Free Linoleic Acid and Oleic Acid Reduce Fat Digestion and Absorption In Vivo as Potent Pancreatic Lipase Inhibitors Derived from Sesame Meal.

Pancreatic lipase catalyzes the cleavage of triacylglycerols at the oil-water interface, and is known as the dominant determiner of dietary fat digestion. Reducing dietary fat digestion and absorption by modulating the activity of pancreatic lipase has become a favorable strategy to tackle obesity. Orlistat is, at present, the only pancreatic lipase inhibitor approved for the treatment of obesity; however, an array of gastrointestinal adverse effects associated with orlistat limits its tolerability. As a safe alternative to orlistat, a number of natural product-derived compounds with varying degrees of pancreatic lipase inhibitory activity have been reported. We herein reported that bioactivity-guided fractionation of sesame meal led to the identification of free linoleic acid and oleic acid as potent inhibitors of porcine pancreatic lipase in vitro with an IC50 of 23.1 µg/mL (82.4 µM) and 11.7 µg/mL (41.4 µM), respectively. In rats, a single oral dose of the mixture of these fatty acids significantly suppressed the elevation of blood triacylglycerol level following fat intake. These results substantiate the role of free linoleic acid and oleic acid as a novel class of natural product-derived functional molecules that act as pancreatic lipase inhibitors, and their potential for healthy, routine-based weight management.

Effects of Free Linoleic Acid and Oleic Acid in Sesame Meal Extract as Pancreatic Lipase Inhibitors on Postprandial Triglyceridemia: A Randomized, Double-Blind, Placebo-Controlled, Crossover Study in Healthy Volunteers.

A great number of chemically diverse pancreatic lipase (PL) inhibitors have been identified to tackle obesity; however, very few of them have entered clinical studies. The ethanolic extract of sesame meal is a potent PL inhibitor, and its activity hinges exclusively on two free fatty acids: linoleic acid and oleic acid, which were proven to reduce postprandial triglyceride excursion in rats. Herein, to investigate the clinical efficacy of the sesame meal extract, in a crossover trial, 30 healthy volunteers were randomized to receive the sesame meal extract containing experimental food or placebo along with a high-fat meal. Treatment with the sesame meal extract significantly lowered the incremental postprandial serum triglyceride concentration and reduced the incremental area under the curve (iAUC) by 16.8% (p-value = 0.03) compared to placebo. Significant decreases in postprandial remnant-like lipoprotein particle cholesterol and low-density lipoprotein particles were also observed, whereas high-density lipoprotein cholesterol was increased. These results suggest that treatment with the sesame meal extract significantly reduced the postprandial excursion of triglycerides and improved the lipidemic profile after high dietary fat intake in healthy individuals, indicating the substantial potential of free linoleic acid and oleic acid and natural products rich in these compounds for the management of obesity and related conditions.

Dietary Gamma-Aminobutyric Acid (GABA) Induces Satiation by Enhancing the Postprandial Activation of Vagal Afferent Nerves.

Gamma-aminobutyric acid (GABA) is present in the mammalian brain as the main inhibitory neurotransmitter and in foods. It is widely used as a supplement that regulates brain function through stress-reducing and sleep-enhancing effects. However, its underlying mechanisms remain poorly understood, as it is reportedly unable to cross the blood-brain barrier. Here, we explored whether a single peroral administration of GABA affects feeding behavior as an evaluation of brain function and the involvement of vagal afferent nerves. Peroral GABA at 20 and 200 mg/kg immediately before refeeding suppressed short-term food intake without aversive behaviors in mice. However, GABA administration 30 min before refeeding demonstrated no effects. A rise in circulating GABA concentrations by the peroral administration of 200 mg/kg GABA was similar to that by the intraperitoneal injection of 20 mg/kg GABA, which did not alter feeding. The feeding suppression by peroral GABA was blunted by the denervation of vagal afferents. Unexpectedly, peroral GABA alone did not alter vagal afferent activities histologically. The coadministration of a liquid diet and GABA potentiated the postprandial activation of vagal afferents, thereby enhancing postprandial satiation. In conclusion, dietary GABA activates vagal afferents in collaboration with meals or meal-evoked factors and regulates brain function including feeding behavior.

Orally administrable peptides derived from egg yolk promote skeletal repair and ameliorate degenerative skeletal disorders in mouse models.

Introduction: Aging, genetic mutations, and other pathological conditions cause impairment of skeletal growth and bone metabolism, which affect activities of daily living and quality of life in all life stages. Although several drugs have been used in clinical settings and new drugs have been developed for the treatment of skeletal degenerative disorders such as osteoporosis and genetic disorders such as osteogenesis imperfecta (OI), there is clear demand for development of new drugs, especially orally available anabolic drugs that are applicable for a wide range of skeletal disorders. Methods: To identify therapeutic candidates for skeletal disorders, peptide screening was performed. To validate the identified peptides, we performed a bone histomorphometric analysis with rat bone tissues and in vitro cell proliferation assays of skeletal cells. To understand the metabolism of the peptides, we performed a biochemical analysis, followed by in vitro assays for proliferation and differentiation of skeletal cells. We examined the therapeutic efficacy of the identified peptides with several mouse models representing skeletal disorders including bone fracture, osteoporosis, and osteogenesis imperfecta. In vivo therapeutic effects of the candidate were assessed with radiological analysis and mechanical property tests. Results: We identified the egg yolk-derived functional peptide PF201. PF201 promoted in vivo bone formation in rodents and enhanced proliferation of osteoblasts and chondrocytes in vitro. D2, a metabolite of PF201, was present and circulated after digestion and absorption in the digestive tract. D2 had positive impacts on the proliferation and differentiation of mesenchymal stem cells and preosteoblasts. Oral administration of D2 accelerated bone healing in a mouse fracture model. D2 also improved bone strength and fracture healing under ovariectomy-induced osteoporotic conditions in mice, and D2 showed a therapeutic effect in a mouse OI model. Conclusion: D2 is likely to be a candidate for an orally available therapeutic for a range of skeletal disorders.

Exosome-Mediated Activation of Neuronal Cells Triggered by γ-Aminobutyric Acid (GABA).

γ-Aminobutyric acid (GABA) is a potent bioactive amino acid, and several studies have shown that oral administration of GABA induces relaxation, improves sleep, and reduces psychological stress and fatigue. In a recent study, we reported that exosomes derived from GABA-treated intestinal cells serve as signal transducers that mediate brain-gut interactions. Therefore, the purpose of this study was to verify the functionality of GABA-derived exosomes and to examine the possibility of improving memory function following GABA administration. The results showed that exosomes derived from GABA-treated intestinal cells (Caco-2) activated neuronal cells (SH-SY5Y) by regulating genes related to neuronal cell functions. Furthermore, we found that exosomes derived from the serum of GABA-treated mice also activated SH-SY5Y cells, indicating that exosomes, which are capable of activating neuronal cells, circulate in the blood of mice orally administered GABA. Finally, we performed a microarray analysis of mRNA isolated from the hippocampus of mice that were orally administered GABA. The results revealed changes in the expression of genes related to brain function. Gene Set Enrichment Analysis (GSEA) showed that oral administration of GABA affected the expression of genes related to memory function in the hippocampus.

Effects of Banafine® , a fermented green banana-derived acidic glycoconjugate, on influenza vaccine antibody titer in elderly patients receiving gastrostomy tube feeding.

Immunosenescence can negatively affect cytokine production in elderly and may impair poor antibody responses to influenza vaccination and infection. Herein, the effects of Banafine® administration on influenza vaccine antibody titer in elderly patients (average age ∼80 years) receiving gastrostomy tube feeding were examined. In the double-blind, single-center, randomized clinical studies, 30 elderly bedridden patients were administered Banafine® or placebo for 8 weeks. At week 4, all patients received influenza vaccination against H1N1, H3N2, B/Yamagata, or B/Victoria. Blood biochemical indices and serum antibody titers were assessed. Banafine® administration significantly increased hemagglutination inhibition titers in response to vaccination against H1N1, H3N2, and B/Yamagata in the elderly patients (P < 0.05). Moreover, the seroconversion rate against H1N1 (47.1%) and H3N2 (29.4%) and seroprotection rate against H1N1 (71.4%) and both B strains (31.3% and 12.5%, respectively) were increased for the Banafine® group. These results suggest that Banafine® administration can increase antibody responses to influenza vaccination in bedridden hospitalized patients, and potentially modulate immune function in the elderly. PRACTICAL APPLICATION: Literature review suggested that most of the synbiotics are based on innate immunity, strain specific (probiotics), and are not consistently observed. Herein, in clinical studies we demonstrate that administration of Banafine® , a plant-based glycoconjugate, can increase antibody levels in bedridden hospitalized elderly patients following influenza vaccination.

Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination.

The aberrant upregulation of protein arginine deiminase 2- (PAD2-) catalyzed citrullination is reported in various autoimmune diseases (rheumatoid arthritis and multiple sclerosis) and several cancers. Currently, there are no anti-PAD2 monoclonal antibodies (mAbs) that can inhibit the citrullination reaction. Here, an epitope 341YLNRGDRWIQDEIEFGY357 was examined as an antigenic site of PAD2. Chickens were immunized with this epitope, and the generated mAbs were screened for its reactivity against the full-length PAD2. Enzyme-linked immunosorbent assay revealed that six mAbs, which were screened from the phage display library, crossreacted with mouse PAD2. Kinetic analysis revealed that mAbs are bound to PAD2 in the nanomolar range, which indicated a strong binding. Results of the in vitro citrullination inhibition assay revealed that the half-maximal effective concentration values of mAbs for the inhibition of histone or benzoyl-L-arginine ethyl ester citrullination were in the range of 6-75 nM which supports strong inhibition capabilities. Alanine scanning of epitope revealed that the peptide fragment 344RGDRWIQDEIEF355 was responsible for generating strong antibody responses that inhibit the PAD2-catalyzed citrullination reaction. These antibodies can aid in understanding the extracellular PAD2 function and treating diseases associated with aberrant citrullination.

γ-Aminobutyric acid (GABA) activates neuronal cells by inducing the secretion of exosomes from intestinal cells.

The oral administration of γ-aminobutyric acid (GABA) has been shown to affect brain functions. However, the molecular mechanisms underlying GABA-induced gut-brain interactions have not yet been fully elucidated. As the blood-brain barrier is impermeable to GABA, we hypothesized that the gut-brain interaction might be stimulated by some secretory factors derived from the gut. Then we focused on exosomes as a secretory mediator. In the present study, we investigated whether exosomes derived from GABA-treated intestinal cells activate neuronal cells. Our results revealed that secretory factors derived from GABA-treated intestinal cells (Caco-2) activated neuronal cells (SH-SY5Y). Further investigation revealed that exosomes derived from GABA-treated Caco-2 cells were responsible for activating the SH-SY5Y cells. These results suggested that GABA-activated intestinal cells induce the secretion of exosomes that activate neuronal cells. MicroRNAs in the exosomes derived from GABA-treated intestinal cells may play a key role in the activation of neuronal cells.

Inhibitory effects of oligopeptides from hen egg white on both human platelet aggregation and blood coagulation.

Egg white proteins have many biological functions and substantial nutritional benefits when used as a food source; however, they also contain allergens such as ovalbumin, ovomucoid, and ovotransferrin. We prepared oligopeptides without allergens from hen egg whites via the use of several proteases, and assessed their effects on platelet aggregation and blood coagulation, known to both of which are known to be major risk factors in thrombogenesis. Egg white oligopeptides (EWOP) inhibited collagen-induced human platelet aggregation in a dose-dependent manner. Additionally, we attempted to determine whether cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), aggregation-inhibiting intracellular molecules, regulate EWOP-inhibited platelet aggregation. EWOP caused an increase in cAMP levels, but did not affect cGMP levels, which suggests that the anti-platelet activity of EWOP operates in a cAMP-dependent manner, rather than via a cGMP-dependent process, in collagen-induced platelet aggregation. In addition, EWOP induced a significantly prolonged prothrombin time (PT) as compared with the controls. These data show that EWOP inhibits the conversion of fibrinogen to fibrin in a plasmatic atmosphere on an extrinsic pathway. Accordingly, these findings suggest that EWOP may be an excellent candidate as a crucial inhibitor of platelet activation, and its anti-platelet effects appear to involve the inhibition of both platelet aggregation and blood coagulation within the cardiovascular system.

Study of the cartilage matrix production-promoting effect of chicken leg extract and identification of the active ingredient.

BACKGROUND/OBJECTIVES: Osteoarthritis (OA) is a major public health issue in Japan and other countries, and foods that prevent or treat OA are in strong demand. Proteins and peptides in chicken meat and bones are known for being rich in functional and nutritional ingredients for the improvement of osteoporosis. We speculated that chicken legs, a food consumed in many regions of the world, may also contain such ingredients. In this study, we aim to (i) evaluate the effect of chicken leg extract (CLE) on the promotion of cartilage matrix production and (ii) identify the active ingredient in CLE that contributes to this function. MATERIALS/METHODS: Artificial CLE digest was prepared, and the acid mucopolysaccharide production-promoting activity of the CLE digest was evaluated by alcian blue staining of ATDC5 cells. CLE was orally administered to rabbits with burr holes in the knee joint of the femur, and the degree of regeneration of cartilage matrix was evaluated. Furthermore, we investigated orally administered CLE-derived peptides in human plasma using LC-MS. From measuring the acid mucopolysaccharide production-promotion activity of these peptides, a molecule considered to be an active ingredient in the CLE digest was identified. RESULTS: CLE digest promoted acid mucopolysaccharide production and facilitated regeneration of cartilage matrix in in vitro and in vivo experiments. Four peptides including phenylalanyl-hydroxyproline (Phe-Hyp) were detected as CLE-derived peptides in human plasma. The effect of CLE was inferred to be due to Phe-Hyp, which was confirmed to be present in the CLE digest. CONCLUSIONS: It was shown that CLE stimulated the production of articular cartilage matrix both in vitro and in vivo, and that CLE could be an effective food for preventing or treating OA. Furthermore, only Phe-Hyp was confirmed as the active compound in the CLE digest, suggesting that the activity of CLE was due to Phe-Hyp.

Oral Supplementation Using Gamma-Aminobutyric Acid and Whey Protein Improves Whole Body Fat-Free Mass in Men After Resistance Training.

BACKGROUND: Oral gamma-aminobutyric acid (GABA) supplementation increases growth hormone (GH) serum levels and protein synthesis. Therefore, post-exercise supplementation using GABA and protein may help enhance training-induced muscle hypertrophy. We evaluated whether GABA with whey protein enhanced muscular hypertrophy in men after progressive resistance training. METHODS: Twenty-one healthy men (26 - 48 years) were randomized to receive whey protein (WP; 10 g) or whey protein + GABA (WP + GABA; 10 g + 100 mg) daily for 12 weeks. Both groups performed resistance training twice per week (three sets of 12 repetitions at 60% of maximal strength; leg press, leg extension, leg curl, chest press, and pull down). Body composition was assessed using dual-energy X-ray absorptiometry. RESULTS: In the WP + GABA group, resting plasma GH concentrations were significantly elevated at 4 and 8 weeks, compared to baseline. However, resting plasma GH concentrations in the WP group were only significantly elevated at 8 weeks. After 12 weeks, the WP + GABA group exhibited significantly greater increase in whole body fat-free mass than the WP group. CONCLUSIONS: The GABA and whey protein combination was more effective for increasing whole body fat-free mass; daily GABA supplementation may help enhance exercise-induced muscle hypertrophy.

Protective effect of lipoic acid against methylglyoxal-induced oxidative stress in LLC-PK(1) cells.

Methylglyoxal (MG), a reactive dicarbonyl compound, is a metabolic byproduct of glycolysis often found at high levels in blood from diabetic patients. The effect of lipoic acid on MG-induced oxidative stress was investigated using LLC-PK(1) renal tubular epithelial cells, which are susceptible to oxidative stress. MG (500 microM) treatment induced LLC-PK(1) cell death to nearly 50% compared with non-treated control cells, but lipoic acid significantly inhibited the MG-induced cytotoxicity in a concentration-dependent manner. In addition, lipoic acid treatment dose-dependently reduced the intracellular reactive oxygen species level increased by 500 microM MG. The nitric oxide level was also increased by 500 microM MG treatment, but it was significantly inhibited by lipoic acid. Furthermore, lipoic acid treatment at 50 microM inhibited the nuclear translocation of nuclear factor-kappa B induced by MG treatment in LLC-PK(1) cells. These findings indicate that lipoic acid has potential as a therapeutic agent against the development of diabetic complications related to MG-induced oxidative stress in diabetes.

Naturally Occurring Hair Growth Peptide: Water-Soluble Chicken Egg Yolk Peptides Stimulate Hair Growth Through Induction of Vascular Endothelial Growth Factor Production.

Alopecia is divided into two categories: androgenic alopecia and nonandrogenic alopecia. An androgen-dependent abnormality of biological functions causes alopecia in males, but the role of androgens is not yet elucidated in female pattern hair loss (FPHL). Modulation of androgenic activity is not effective in certain kinds of androgenic alopecia in females, as well as in cases of nonandrogenic alopecia in males and females. The hair growth drug, minoxidil, stimulates vascular endothelial growth factor (VEGF) production as well as vascularization and hair growth in females. Yet, because minoxidil has side effects with long-term use, a safe alternative hair growth agent is needed. Whereas hair develops after birth in mammalian species, hair mostly grows in a precocial bird, in the chicken, between hatching days 14 and 15. Therefore, we hypothesized that the chicken egg contains a key hair growth factor. In this study, we demonstrated that water-soluble peptides derived from the egg yolk stimulate VEGF production and human hair follicle dermal papilla cell growth. We also found that these peptides enhance murine hair growth and improve hair growth in FPHL. Finally, we characterized that water-soluble egg yolk peptides induce VEGF expression through insulin growth factor-1 receptor activation-induced hypoxia-inducible factor-1α transcription pathway. We have given the name "hair growth peptide (HGP)" to this water-soluble egg yolk peptide.

Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach.

C-type lysozyme (cLZ) is an antimicrobial enzyme that plays a major defense role in many human secretions. Recently, we have identified a helix-loop-helix antimicrobial peptide fragment of cLZ. This finding suggests that processing by coexisting proteases might be a relevant physiological process for generating peptides that contribute to the in vivo mucosal defense role of cLZ. In this study, we found that pepsin, under condition relevant to the newborn stomach (pH 4.0), generated various peptides from cLZ with potent bactericidal activity against several strains of Gram-negative and Gram-positive bacteria. Microsequencing and mass spectral analysis revealed that pepsin cleavage occurred at conserved loops within the alpha-domain of cLZ. We found that the bactericidal domain, which was isolated by gel filtration and reversed-phase HPLC, contains two cationic alpha-helical peptides generated from a helix-loop-helix domain (residues 1-38 of cLZ) by nicking at leucine17. A third peptide consisting of an alpha-helix (residues 18-38) and a two-stranded beta-sheet (residues 39-56) structure was also identified. These peptides share structural motifs commonly found in different innate immune defenses. Functional cellular studies with outer membrane-, cytoplasmic membrane vitality- and redox-specific fluorescence dyes revealed that the lethal effect of the isolated antimicrobial peptides is due to membrane permeabilization and inhibition of redox-driven bacterial respiration. The results provide the first demonstration that pepsin can fine-tune the antimicrobial potency of cLZ by generating multiple antimicrobial peptide motifs, delineating a new molecular switch of cLZ in the mucosal defense systems. Finally, this finding offers a new strategy for the design of antibiotic peptide drugs with potential use in the treatment of infectious diseases.

Protective role of gamma-aminobutyric acid against chronic renal failure in rats.

The protective effect of gamma-aminobutyric acid (GABA) against chronic renal failure (CRF) was investigated using a remnant kidney model with 5/6 nephrectomized rats. Nephrectomy led to renal dysfunction, which was evaluated via several parameters including serum urea nitrogen, creatinine (Cr) and Cr clearance. However, the administration of GABA ameliorated renal dysfunction, and a longer administration period of GABA increased its protective effect. In addition, nephrectomized control rats showed an elevation in the fractional excretion of sodium (FE(Na)) with an increase in urinary sodium, while GABA led to a significant decline in FE(Na). Moreover, nephrectomy resulted in a decrease of serum albumin and an increase of urinary protein with a change in the urinary protein pattern, whereas the rats administered GABA showed improvement in these changes associated with CRF caused by nephrectomy. This suggests that GABA would inhibit the disease progression and have a protective role against CRF. As one of the risk factors for CRF progression, hypertension was also regulated by GABA. The results also indicate that GABA may play a protective role against CRF through improvement of the serum lipid profile, with reductions in triglyceride and total cholesterol. Furthermore, nephrectomy led to renal oxidative stress with a decrease in the activity of antioxidative enzymes and elevation of lipid peroxidation. The administration of GABA attenuated oxidative stress induced by nephrectomy through an increase in superoxide dismutase and catalase, and decrease in lipid peroxidation. The histopathological lesions, including glomerular, tubular and interstitial lesions, under nephrectomy were also improved by GABA with the inhibition of fibronectin expression. This study demonstrated that GABA attenuated renal dysfunction via regulation of blood pressure and lipid profile, and it also ameliorated the oxidative stress induced by nephrectomy, suggesting the promising potential of GABA in protecting against renal failure progression.

Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans.

The effect of orally administrated gamma-aminobutyric acid (GABA) on relaxation and immunity during stress has been investigated in humans. Two studies were conducted. The first evaluated the effect of GABA intake by 13 subjects on their brain waves. Electroencephalograms (EEG) were obtained after 3 tests on each volunteer as follows: intake only water, GABA, or L-theanine. After 60 minutes of administration, GABA significantly increases alpha waves and decreases beta waves compared to water or L-theanine. These findings denote that GABA not only induces relaxation but also reduces anxiety. The second study was conducted to see the role of relaxant and anxiolytic effects of GABA intake on immunity in stressed volunteers. Eight acrophobic subjects were divided into 2 groups (placebo and GABA). All subjects were crossing a suspended bridge as a stressful stimulus. Immunoglobulin A (IgA) levels in their saliva were monitored during bridge crossing. Placebo group showed marked decrease of their IgA levels, while GABA group showed significantly higher levels. In conclusion, GABA could work effectively as a natural relaxant and its effects could be seen within 1 hour of its administration to induce relaxation and diminish anxiety. Moreover, GABA administration could enhance immunity under stress conditions.

Effect of oral γ-aminobutyric acid (GABA) administration on sleep and its absorption in humans.

The effects of γ-aminobutyric acid (GABA) on sleep and its levels in blood after oral administration were investigated in humans. A randomized, single-blind, placebo-controlled crossover-designed study was conducted to evaluate the effect of GABA on sleep. Sleep was evaluated by electroencephalography (EEG) after oral GABA administration. GABA significantly shortened sleep latency and increased the total non-rapid eye movement (non-REM) sleep time. Questionnaires showed that subjects receiving GABA realized its effects on sleep. In addition, the blood level of GABA after administration was investigated, and the absorption and metabolism rates of GABA were determined. GABA was quickly absorbed, and the blood level of GABA was the highest 30 min after oral administration, with a subsequent decrease in concentration. As GABA strongly affected the early stage of sleep, the effect of GABA on sleep may be connected to its levels in blood.

The Improvement of Sleep by Oral Intake of GABA and Apocynum venetum Leaf Extract.

The effects of two food materials, γ-aminobutyric acid (GABA) produced by natural fermentation and Apocynum venetum leaf extract (AVLE), on the improvement of sleep were investigated in humans. The electroencephalogram (EEG) test revealed that oral administration of GABA (100 mg) and AVLE (50 mg) had beneficial effects on sleep. GABA shortened sleep latency by 5.3 min and AVLE increased non-rapid eye movement (REM) sleep time by 7.6%. Simultaneous intake of GABA and AVLE shortened sleep latency by 4.3 min and increased non-REM sleep time by 5.1%. The result of questionnaires showed that GABA and AVLE enabled subjects to realize the effects on sleep. These results mean that GABA can help people to fall asleep quickly, AVLE induces deep sleep, and they function complementarily with simultaneous intake. Since both GABA and AVLE are materials of foods and have been ingested for a long time, they can be regarded as safe and appropriate for daily intake in order to improve the quality of sleep.

Effects of food enriched with egg yolk hydrolysate (bone peptide) on bone metabolism in orchidectomized dogs.

We examined the effects of chicken egg hydrolysate (also known as "bone peptide" or BP) on bone metabolism in 5- to 8-month-old orchidectomized dogs. The bone formation marker serum bone alkaline phosphatase (BAP) and the bone resorption marker urine deoxypyridinoline (DPD) were used as indicators to measure changes in bone metabolism. The following results were observed that Serum BAP was higher in dogs fed BP-enriched food throughout the clinical investigation. Serum BAP was statistically significantly higher in dogs fed BP-enriched food than in dogs fed non-BP-enriched food at 2 months after orchidectomy. This suggests that BP promoted bone formation immediately after orchidectomy.

Activity of (-)-epigallocatechin 3-O-gallate against oxidative stress in rats with adenine-induced renal failure.

Methylguanidine (MG) is widely recognized as a strong uremic toxin. The hydroxyl radical (*OH) specifically plays an important role in the pathway of MG production from creatinine (Cr). In this study, we investigated whether oral administration of (-)-epigallocatechin 3-O-gallate (EGCg) suppresses MG production in rats with chronic renal failure after intraperitoneal Cr injection. MG production from Cr was significantly increased in rats with adenine-induced renal failure, which was more vulnerable to oxidative stress, compared with that in normal rats. However, oral administration of EGCg 30 min before and after Cr injection effectively inhibited MG production. Our findings suggest that EGCg, an excellent antioxidant from green tea, exerts protective activity in rats with chronic renal failure, resulting in suppression of Cr oxidation influenced by *OH.