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.

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.

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.

Trigonelline ameliorates oxidative stress in type 2 diabetic Goto-Kakizaki rats.

Previously, we showed that trigonelline (TRG) exerts antidiabetic effects in type 2 diabetic Goto-Kakizaki (GK) rats and also lowers blood and liver thiobarbituric acid reactive substances and urinary 8-hydroxydeoxyguanosine when compared with those levels in GK control rats without TRG. These results suggested that TRG also mitigates oxidative stress, which accelerates diabetes. In this study, the mechanisms of TRG prevention of oxidative stress were determined by measuring erythrocyte and liver antioxidant enzyme activities, and expressions of genes associated with reactive oxygen species production, and carbohydrate and lipid metabolisms by DNA microarray. Erythrocyte and liver glutathione peroxidase, and liver catalase activities in the GK rats fed with TRG were significantly lower than those of the GK control rats. TRG downregulated the gene expressions involved with NADPH oxidase and mitochondrial electron transfer system when compared with those of the GK control group. These results suggested that mitigation of diabetes by TRG is mediated by its ameliorating effects on oxidative stress.

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.

Anti-diabetic effect of pyroglutamic acid in type 2 diabetic Goto-Kakizaki rats and KK-Ay mice.

With the rapidly increasing prevalence of type 2 diabetes mellitus (T2DM), specific dietary components with anti-diabetic efficacy could be one strategy with therapeutic potential. In the present study, the anti-diabetic effects of an amino acid, pyroglutamic acid (PA), found in vegetables and fruits were investigated in T2DM models using Goto-Kakizaki (GK) rats and KK-Ay mice by measuring glucose tolerance and other markers of diabetes. Moreover, the effect of PA on gene expression in GK rats was measured by DNA microarray analysis. Oral glucose tolerance and serum insulin levels were reduced by PA in both animal models. Serum and liver total cholesterol levels were also improved by PA. Expression of genes involved with gluconeogenesis and those involved with its related transcription factor were down-regulated by feeding PA. In KK-Ay mice, the glucokinase:glucose-6-phosphatase (G6Pase) activity ratio increased. From these results, it is suggested that dietary PA beneficially modifies glucose and lipid metabolism in diabetic animals, and can potentially contribute to the mitigation of T2DM.

Anti-diabetic effects of pumpkin and its components, trigonelline and nicotinic acid, on Goto-Kakizaki rats.

The effects of a pumpkin paste concentrate and its components on oral glucose tolerance and serum lipid levels were determined in non-obese type 2 diabetic Goto-Kakizaki (GK) rats. In the oral glucose tolerance test, the pumpkin paste concentrate-fed group maintained a lower glucose level than the control group between 15 and 60 min. The compounds considered to be effective in improving glucose tolerance and contained in the methanol extract of the pumpkin in relatively abundant amounts were isolated and identified as trigonelline (TRG) and nicotinic acid (NA).Feeding a diet containing TRG and NA respectively improved and tended to improve glucose tolerance. The insulin level increased after 15 min in the TRG-fed GK rats and then gradually decreased over the next 120 min. In contrast, a gradual increase was seen in the insulin level over 120 min in the control GK rats not fed with TRG, suggesting that TRG could improve the insulin resistance. The serum and liver triglyceride (TG) levels in the TRG- and NA-fed GK rats were lower than those in the control GK rats. Lower activity of liver fatty acid synthase (FAS), and higher activity of liver carnitine palmitoyl transferase (CPT) and glucokinase (GLK) in the TRG- and NA-fed GK rats than in the control GK rats were observed. This suggests that the regulation of these enzyme activities by TRG and NA was closely related to the suppression of both TG accumulation and the progression of diabetes.

Effects of dietary catechins on glucose tolerance, blood pressure and oxidative status in Goto-Kakizaki rats.

Effects of green tea catechins comprising EGCg, EGC, ECg, EC, GCg, GC, Cg, and C were determined on blood glucose tolerance and oxidative stress status in type 2 diabetic Goto-Kakizaki (GK) rats. GK rats fed the catechin-containing diet tended to maintain blood glucose and systolic blood pressure at lower levels in the latter stages of the feeding period of 76 d, compared to those not receiving dietary catechins (control group). The blood glucose tolerance test performed on days 48-49 showed that GK rats fed the catechins had lower blood glucose levels than GK rats not fed catechins during the 120 min after glucose loading. In catechin-fed rats, amounts of 8-OH dG and albumin excreted into the urine determined on days 71-72, and kidney ACE activity determined on day 76, were lower than those in control rats. From these results it is concluded that dietary catechins may be effective in delaying the progression of diabetes and the associated oxidative stress.