient with cystathioninuria using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system.

Perhydro-1,4-thiazepine-4,5-dicarboxylic acid sulfoxide (cyclic cystathionine sulfoxide [cyclic cystaSO]) and N-acetylperhydro-1,4-thiazepine-3,5-dicarboxylic acid (NAc-cyclic cysta) have been identified in the urine of a patient with cystathioninuria as new metabolites of cystathionine for the first time using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system (LC/APCI-MS). The concentrations of cyclic cystaSO and NAc-cyclic cysta in the urine of a patient with cystathioninuria have also been determined for the first time using this method: 18.24 +/- 0.79 and 25.23 +/- 0.83 mg/g creatinine, respectively.

Identification of perhydro-1,4-thiazepine-3,5-dicarboxylic acid, cystathionine mono-oxo acids, cystathionine ketimines, cystathionine sulfoxide and N-acetylcystathionine sulfoxide in the urine sample of D,L-propargylglycine treated rats.

Novel cystathionine metabolites, perhydro-1,4-thiazepine-3,5-dicarboxylic acid (PHTZDC), cystathionine mono-oxo acids [S-(3-oxo-3-carboxy-n-propyl)cysteine and S-(2-oxo-2-carboxyethyl)homocysteine], cystathionine ketimines, cystathionine sulfoxide and N-acetylcystathionine sulfoxide were identified previously in the urine of patients with cystathioninuria. We have identified these compounds for the first time in the urine of D,L-propargylglycine-treated rats using LC/APCl-MS (liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system) and an amino acid analyzer. Cystathionine mono-oxo acids and cystathionine ketimines were easily interconvertible depending on the pH of the solution. The excretion of PHTZDC, total cystathionine ketimine (cystathionine mono-oxo acids plus cystathionine ketimines), cystathionine sulfoxide and Nac-cystathionine sulfoxide in the rat urine increased in proportion to that of cystathionine content after D,L-propargylglycine administration.

Identification of N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine in the urine of a patient with cystathioninuria using LC/APCI-MS.

A new cystathionine metabolite has been identified in the urine of a patient with cystathioninuria using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system (LC/APCI-MS). By this method a very intense quasi-molecular ion was observed as a base peak of synthetic N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine (NAc-OCPC). The quasimolecular ion [M + H]+ of NAc-OCPC observed in the urine of a patient with cystathioninuria was the same as that of the authentic compound (m/z 264). The retention time and Rf value on paper chromatography of the synthetic compound were the same as those of the urinary compound from the patient with cystathioninuria. From these results, this new cystathionine metabolite was identified as N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine.

Increase in cystathionine content in rat liver mitochondria after D,L-propargylglycine administration.

Intraperitoneal administration of D,L-propargylglycine to rats resulted in an increase in the cystathionine content of whole liver and liver mitochondria. Cystathionine in mitochondria was identified by amino acid analysis, thin layer chromatography, high-voltage paper electrophoresis and liquid chromatography-mass spectrometry. The cystathionine content of whole liver was 5.37 ± 1.59µmol per g of fresh liver at 14 h after the administration of 50 mg of D,L-propargylglycine per kg of body weight, while 0.07 ± 0.02µmol of cystathionine per g of fresh liver was detected in the control rats. The cystathionine content of liver mitochondria from both groups of rats was 9.40 ± 1.20 and 0.19 ± 0.04 nmol of cystathionine per mg of protein, respectively. The mitochondrial cystathionine increased dose-dependently with the increase of D,L-propargylglycine administered. The increase was proportional to the time after the administration up to 12 h, and then decreased. The increase of cystathionine in the liver mitochondria was linearly proportional to that in the whole liver. These results suggest that cystathionine in liver mitochondria is in an equilibrium with that in the cytosol.

Increase in tissue cysteine level and excretion of sulfate and taurine after intragastric administration ofL-2-oxothiazolidine-4-carboxylate in rats.

Five mmol ofL-2-oxothiazolidine-4-carboxylate (OTC)/kg of body weight was administered into the stomach of rats, and cysteine levels in tissues and sulfate and taurine excreted in the urine were determined. The cysteine (plus cystine expressed as cysteine) concentration in the liver increased to 170-200% of the original level at 30 min and that in the blood to 160% at 60 min after the OTC administration. These high levels were maintained until 8 h after the administration and decreased gradually thereafter. Excretion of sulfate and taurine increased after the OTC administration and the increase corresponded to 26% and 15%, respectively, of the OTC administered. These findings suggest that at least about 40% of the OTC administered into the stomach was taken up and converted to cysteine, which was metabolized to sulfate and taurine.

High-performance liquid chromatographic determination of taurine and hypotaurine using 3,5-dinitrobenzoyl chloride as derivatizing reagent.

A method for the determination of taurine and hypotaurine in biological samples involving the preparation of their 3,5-dinitrobenzoyl derivatives followed by HPLC was established. Taurine and hypotaurine in aqueous media were reacted with 3,5-dinitrobenzoyl chloride in the presence of triethylamine to prepare 3,5-dinitrobenzoyl derivatives. These derivatives were separated on a C18 reversed-phase column and detected by recording the absorbance at 254 nm. Derivatives of taurine and hypotaurine were obtained in yields of 91.4 +/- 3.3 and 85.6 +/- 2.6%, respectively. The calibration graphs for taurine and hypotaurine were linear between 2.5 and 500 microM with correlation coefficients of 0.999. The method was applied to the determination of taurine and hypotaurine in human and rat urine and blood and in rat liver and heart.