Combined citicoline and docosahexaenoic acid treatment improves cognitive dysfunction following transient brain ischemia.

Phospholipids are structural components of cellular membranes that play important roles as precursors for various signaling pathways in modulating neuronal membrane function and maintenance of the intracellular environment. Phosphatidylcholine (PtdCho) is the most abundant cellular phospholipid. Citicoline and docosahexaenoic acid (DHA) are essential intermediates in the synthesis of PtdCho. Both PtdCho intermediates have independently shown neuroprotective effects in cerebral ischemia, but their combined effect is unknown. This study aimed to investigate the combined effect of oral citicoline and DHA treatment on improvement of cognitive deficits following cerebral ischemia using a 20-min bilateral common carotid artery occlusion (BCCAO) mouse model. BCCAO ischemic mice were treated for a total of 11 days with a combination of citicoline (40 mg/kg body weight/day) and DHA (300 mg/kg body weight/day) or each alone. Combined citicoline and DHA synergistically and significantly improved learning and memory ability of ischemic mice compared with either alone. Further, citicoline and DHA treatment significantly prevented neuronal cell death, and slightly increased DHA-containing PtdCho in the hippocampus, albeit not significantly. Taken together, these findings suggest that combined citicoline and DHA treatment may have synergistic benefits for partially improving memory deficits following transient brain ischemia.

Protective effects of oral glutathione on fasting-induced intestinal atrophy through oxidative stress.

AIM: To determine whether oral glutathione (GSH) administration can alleviate the effects of fasting-induced intestinal atrophy in the small intestinal mucosa. METHODS: Rats were divided into eight groups. One group was fed ad libitum, another was fed ad libitum and received oral GSH, and six groups were administrated saline (SA) or GSH orally during fasting. Mucosal height, apoptosis, and cell proliferation in the jejunum were histologically evaluated. iNOS protein expression (by immunohistochemistry), nitrite levels (by high performance liquid chromatography, as a measure of NO production), 8-hydroxydeoxyguanosine formation (by ELISA, indicating ROS levels), glutathione/oxidized glutathione (GSH/GSSG) ratio (by enzymatic colorimetric detection), and γ-glutamyl transpeptidase (Ggt1) mRNA levels in the jejunum (by semi-quantitative RT-PCR) were also estimated. RESULTS: Oral GSH administration was demonstrated to drastically reduce fasting-induced intestinal atrophy in the jejunum. In particular, jejunal mucosal height was enhanced in GSH-treated animals compared to SA-treated animals [527.2 ± 6.9 for 50 mg/kg GSH, 567.6 ± 5.4 for 500 mg/kg GSH vs 483.1 ± 4.9 (µm), P < 0.01 at 72 h]. This effect was consistent with decreasing changes in GSH-treated animals compared to SA-treated animals for iNOS protein staining [0.337 ± 0.016 for 50 mg/kg GSH, 0.317 ± 0.017 for 500 mg/kg GSH vs 0.430 ± 0.023 (area of staining part/area of tissue), P < 0.01 at 72 h] and NO [2.99 ± 0.29 for 50 mg/kg GSH, 2.88 ± 0.19 for 500 mg/kg GSH vs 5.34 ± 0.35 (nmol/g tissue), P < 0.01 at 72 h] and ROS [3.92 ± 0.46 for 50 mg/kg GSH, 4.58 ± 0.29 for 500 mg/kg GSH vs 6.42 ± 0.52 (8-OHdG pg/µg DNA), P < 0.01, P < 0.05 at 72 h, respectively] levels as apoptosis mediators in the jejunum. Furthermore, oral GSH administration attenuated cell proliferation decreases in the fasting jejunum [182.5 ± 1.9 for 500 mg/kg GSH vs 155.8 ± 3.4 (5-BrdU positive cells/10 crypts), P < 0.01 at 72 h]. Notably, both GSH concentration and Ggt1 mRNA expression in the jejunum were also attenuated in rats following oral administration of GSH during fasting as compared with fasting alone [0.45 ± 0.12 vs 0.97 ± 0.06 (nmol/mg tissue), P < 0.01; 1.01 ± 0.11 vs 2.79 ± 0.39 (Ggt1 mRNA/Gapdh mRNA), P < 0.01 for 500 mg/kg GSH at 48 h, respectively]. CONCLUSION: Oral GSH administration during fasting enhances jejunal regenerative potential to minimize intestinal mucosal atrophy by diminishing fasting-mediated ROS generation and enterocyte apoptosis and enhancing cell proliferation.

l-Citrulline ameliorates cerebral blood flow during cortical spreading depression in rats: Involvement of nitric oxide- and prostanoids-mediated pathway.

l-Citrulline is a potent precursor of l-arginine, and exerts beneficial effect on cardiovascular system via nitric oxide (NO) production. Migraine is one of the most popular neurovascular disorder, and imbalance of cerebral blood flow (CBF) observed in cortical spreading depression (CSD) contributes to the mechanism of migraine aura. Here, we investigated the effect of l-citrulline on cardiovascular changes to KCl-induced CSD. in rats. Intravenous injection of l-citrulline prevented the decrease in CBF, monitored by laser Doppler flowmetry, without affecting mean arterial pressure and heart rate during CSD. Moreover, l-citrulline attenuated propagation velocity of CSD induced by KCl. The effect of l-citrulline on CBF change was prevented by l-NAME, an inhibitor of NO synthase, but not by indomethacin, an inhibitor of cyclooxygenase. On the other hand, attenuation effect of l-citrulline on CSD propagation velocity was prevented not only by l-NAME but also by indomethacin. In addition, propagation velocity of CSD was attenuated by intravenous injection of NOR3, a NO donor, which was diminished by ODQ, an inhibitor of soluble guanylyl cyclase. These results suggest that NO/cyclic GMP- and prostanoids-mediated pathway differently contribute to the effect of l-citrulline on the maintenance of CBF.

The effects on plasma L-arginine levels of combined oral L-citrulline and L-arginine supplementation in healthy males.

We investigated the effects of combining 1 g of l-citrulline and 1 g of l-arginine as oral supplementation on plasma l-arginine levels in healthy males. Oral l-citrulline plus l-arginine supplementation more efficiently increased plasma l-arginine levels than 2 g of l-citrulline or l-arginine, suggesting that oral l-citrulline and l-arginine increase plasma l-arginine levels more effectively in humans when combined.

Oral L-citrulline supplementation enhances cycling time trial performance in healthy trained men: Double-blind randomized placebo-controlled 2-way crossover study.

BACKGROUND: Many human studies report that nitric oxide (NO) improves sport performance. This is because NO is a potential modulator of blood flow, muscle energy metabolism, and mitochondrial respiration during exercise. L-Citrulline is an amino acid present in the body and is a potent endogenous precursor of L-arginine, which is a substrate for NO synthase. Here, we investigated the effect of oral L-citrulline supplementation on cycling time trial performance in humans. METHODS: A double-blind randomized placebo-controlled 2-way crossover study was employed. Twenty-two trained males consumed 2.4 g/day of L-citrulline or placebo orally for 7 days. On Day 8 they took 2.4 g of L-citrulline or placebo 1 h before a 4-km cycling time trial. Time taken to complete the 4 km cycle, along with power output/VO2 ratio (PO/VO2), plasma nitrite and nitrate (NOx) and amino acid levels, and visual analog scale (VAS) scores, was evaluated. RESULTS: L-Citrulline supplementation significantly increased plasma L-arginine levels and reduced completion time by 1.5 % (p < 0.05) compared with placebo. Moreover, L-citrulline significantly improved subjective feelings of muscle fatigue and concentration immediately after exercise. CONCLUSIONS: Oral L-citrulline supplementation reduced the time take to complete a cycle ergometer exercise trial. TRIAL REGISTRATION: Current Controlled Trials UMIN000014278.

Effects of Orally Administered Pyrroloquinoline Quinone Disodium Salt on Dry Skin Conditions in Mice and Healthy Female Subjects.

Pyrroloquinoline quinone (PQQ) is a coenzyme involved in the redox-cycling system. The supplemental use of PQQ has been examined based on its properties as an antioxidant and redox modulator. Although an animal study on deficiency of PQQ suggested that PQQ contributes to skin conditions, its efficacy in humans has not been reported. The present study aimed to investigate the effects of orally administered PQQ on skin moisture, viscoelasticity, and transepidermal water loss (TEWL) both in dry skin mouse models and in healthy female subjects with a subjective symptom of dry skin. In our dry skin mouse model study, oral intake of PQQ (0.0089%, w/w, in the diet for 6 wk) significantly decreased the number of mast cells in the dermis and the number of CD3⁺ T-cells in the epidermis. In our human study, oral intake of PQQ (20 mg/d for 8 wk) significantly inhibited the increase in TEWL on the forearm. Finally, subject questionnaires showed positive impressions for the improvement of skin conditions. These results suggest that oral intake of PQQ improves skin conditions both in female subjects with dry skin and in mice with a compromised skin barrier function.

Skin-whitening and skin-condition-improving effects of topical oxidized glutathione: a double-blind and placebo-controlled clinical trial in healthy women.

PURPOSE: Glutathione is a tripeptide consisting of cysteine, glycine, and glutamate and functions as a major antioxidant. It is synthesized endogenously in humans. Glutathione protects thiol protein groups from oxidation and is involved in cellular detoxification for maintenance of the cell environment. Reduced glutathione (GSH) has a skin-whitening effect in humans through its tyrosinase inhibitory activity, but in the case of oxidized glutathione (GSSG) this effect is unclear. We examined the skin-whitening and skin-condition effects of topical GSSG in healthy women. SUBJECTS AND METHODS: The subjects were 30 healthy adult women aged 30 to 50 years. The study design was a randomized, double-blind, matched-pair, placebo-controlled clinical trial. Subjects applied GSSG 2% (weight/weight [w/w]) lotion to one side of the face and a placebo lotion to the other side twice daily for 10 weeks. We objectively measured changes in melanin index values, moisture content of the stratum corneum, smoothness, wrinkle formation, and elasticity of the skin. The principal investigator and each subject also used subjective scores to investigate skin whitening, wrinkle reduction, and smoothness. Analysis of variance was used to evaluate differences between groups. RESULTS: The skin melanin index was significantly lower with GSSG treatment than with placebo from the early weeks after the start of the trial through to the end of the study period (at 10 weeks, P<0.001). In addition, in the latter half of the study period GSSG-treated sites had significant increases in moisture content of the stratum corneum, suppression of wrinkle formation, and improvement in skin smoothness. There were no marked adverse effects from GSSG application. CONCLUSION: Topical GSSG is safe and effectively whitens the skin and improves skin condition in healthy women.

Low-dose menaquinone-4 improves γ-carboxylation of osteocalcin in young males: a non-placebo-controlled dose-response study.

BACKGROUND: Menaquinone-4 is a type of vitamin K that has a physiological function in maintaining bone quality via γ-carboxylation of osteocalcin. However, little is known about the beneficial effect of intake of dosages below1500 µg/day. FINDINGS: Fifteen healthy males aged 25.0 years (median) participated in a non-placebo-controlled dose-examination study. They received menaquinone-4 daily for 5 weeks at 0, 300, 600, 900, and 1500 µg/day in weeks 1, 2, 3, 4, and 5, respectively. Compared with baseline, serum γ-carboxylated osteocalcin levels were significantly greater at an intake of 900 µg/day or more; serum undercarboxylated osteocalcin levels and the ratio of serum undercarboxylated osteocalcin to γ-carboxylated osteocalcin were significantly lower than baseline at doses of 600 µg/day or more. CONCLUSIONS: This preliminary graded-dose study suggested that menaquinone-4 supplementation at 600 µg/day or more is likely to be important in terms of vitamin K requirements for bone health.

Topical effects of N-acetyl-L-hydroxyproline on ceramide synthesis and alleviation of pruritus.

PURPOSE: N-acetyl-l-hydroxyproline (AHYP) is an acetylated form of l-hydroxyproline that is used to treat skin ulcers and porphyria cutanea tarda. Its other biological and physiological effects on the skin have not been elucidated. We investigated the effects of AHYP on the skin-barrier function, focusing on ceramide synthesis and the effects of topical AHYP on atopic dermatitis. MATERIALS AND METHODS: AHYP was applied to a three-dimensional cultured skin model. Ceramides were quantified by high-performance thin-layer chromatography. Serine palmitoyltransferase (SPT) is the rate-limiting enzyme in de novo ceramide synthesis, and the mRNA of its long-chain base subunit 1 (SPTLC1) was evaluated by quantitative reverse-transcription polymerase chain reaction. A clinical trial in the form of an intraindividual, comparative, double-blind, randomized, vehicle-controlled test involving 15 female subjects suffering from slight atopic dermatitis was performed. Subjects applied 1% (w/w) AHYP cream to one forearm and a control cream to the other forearm twice daily for 4 weeks. Skin condition was evaluated by measuring transepidermal water loss (TEWL). Dermatological observations were made by a dermatologist, and subjects evaluated their own pruritus intensity before beginning treatment and 4 weeks after the start of treatment. RESULTS: SPTLC1 expression and ceramide synthesis were significantly increased in an AHYP-treated skin model (P < 0.05). In the clinical trial, no adverse effects were observed in any subjects. TEWL was increased in the control-treated region of the forearm (P < 0.05) after 4 weeks' application, whereas there was no change in the AHYP-treated region of the forearm. Pruritus intensity declined in the AHYP-treated forearms between 0 and 4 weeks (P < 0.05), but there was no change in the control-treated forearms. CONCLUSION: AHYP increased ceramide synthesis by upregulating SPTLC1 in a three-dimensional cultured skin model, and it prevented TEWL increase and alleviated pruritus in human subjects with slight atopic dermatitis.

Increasing effect of an oral intake of L-hydroxyproline on the soluble collagen content of skin and collagen fragments in rat serum.

We examined the effects of oral L-hydroxyproline (Hyp) on collagen in the body. After 2 weeks' administration of Hyp (0.5 or 1 g/kg) to F344 male rats, the soluble collagen content of the skin had increased, and the serum concentration of collagen peptides was correlated with the skin content of soluble collagen. This result suggests that oral Hyp augmented collagen metabolism.

Investigation of the topical application of procyanidin oligomers from apples to identify their potential use as a hair-growing agent.

BACKGROUND: Procyanidins are a family of condensed tannins, which have been shown to possess hair-growing activity in both the in vitro and in vivo murine models. AIMS: We report a 12-month clinical study aimed at treating male pattern baldness by external application of 0.7% apple procyanidin oligomers. PATIENTS/METHODS: A double-blind clinical test involving a total of 43 subjects was performed. Twenty-one men in the procyanidin group and 22 men in the placebo control group were subjected to analysis. In the first 6 months, we compared the procyanidin and the placebo groups to assess the medicinal effects of procyanidin oligomers. The application time of the procyanidin group was subsequently extended to 12 months, and the time course of its remedial value was examined. RESULTS: The increase in total number of hairs in a designated scalp area of the procyanidin group subjects after the 6-month trial was significantly greater than that of the placebo control group subjects (procyanidin, 3.3 +/- 13.0 (mean +/- SD)/0.50 cm(2); placebo, -3.6 +/- 8.1/0.50 cm(2); P < 0.001, two-sample t-test). Time course-dependent improvement in hair density was observed in the procyanidin subjects. No adverse side effects were observed in any of the subjects. Procyanidin therapy thus shows potential hair-growing activity.

Phosphatidic acid has a potential to promote hair growth in vitro and in vivo, and activates mitogen-activated protein kinase/extracellular signal-regulated kinase kinase in hair epithelial cells.

Phospholipids have recently been discovered to play an important role in cellular regulation. In this study, we focused on phosphatidic acid and lysophosphatidic acid, which are phospholipids known to possess growth-hormonal effects on several types of cells, and examined their growth-promoting effects on murine hair epithelial cells. We discovered that phosphatidic acid possesses intensive growth-promotional effects on hair epithelial cells and epidermal keratinocytes. In contrast, lyso-phosphatidic acid showed lower growth-promoting effects on hair epithelial cells relative to phosphatidic acid and showed minimal or no growth-promoting activity on epidermal keratinocytes. Phosphatidic acid was also shown to have hair-growing activity to induce the anagen phase of the hair cycle in the in vivo murine model. For the purpose of examining the hair-growing mechanisms of phosphatidic acid, we examined its relationship to the mitogen-activated protein kinase cascade linked to cell proliferation and the transforming growth factor beta signal pathway known to be a regulator of catagen induction. We confirmed that phosphatidic acid activates MEK-1/2 and upregulates the expression of MEK-1/2 in cultured murine hair epithelial cells. Addition of transforming growth factor beta1 to hair epithelial cell cultures concentration-dependently decreased cell growth and induced apoptosis; however, addition of phosphatidic acid to the culture neutralized the growth-inhibiting effects of transforming growth factor beta1 and protected the cells from apoptosis. We speculate that the hair-growing activity of phosphatidic acid is at least linked to its growth-promoting effects on hair epithelial cells that follow mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activation and its protective action on transforming-growth-factor-beta1-induced apoptosis that is assumed to trigger catagen induction in the hair cycle.

Procyanidin B-3, isolated from barley and identified as a hair-growth stimulant, has the potential to counteract inhibitory regulation by TGF-beta1.

With the aim of identifying natural products, which possess hair-growing activity, we examined more than 1000 plant extracts with respect to their growth-promoting effects on hair epithelial cells. We discovered intensive growth-promoting activity, about 140% relative to controls, in barley extract. Our strategy for identifying active compounds in barley extract involved subjecting it to column chromatography using HP-20 resin columns, an LH-20 resin column, and preparative high-performance liquid chromatography (HPLC) using an ODS column. The 60% (v/v) aqueous methanol eluted fraction from the HP-20 column and the 75% (v/v) aqueous methanol eluted fraction from the subsequent LH-20 column showed high hair-growing activity in vivo. We isolated two major substances from the LH-20 active fraction using preparative HPLC. By means of mass spectrometry, 1H-NMR, and 13C-NMR analyses, one substance was revealed to be procyanidin B-3 and the other substance was identified as (+)-catechin. Purified procyanidin B-3 showed high hair-growing activity in the form of in vitro hair epithelial cell growth-promoting activity and in vivo anagen-inducing activity; however (+)-catechin showed no hair-growing activity. For the purpose of examining the hair-growing mechanisms of procyanidin B-3, we examined its relationship to the TGF-beta signal pathway, which is known to be a regulator of catagen induction. Addition of TGF-beta1 to hair epithelial cell cultures dose-dependently decreased the cell growth, and addition of procyanidin B-3 to the culture neutralized the growth-inhibiting effect of TGF-beta1. From these results, it is concluded that procyanidin B-3 can directly promote hair epithelial cell growth in vitro, has the potential to counteract the growth-inhibiting effect caused by TGF-beta1 in vitro, and has potential to stimulate anagen induction in vivo.