Safety assessment of a novel, dietary pyrroloquinoline quinone disodium salt (mnemoPQQ®).

Pyrroloquinoline quinone (PQQ), a potent coenzyme antioxidant naturally occurring in foods, has been demonstrated to protect brain cells by enhancing the expression of nerve growth factors (NGF) and NGF receptors, and suppressing the fibril formation and aggression of amyloid ß. We developed mnemoPQQ®, a novel PQQ disodium salt and assessed its safety in GLP compliant toxicity studies. Acute toxicity studies of mnemoPQQ® in Wistar rats revealed that its LD50 was 1825- and 1410 mg/kg body weight (bw) in male and female rats, respectively, whereas its acute dermal LD50 was >2000 mg/kg bw. mnemoPQQ® was found to be nonirritant to the skin of rabbit in an acute dermal irritation/corrosion study, and classified mnemoPQQ® as a nonirritant to the eye of rabbit in an acute eye irritation/corrosion study. Ames bacterial reverse mutation assay and in vitro Mammalian cell gene mutation test exhibited its non-mutagenic potential. In mammalian in vivo erythrocyte micronucleus test, mnemoPQQ® was classified as non-clastogenic and non-mutagenic. A 90-day sub-chronic toxicity study, conducted at and up to the highest daily dose of 600 mg/kg body weight, revealed no evidence of systemic toxicity. All rats survived the treatment without any significant abnormal clinical signs and alterations in hematology, clinical chemistry, neurological evaluation, thyroid functions, reproductive hormone levels, sperm evaluations, vaginal cytology, endocrine functions, organ weight and gross and microscopic pathology findings. No observed adverse effect level (NOAEL) of mnemoPQQ® was found to be greater than 600 mg/kg body weight. These studies affirm that mnemoPQQ® has broad spectrum safety for human consumption.

An overview of a novel, water-soluble undenatured type II collagen (NEXT-II).

BACKGROUND: Osteoarthritis, the most common form of arthritis, is a crippling, chronic debilitating bone disease that commonly affects humans, dogs, and horses. Inflammation and inflammatory responses are key factors for causing swelling, redness, pain, and loss of movement in arthritic animals and humans. METHODS AND RESULTS: We developed a novel, water-soluble, undenatured type II collagen (NEXT-II) for osteoarthritis. NEXT-II demonstrated broad-spectrum safety and nonmutagenicity. NEXT-II exhibited significant efficacy in ameliorating pain and inflammation in collagen-induced arthritis in mice. NEXT-II enhanced the proportion of CD4+CD25+T cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cell such as forkhead box p3, transforming growth factor-ß1, and CD25. Furthermore, NEXT-II was assessed in moderately arthritic dogs receiving either placebo or 10 mg NEXT-II over a period of 150 days. NEXT-II exhibited a significant reduction in overall pain, pain after limb manipulation, and pain after physical exertion compared to the control dogs. Physical health and serum chemistry (alanine aminotransferase, blood urea nitrogen, and creatine kinase) were not altered when these arthritic dogs were treated over a period of 150 days. CONCLUSIONS: These results demonstrate the broad-spectrum safety and efficacy of NEXT-II in ameliorating the symptoms of arthritis. Key Teaching Points: •A novel, water-soluble, undenatured type II collagen (NEXT-II) was developed for osteoarthritis. •The safety studies including acute oral and dermal toxicity, primary dermal and primary eye irritation, Ames' bacterial reverse mutation assay, mouse lymphoma assay, and 150-day long-term safety studies were conducted. •NEXT-II exhibited significant efficacy in ameliorating pain and inflammation in collagen-induced arthritis in mice. •NEXT-II exhibited a significant reduction in overall pain in moderately arthritic dogs without changing physical parameters.

Hepatoprotective effect of germanium-containing Spirulina in rats with (D)-galactosamine- and lipopolysaccharide-induced hepatitis.

In the present study, the protective effects of dietary Spirulina (SP) and germanium-containing Spirulina (GeSP) were compared in rats with liver injury induced by an intraperitoneal injection of d-galactosamine and lipopolysaccharide (GalN/LPS). Wistar rats were fed one of the following diets: the basal diet (GalN/LPS-CON group; n 6), the basal diet supplemented with 5 % SP or GeSP (GalN/LPS-SP and GalN/LPS-GeSP group, respectively; n 7 each). After administering these diets for 7 d, each rat was intraperitoneally injected with GalN/LPS. Increases in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were suppressed in the GalN/LPS-GeSP group (GalN/LPS-CON v. GalN/LPS-GeSP: ALT 1052 (sem 187) v. 509 (sem 88) IU/l and AST 2183 (sem 368) v. 1170 (sem 196) IU/l) following the injection of GalN/LPS. Plasma levels of interferon-γ (IFN-γ) and TNF-α in GeSP-fed rats were significantly lower when compared with those in the GalN/LPS-CON group (GalN/LPS-CON v. GalN/LPS-GeSP: IFN-γ 142·8 (sem 17·5) v. 66·8 (sem 9·7) pg/ml and TNF-α 72·3 (sem 15·4) v. 31·2 (sem 6·8) pg/ml). However, the decrease in these levels observed in the GalN/LPS-SP group was not as prominent as those observed in the GalN/LPS-GeSP group. Furthermore, the increase in liver catalase (CAT) and glutathione peroxidase (GPx) activities, as well as the level of oxidised glutathione (GSSG), was more suppressed in GeSP-fed rats (GalN/LPS-CON v. GalN/LPS-GeSP: CAT 457 (sem 47) v. 262 (sem 54) U/mg liver protein; GPx 1·30 (sem 0·11) v. 0·53 (sem 0·09) U/mg liver protein; GSSG 2·18 (sem 0·33) v. 1·31 (sem 0·24) mmol/kg liver) after the injection of GalN/LPS. These changes were more pronounced in the GalN/LPS-GeSP group than in the GalN/LPS-SP group. These results suggest that GeSP could afford a significant protective effect in the alleviation of GalN/LPS-induced hepatic damage. In addition, the results indicate that GeSP is more effective than SP.

Safety and toxicological evaluation of a novel, water-soluble undenatured type II collagen.

This study was conducted to determine the broad-spectrum safety of a novel, water-soluble undenatured type II collagen (NEXT-II) derived from chicken sternum cartilage. The presence of epitope in NEXT-II was confirmed by using a commercial kit. The acute oral LD50 of NEXT-II was found to be greater than 5000 mg/kg bw in rats, while the single-dose acute dermal LD50 was greater than 2000 mg/kg bw. The primary dermal irritation index (PDII) of NEXT-II was found to be 1.8 and classified as slightly irritating to the skin. In primary eye irritation studies, the maximum mean total score (MMTS) of NEXT-II was observed to be 7.3 and classified as minimally irritating to the eye. Long-term safety studies were conducted in dogs over a period of 150 d, and no significant changes were observed in body weight, heart rate, respiration rate and blood chemistry. NEXT-II does not induce mutagenicity in the bacterial reverse mutation test in five Salmonella typhimurium strains either with or without metabolic activation. Furthermore, two experiments were conducted to assess the potential of NEXT-II to induce mutations with and without metabolic activation at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. No biologically relevant increase of mutants was observed. Also, no dose-dependent toxicity was observed. Furthermore, colony sizing showed no clastogenic effects induced by NEXT-II under the experimental conditions. These studies demonstrated the broad spectrum of safety of NEXT-II.

Efficacy and Safety of Dietary Undenatured Type II Collagen on Joint and Motor Function in Healthy Volunteers: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study.

Prevalence of osteoarthritis (OA) is increasing alarmingly worldwide. Slowing down the progression of OA and diverse locomotive organ disorders is gaining interest in improving the quality of life (QOL) and extending healthy life-span. In a pilot study, intake of a small amount of undenatured type II collagen exhibited suppression of damage to the articular cartilage via oral immune tolerance. It also demonstrated improvement of knee and joint flexibility and mobility with continued intake of undenatured type II collagen (NEXT-II®) derived from chicken sternum cartilage. This randomized, double-blind, placebo-controlled, parallel-group clinical investigation (RCT) evaluated the efficacy and safety of 12 weeks of regular intake of NEXT-II® on joint and motor function in healthy Japanese male and female participants (aged 20 to <75 years).Sixty-four participants were randomized to receive either NEXT-II® (undenatured type II collagen 3.2 mg/d) or placebo over a period of 12 consecutive weeks. Efficacy on joint and motor functions were evaluated measuring knee passive range of motion as the primary outcome; the Japan Knee Osteoarthritis Measure (JKOM), Visual Analog Scale (VAS) for knee discomfort, and motor functions (10-meter walking and stair-climbing test) as the secondary outcomes; and Japan Low back pain Evaluation Questionnaire (JLEQ) and VAS for lower back discomfort as the exploratory outcomes.Fifty-eight participants (placebo = 28; NEXT-II® group = 30) completed the study. In the assessment of knee passive range of motion, significant improvements in "flexion" and "flexible angle (range)" were observed in the NEXT-II® group at 4, 8, and 12 weeks of treatment. NEXT-II® induced significant improvements in JKOM, VAS for knee and lower back discomfort, 10-meter walking test, stair-climbing test, and JLEQ.Results demonstrate that undenatured type II collagen is safe and efficacious in improving knee flexibility and mobility, reducing knee and lower back pain, and enhancing motor function.

Effect of Dietary Pyrroloquinoline Quinone Disodium Salt on Cognitive Function in Healthy Volunteers: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study.

BACKGROUND: Cognitive dysfunctions are increasing alarmingly around the world, and researchers are exploring preventive measures for improving brain performance. Pyrroloquinoline quinone (PQQ), a naturally occurring coenzyme in foods, exhibits potent antioxidant activity, and improves diverse functions which include mitochondrial activation, growth, repair, protection of nerve cells by increased expression of nerve growth factor (NGF) and NGF receptors; and suppression of fibril formation and aggregation of amyloid ß. OBJECTIVE: This randomized, double-blind, placebo-controlled, parallel-group clinical investigation (RCT) evaluated the efficacy and safety of PQQ disodium salt powder (mnemoPQQ®) for improved cognitive function after 12 weeks of supplementation in healthy Japanese male and female (age 40 to <80 Y). METHODS: 64 healthy subjects were randomly assigned to receive either mnemoPQQ® (PQQ disodium salt: 21.5 mg/day) or a placebo over a period of 12 weeks. The efficacy of mnemoPQQ® on cognitive performance (memory, attention, judgment, and cognitive flexibility) was examined using Cognitrax as the primary outcome (primary endpoint), and forgetfulness questionnaire (DECO: Deterioration Cognitive Observee) and Mini-Mental State Examination-Japanese (MMSE-J) as the secondary outcome (secondary endpoint). RESULTS: A total of 58 subjects (placebo = 31; Age = 70.91 ± 3.06 Y; mnemoPQQ® group = 27; Age = 72.10 ± 3.77 Y) completed the study over a period of 12 weeks of supplementation. Significant improvements were observed on the Cognitrax's cognitive function domain score on "composite memory", "verbal memory", "reaction time", "complex attention", "cognitive flexibility", "executive function", and "motor speed" in the mnemoPQQ® group as compared to the placebo group. The DECO and the MMSE-J scores were also significantly improved in the mnemoPQQ® group. No adverse events were observed. CONCLUSIONS: Study demonstrates that supplementation of PQQ disodium salt is useful in improving memory, attention, judgment, and cognitive function, in middle-aged to elderly population, who feel they have become more forgetful because of aging.

Water-soluble undenatured type II collagen ameliorates collagen-induced arthritis in mice.

Earlier studies have reported the efficacy of type II collagen (C II) in treating rheumatoid arthritis (RA). However, a few studies have investigated the ability of the antigenic collagen to induce oral tolerance, which is defined as active nonresponse to an orally administered antigen. We hypothesized that water-soluble undenatured C II had a similar effect as C II in RA. The present study was designed to examine the oral administration of a novel, water-soluble, undenatured C II (commercially known as NEXT-II) on collagen-induced arthritis (CIA) in mice. In addition, the underlying mechanism of NEXT-II was also identified. After a booster dose (collagen-Freund's complete adjuvant), mice were assigned to control CIA group, or NEXT-II treatment group, to which saline and NEXT-II were administered, respectively. The arthritis index in the NEXT-II group was significantly lower compared with the CIA group. Serum IL-6 levels in the NEXT-II group were significantly lower compared with the CIA group, while serum IL-2 level was higher. Furthermore, oral administration of NEXT-II enhanced the proportion of CD4+CD25+T (Treg) cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cells such as forkhead box p3 (Foxp3), transforming growth factor (TGF)-ß1, and CD25. These results demonstrated that orally administered water-soluble undenatured C II (NEXT-II) is highly efficacious in the suppression of CIA by inducing CD4+CD25+ Treg cells.

Anti-obesity effects of onion extract in Zucker diabetic fatty rats.

Anti-obesity effects of onion extract were determined in obesity and diabetes-prone Zucker diabetic fatty rats by measuring the efficacy of markers concerned with diabetes and obesity. Body and adipose tissue weights in 5% of onion extract-fed group were found to be significantly lower than the control group without onion extract. Fasting blood glucose and HOMA-IR levels were also improved, although the serum insulin and leptin levels did not show any remarkable difference. Serum triglyceride and free fatty acid levels in both the 3% and 5%-fed group were found to be reduced compared to the control group. Additionally the feeding of the onion extract increased the glucose tolerance. These results suggest that dietary onion extract is beneficial for improving diabetes by decreasing lipid levels. We also examined differentiation ability of rat white preadipocyte cells using the onion extract and its sulfur-containing components. Cycloalliin, S-methyl-L-cysteine, S-propyl-L-cysteine sulfoxide, dimethyl trisulfide, especially S-methyl-L-cysteine sulfoxide were reported to be effective in inhibiting formation of oil drop in the cells, suggesting that these compounds may be involved in the anti-obesity effect of the onion extract.