Safety evaluation of vitamin K2 (menaquinone-7) via toxicological tests.

This study aims to evaluate the safety of MK-7 produced by fermentation process using a Bacillus subtilis var. natto strain for human ingestion via acute oral toxicity, repeated dose 90-day oral toxicity, 28-day recovery test, and genotoxicity tests. The acute oral toxicity test results indicated that all subjects survived at the dose of 5000 mg/kg with no toxic effects. For the repeated dose 90-day oral toxicity test, MK-7 was administered to rats at 500, 1500, and 4500 mg/kg for 90 d. No abnormal findings were detected in clinical observations or in clinical pathological and histopathological examinations. The no-observed-adverse-effect level(NOAEL) was determined to be 4500 mg/kg/d, the maximum dose tested. For the evaluation of genotoxicity, reverse mutation, chromosomal aberration, and micronucleus tests were performed. In the reversion mutation test, vitamin K2 did not induce reversion in bacterial strains, and no chromosomal abnormality was observed in the chromosomal abnormality test using Chinese hamster lung cells. In the micronucleus test, micronuclei were not induced using ICR mouse bone marrow cells. All the toxicity test results suggest that vitamin K2 produced by fermentation processes using Bacillus subtilis var. natto induced no toxicological changes under the experimental conditions.

Menaquinone-7 ameliorates cerebrovascular calcification-associated memory decline in aged mice.

Menaquinone (MK)-7 is a vitamin K2 analog that functions as a cofactor of γ-glutamyl carboxylase involved in the activation of vitamin K (VK)-dependent proteins. The present study aimed to evaluate the effect of MK-7 on memory and cognitive function in aged C57BL/6 mice. Eighteen-month-old mice were raised for a further 4 months, fed on a standard or calcium-rich diet (3 % [w/w]), and were orally given MK-7 (40 and 400 µg/day/mouse) five times per week during the same period. The Morris water maze (MWM) test was performed at 19 and 22 months. The aged mice showed noticeable memory declines in the MWM test at all time points compared with 6-week-old mice, and this memory loss was significantly restored by the daily administration of high-dose MK-7 for 4 months. MK-7 administration also improved micro-computed tomography-based cerebrovascular calcification and aging-associated declines in growth arrest-specific 6, total and carboxylated matrix Gla proteins, and ganglioside levels in the brain of aged mice. It serologically reduced phosphorous levels in the blood, but not the urea, cholesterol, and calcium. Taken together, the long-term administration of MK-7 significantly improved age-related memory and cognitive impairments, possibly through inhibition of cerebrovascular calcification in aged mice, indicating that it can be used to develop new drugs for improving memory and cognitive function in older adults.

Ex Vivo Live Full-Thickness Porcine Skin Model as a Versatile In Vitro Testing Method for Skin Barrier Research.

Since the European Union (EU) announced their animal testing ban in 2013, all animal experiments related to cosmetics have been prohibited, creating a demand for alternatives to animal experiments for skin studies. Here, we investigated whether an ex vivo live porcine skin model can be employed to study the safety and skin barrier-improving effects of hydroxyacids widely used in cosmetics for keratolytic peels. Glycolic acid (1-10%), salicylic acid (0.2-2%), and lactobionic acid (1.2-12%) were used as representative substances for α-hydroxyacid (AHA), ß-hydroxyacid (BHA), and polyhydroxyacid (PHA), respectively. When hydroxyacids were applied at high concentrations on the porcine skin every other day for 6 days, tissue viability was reduced to 50-80%, suggesting that the toxicity of cosmetic ingredients can be evaluated with this model. Based on tissue viability, the treatment scheme was changed to a single exposure for 20 min. The protective effects of a single exposure of hydroxyacids on skin barrier function were evaluated by examining rhodamine permeability and epidermal structural components of barrier function using immunohistochemistry (IHC) and immunofluorescence (IF) staining. Lactobionic acid (PHAs) improved skin barrier function most compared to other AHAs and BHAs. Most importantly, trans-epidermal water loss (TEWL), an important functional marker of skin barrier function, could be measured with this model, which confirmed the significant skin barrier-protective effects of PHAs. Collectively, we demonstrated that the ex vivo live full-thickness porcine skin model can be an excellent alternative to animal experiments for skin studies on the safety and efficacy of cosmetic ingredients.

Efficient production of lactobionic acid using genetically engineered Pseudomonas taetrolens as a whole-cell biocatalyst.

Lactobionic acid (LBA) has been widely used in the food, pharmaceutical, and cosmetic industries. Pseudomonas taetrolens is an efficient LBA-producing bacterium. To improve the LBA-production ability of P. taetrolens, we modified the strain by genetic engineering. We performed homologous expression of the quinoprotein glucose dehydrogenase gene in P. taetrolens and measured the intracellular lactose-oxidizing activity and LBA production titer. In flask cultures at 12 h of incubation, the intracellular lactose oxidizing activity (0.159 U/g dry weight cell) and LBA production titer (77.2 g/L) of the recombinant P. taetrolens were approximately 118 % and 69 % higher than those (0.073 U/g dry weight cell and 45.8 g/L, respectively) of wild-type P. taetrolens. Using this recombinant strain as a whole-cell biocatalyst (WCB), the effects of reaction parameters, such as reaction temperature, cell density, and cell harvest time, were investigated on LBA production. Under optimized reaction conditions, the LBA production titer, yield, and productivity of WCB were 200 g/L, 95.6 %, and 16.7 g/L/h, respectively. Compared with our previous study, LBA production titer, yield, and productivity, which are key factors for industrial LBA production, were significantly improved by fermentation of wild-type P. taetrolens. Moreover, the reaction for LBA production could be performed up to seven times without a significant reduction in productivity, implying that this WCB was rather robust. Our results suggest that the utilization of whole-cell biocatalysis using recombinant P. taetrolens provides a potential solution to achieve economically feasible production of LBA.

Enhancement of Lactobionic Acid Productivity by Homologous Expression of Quinoprotein Glucose Dehydrogenase in Pseudomonas taetrolens.

This is the first study on improving lactobionic acid (LBA) production capacity in Pseudomonas taetrolens by genetic engineering. First, quinoprotein glucose dehydrogenase (GDH) was identified as the lactose-oxidizing enzyme of P. taetrolens. Of the two types of GDH genes in P. taetrolens, membrane-bound (GDH1) and soluble (GDH2), only GDH1 showed lactose-oxidizing activity. Next, the genetic tool system for P. taetrolens was developed based on the pDSK519 plasmid for the first time, and GDH1 gene was homologously expressed in P. taetrolens. Recombinant expression of the GDH1 gene enhanced intracellular lactose-oxidizing activity and LBA production of P. taetrolens in flask culture. In batch fermentation of the recombinant P. taetrolens using a 5 L bioreactor, the LBA productivity of the recombinant P. taetrolens was approximately 17% higher (8.70 g/(L h)) than that of the wild type (7.41 g/(L h)). The LBA productivity in this study is the highest ever reported using bacteria as production strains for LBA.

Surgical excision of osteochondroma on mandibular condyle via preauricular approach with zygomatic arch osteotomy.

BACKGROUND: Osteochondroma is a benign tumor that tends to develop in mandibular condyle and coronoid process in the craniofacial region. If tumor mass has grown from condyle into the infratemporal space with zygomatic arch obstructing the access, there are risks associated with surgical exposure and local resection of these masses. CASE PRESENTATION: This study reports on a case of osteochondroma on mandibular condylar head where we treated with surgical excision via preauricular approach with 3D analysis. After the local resection, there were no surgical and post-operative complications until 8-month follow-up period. CONCLUSIONS: In local excision of osteochondroma, our method is a minimally invasive method. It is a good example of osteochondroma treatment.

Glucosylceramide attenuates the inflammatory mediator expression in lipopolysaccharide-stimulated RAW264.7 cells.

The positive effect of glucosylceramide (GlcCer) on skin conditions is well known. Recently, there has been increasing interest in the potential antiinflammatory effects of GlcCer due to its efficacy in relieving atopic skin symptoms. However, the role of GlcCer in inflammation has not been investigated completely. Thus, we hypothesized that GlcCer might exhibit the antiinflammatory effects through the inhibition of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. To test this hypothesis, the antiinflammatory effects and signaling mechanisms of GlcCer were investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. We report that GlcCer inhibited messenger RNA and protein expression of tissue necrosis factor α and interleukin 1ß without cytotoxicity. However, it did not affect interleukin 6 production in LPS-stimulated RAW 264.7 macrophages. Glucosylceramide also suppressed prostaglandin E2 but not nitric oxide production, consistent with its inhibition of cyclooxygenase 2 but not of inducible nitric oxide synthase expression. The molecular mechanism of GlcCer-mediated inhibition of LPS-induced inflammation in RAW 264.7 cells is closely related to suppression of NF-κB p65 subunit nuclear translocation as well as to phosphorylation of extracellular signal-regulated kinase and, in particular, p38 MAPK. In addition, GlcCer did not affect c-Jun N-terminal kinase phosphorylation. In conclusion, GlcCer inhibits LPS-induced inflammation by blocking the nuclear translocation of NF-κB and inhibiting the phosphorylation of extracellular signal-regulated kinase/p38 MAPK pathways in macrophages, suggesting that it might be a promising potential drug candidate for various inflammatory diseases.

Oral administration of squid lecithin-transphosphatidylated phosphatidylserine improves memory impairment in aged rats.

Recently, lecithin-derived phosphatidylserine (PS), which originates from marine life, has received much attention as a viable alternative to bovine cerebral cortex PS. In this study, the use of squid phosphatidylcholine-transphosphatidylated PS (SQ-PS) was evaluated through examination of its ameliorating effects on age-associated learning and memory deficits in rats. Aged rats were orally administered SQ-PS (10, 20, or 50 mg/kg per day) once a day for seven days 30 min prior to behavioral assessment in a Morris water maze. SQ-PS administration produced significant dose-dependent improvements in escape latency for finding the platform in the Morris water maze in the aged rats even though Soy-PS administration also exhibited comparable improvements with SQ-PS. Biochemical alterations in the hippocampal cholinergic system, including changes in choline acetyltransferase and acetylcholinesterase immunoreactivity, were consistent with the behavioral results. In addition, SQ-PS treatment significantly restored age-associated decreases of choline transporter and muscarinic acetylcholine receptor type 1 mRNA expression in the hippocampus. These results demonstrate that orally administered SQ-PS dose-dependently aids in the improvement of memory deficits that occur during normal aging in rats. This suggests that SQ-PS may be a useful therapeutic agent in the treatment of diminished memory function in elderly people.

Administration of glucosylceramide ameliorated the memory impairment in aged mice.

The function and the role of glucosylceramide have not been well studied in the central nervous system. This study was aimed to investigate the possible roles of glucosylceramide in memory function in aged mice. Glucosylceramide (50 mg/kg, p.o.) showed memory enhancing activity after 3-month treatment in the aged mice (C56BL/6, 18-20 months old) through Y-maze, novel objective test, and Morris water maze test. Long-term treatment of glucosylceramide decreased the expression of iNOS and COX-2 in the brain of aged mice. The LPS-induced mRNA level of iNOS, COX-2, IL-1 ß , and TNF- α was reduced by the acute treatment with glucosylceramide in adult mice. These results suggest that glucosylceramide plays an important role in anti-inflammatory and memory enhancement, and it could be a potential new therapeutic agent for the treatment of neurodegenerative diseases such as Alzheimer's disease.

Phosphatidylserine inhibits inflammatory responses in interleukin-1ß-stimulated fibroblast-like synoviocytes and alleviates carrageenan-induced arthritis in rat.

Recently, phosphatidylserine (PS) has received attention for its anti-inflammatory effect; however, the molecular mechanisms of its action have not been fully understood. Thus, we hypothesized that PS might have antiarthritic and anti-inflammatory effects. To test this hypothesis, the in vitro anti-inflammatory effect of soybean-derived PS was tested on interleukin (IL)-1ß-stimulated fibroblast-like synoviocytes from rheumatoid arthritis patients (RA-FLS) by measuring the levels of IL-6, IL-8, prostaglandin E(2), and vascular endothelial growth factor by enzyme-linked immunosorbent assay. The analgesic and antiarthritic activities of PS were investigated in rat models of carrageenan-induced acute paw pain and arthritis. The former was evaluated with a paw pressure test; the latter, by measuring paw volume and weight distribution ratio. In addition, the participation of mitogen-activated protein kinase signaling in the anti-inflammatory and antiarthritic effects of PS was investigated in RA-FLS. Phosphatidylserine inhibited the production of inflammatory mediators IL-6; IL-8; vascular endothelial growth factor; and, in particular, prostaglandin E(2) in IL-1ß-stimulated RA-FLS. These effects were associated with abrogation of inhibitor of nuclear factor-κBα phosphorylation and suppression of p38 and c-jun amino terminal kinase but not extracellular signal-regulated kinase 1/2 phosphorylation. In rats, PS also showed a significant inhibitory effect on arthritic and nociceptive symptoms induced by carrageenan. These findings suggest that PS has anti-inflammatory and antiarthritic effects in vitro and in in vivo animal models; thus, PS should be further studied to determine its potential use as either a pharmaceutical or dietary supplement for alleviating arthritic symptoms.

Enhanced learning and memory of normal young rats by repeated oral administration of Krill Phosphatidylserine.

OBJECTIVE: Phosphatidylserine, a major acidic phospholipid in the brain, has been studied extensively in regard to its actions on brain functions. The present study examined the effects of Krill phosphatidylserine (Krill-PS) on the learning and memory function and the neural activity in the normal young rats. METHODS: The rats were administered saline or Krill-PS (Krill-PS 50, 100 mg/kg, per oral) daily for 30 days. The cognitive improvement effect of Krill-PS on the normal young rats was investigated by assessing the Morris water maze (MWM) test and by insulin-like growth factor (IGF) and brain-derived neurotrophic factor (BDNF) immunohistochemistry. A positron emission tomography (PET) scan was also performed. RESULTS: Treatment with Krill-PS (100 mg/kg) produced a significant improvement of the escape latency to find the platform in the MWM at the 3rd day compared to that of the normal group. In the retention test, the Krill-PS100 group showed markedly increased time spent, distance, and crossing number around the platform compared to that of the normal group. Consistent with the behavioral data, the Krill-PS 100 group was significantly enhanced the BDNF and IGF immuno-positive neurons in the hippocampal CA1. In the PET analysis, the glucose uptake of the Krill-PS-treated groups was increased in the frontal lobe and hippocampus. These results suggest that repeated Krill-PS treatment may be useful for improving the cognitive function via regulation of neuronal growth factor activity.

Oral administration of glucosylceramide ameliorates inflammatory dry-skin condition in chronic oxazolone-induced irritant contact dermatitis in the mouse ear.

BACKGROUND: Irritant contact dermatitis (ICD) is an inflammatory skin disease triggered by exposure to a chemical that is toxic or irritating to the skin. A major characteristic of chronic ICD is an inflammatory dry-skin condition with associated itching. Although glucosylceramide (GlcCer) is known to improve the skin barrier function, its mechanism of action is unknown. OBJECTIVES: Using a mouse model of oxazolone-induced chronic ICD, this study investigated the effects of oral administration of GlcCer on inflammatory dry skin. METHODS: Chronic ICD was induced by repeated application of oxazolone in mice. GlcCer was orally administered once daily throughout the elicitation phase. The beneficial efficacy of GlcCer on cutaneous inflammation was evaluated by assessing ear thickness, lymph node weight, histological findings, and mRNA expression of pro-inflammatory cytokines such as IL-1ß and IL-6. Additionally, parameters of the itch-associated response, including scratching behavior, water content of the skin, and aquaporin-3 levels in the lesional ear, were measured. RESULTS: Oral GlcCer administration significantly suppressed mRNA expression of the pro-inflammatory cytokines IL-1ß and IL-6. GlcCer also suppressed ear swelling, lymph node weight gains, and infiltration of leukocytes and mast cells in ICD mice. In oxazolone-induced ICD mice, GlcCer significantly inhibited irritant-related scratching behavior and dehydration of the stratum corneum, and decreased aquaporin-3 expression. CONCLUSIONS: Our results indicate that GlcCer suppressed inflammation not only by inhibiting cytokine production but also by repairing the skin barrier function, suggesting a potential beneficial role for GlcCer in the improvement of chronic ICD.

Krill-Derived Phosphatidylserine Improves TMT-Induced Memory Impairment in the Rat.

The present study examined the effects of krill-derived phosphatidylserine (Krill-PS) on the learning and memory function and the neural activity in rats with trimethyltin (TMT)-induced memory deficits. The rats were administered vehicle (medium-chain triglyceride: MCT) or Krill-PS (50, 100 mg/kg, p.o.) daily for 21 days. The cognitive improving efficacy of Krill-PS in TMT-induced amnesic rats was investigated by assessing the Morris water maze test and by performing choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and cAMP responsive element binding protein (CREB) immunohistochemistry. The rats with TMT injection showed impaired learning and memory of the tasks and treatment with Krill-PS produced a significant improvement of the escape latency to find the platform in the Morris water maze at the 2(nd) and 4(th) day compared to that of the MCT group (p<0.05). In the retention test, the Krill-PS+MCT groups showed increased time spent around the platform compared to that of the MCT group. Consistent with the behavioral data, Krill-PS 50+MCT group significantly alleviated the loss of acetylcholinergic neurons in the hippocampus and medial septum compared to that of the MCT group. Treatment with Krill-PS significantly increased the CREB positive neurons in the hippocampal CA1 area as compared to that of the MCT group. These results suggest that Krill-PS may be useful for improving the cognitive function via regulation of cholinergic marker enzyme activity and neural activity.

Krill phosphatidylserine improves learning and memory in Morris water maze in aged rats.

The ameliorating effect of phosphatidylserine (PS) isolated from krill (KR-PS) on the learning and memory deficits associated with normal aging in rats was investigated, as compared with soybean PS (SOY-PS). Rats were orally administered with KR-PS (20, 50 mg kg-1) and SOY-PS (50 mg kg-1) daily, for 7 days, 30 min before behavioral assessment using the Morris water maze (MWM). Changes in the cholinergic system were examined by measuring choline acetyltransferase (ChAT) and acetylcholinesterase (AchE) immunoreactivity in the hippocampus. The daily administration of KR-PS produced a significant improvement in the escape latency for finding the platform in the MWM, as compared with SOY-PS. Consistent with the behavioral results, KR-PS treatments significantly alleviated age-associated losses of cholinergic immunoreactivity, and muscarinic acetylcholine receptor type 1 (mAChR-M1) and choline transporter (CHT) mRNA expression in the hippocampus. These findings demonstrate that KR-PS showed significant neuroprotective activity against the neuronal and cognitive impairments that occur with normal aging in rats; comparable results were obtained with SOY-PS. These data indicate that oral administration of PS derived from marine life could substitute for bovine cerebral cortex PS (BC-PS) as therapy for the improvement of diminished memory function in elderly people.

Phosphatidylserine prevents UV-induced decrease of type I procollagen and increase of MMP-1 in dermal fibroblasts and human skin in vivo.

In an effort to find topical agents that prevent or retard cutaneous aging, seven functional lipids were screened for their procollagen-upregulating and matrix metalloproteinase (MMP)-1-downregulating activities in human dermal fibroblasts by Western blotting. The preventive effect on ultraviolet (UV)-induced decrease of procollagen was demonstrated in phosphatidylserine (PS), lysophosphatidylserine (LPS), lysophosphatidic acid (LPA), N-acetyl phytosphingosine (NAPS), and tetraacetyl phytosphingosine (TAPS). Furthermore, PS, LPS, and LPA upregulated procollagen expression in unirradiated basal conditions. The inhibitory effect on UV-induced MMP-1 expression was seen in NAPS, TAPS, LPA, PS, lysophosphatidylglycerol, and LPS. PS was chosen as the most suitable candidate anti-aging chemical for the subsequent in vivo studies. We investigated the effects of PS on acute UV response and chronologic skin aging by topically applying it to young skin before UV irradiation and to aged human skin, respectively. Real-time PCR and Western blot revealed that in the young skin, PS treatment prevented UV-induced reduction in procollagen expression and inhibited UV-induced MMP-1 expression. PS also blocked UV-induced IL-6 and COX-2 gene expression in cultured fibroblasts dose-dependently. In the aged skin, PS caused increased procollagen transcription and procollagen immunostaining in the upper dermis, and a significant decrease in MMP-1 expression at both mRNA and protein levels. These results indicate that topical PS has anti-skin-aging properties and point to the potential use of PS as a therapeutic agent in the prevention and treatment of cutaneous aging.