Effect of Long-Term Green Tea Polyphenol Supplementation on Bone Architecture, Turnover, and Mechanical Properties in Middle-Aged Ovariectomized Rats.

We investigated the effects of 6-month green tea polyphenols (GTP) supplementation on bone architecture, turnover, and mechanical properties in middle-aged ovariectomized (OVX) rats. Female rats were sham-operated (n = 39, 13/group) or OVX (n = 143, 13/group). Sham-control and OVX-control rats (n = 39) receiving no GTP were assigned for sample collection at baseline, 3, or 6 months. The remaining OVX rats (n = 104) were randomized to 0.15%, 0.5%, 1%, and 1.5% (g/dL) GTP for 3 or 6 months. Blood and bone samples were collected. Relative to the OVX-control group, GTP (1% and 1.5%) lowered serum procollagen type 1 N-terminal propeptide at 3 and 6 months, C-terminal telopeptides of type I collagen at 3 months, and insulin-like growth factor-I at 6 months. GTP did not affect bone mineral content and density. At 6 months, no dose of GTP positively affected trabecular bone volume based on microCT, but a higher cortical thickness and improved biomechanical properties of the femur mid-diaphysis was observed in the 1.5% GTP-treated group. At 3 and 6 months, GTP (0.5%, 1%, and 1.5%) had lower rates of trabecular bone formation and resorption than the OVX-control group, but the inhibitory effects of GTP on periosteal and endocortical bone mineralization and formation at the tibial midshaft were only evident at 3 months. GTP at higher doses suppressed bone turnover in the trabecular and cortical bone of OVX rats and resulted in improved cortical bone structural and biomechanical properties, although it was not effective in preventing the ovariectomy-induced dramatic cancellous bone loss.

A 12-week evaluation of annatto tocotrienol supplementation for postmenopausal women: safety, quality of life, body composition, physical activity, and nutrient intake.

BACKGROUND: Evidence suggests that tocotrienols may benefit bone health in osteopenic women. However, their safety in this population has never been investigated. This study was to evaluate the safety of a 12-week supplementation of annato tocotrienol in postmenopausal osteopenic women, along with effects of the supplementation on quality of life, body composition, physical activity, and nutrient intake in this population. METHODS: Eighty nine postmenopausal osteopenic women were randomly assigned to 3 treatment arms: (1) Placebo (430 mg olive oil/day), (2) Low tocotrientol (Low TT) (430 mg tocotrienol/day from DeltaGold 70 containing 300 mg tocotrienol) and (3) High tocotrienol (High TT) (860 mg tocotrienol/day from DeltaGold 70 containing 600 mg tocotrienol) for 12 weeks. DeltaGold 70 is an extract from annatto seed with 70% tocotrienol consisting of 90% delta-tocotrienol and 10% gamma-tocotrienol. Safety was examined by assessing liver enzymes (aspartate aminotransferase, alanine aminotransferase), alkaline phosphatase, bilirubin, kidney function (blood urea nitrogen and creatinine), electrolytes, glucose, protein, albumin, and globulin at 0, 6, and 12 weeks. Serum tocotrienol and tocopherol concentrations were assessed and pills counted at 0, 6, and 12 weeks. Quality of life, body composition, physical activity, and dietary macro- and micro-nutrient intake were evaluated at 0 and 12 weeks. A mixed model of repeated measures ANOVA was applied for analysis. RESULTS: Eighty seven subjects completed the study. Tocotrienol supplementation did not affect liver or kidney function parameters throughout the study. No adverse event due to treatments was reported by the participants. Tocotrienol supplementation for 6 weeks significantly increased serum delta-tocotrienol level and this high concentration was sustained to the end of study. There was no difference in serum delta-tocotrienol levels between the Low TT and the High TT groups. No effects of tocotrienol supplementation were observed on quality of life, body composition, physical activity, and nutrient intake. CONCLUSIONS: Annatto-derived tocotrienol up to 600 mg per day for 12 weeks appeared to be safe in postmenopausal osteopenic women, particularly in terms of liver and kidney functions. Tocotrienol supplementation for 12 weeks did not affect body composition, physical activity, quality of life, or intake of macro- and micro-nutrients in these subjects. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02058420 . TITLE: Tocotrienols and bone health of postmenopausal women.

Effect of annatto-extracted tocotrienols and green tea polyphenols on glucose homeostasis and skeletal muscle metabolism in obese male mice.

Skeletal muscle is the major site for glucose uptake and thus plays an important role in initiating insulin resistance in type 2 diabetes mellitus. This study evaluated the effects of tocotrienols (TT) and green tea polyphenols (GTP) individually or in combination on glucose homeostasis and skeletal muscle metabolism in obese mice with insulin resistance and elevation of blood glucose. Forty-eight male mice were fed a high-fat diet and assigned to 4 groups in a 2 (no TT vs. 400 mg TT/kg diet) × 2 (no GTP vs. 0.5% vol/wt GTP in water) for 14 weeks. Both GTP and TT improved area under curve of insulin intolerance; while GTP increased serum insulin levels in obese mice, probably due to the addition of sweetener in drinking water. An interaction (TT×GTP) was observed in glucose tolerance test, total pancreas insulin concentration, and citrate synthase activity of soleus in mice. Neither TT nor GTP affected insulin and glucagon protein expression in pancreas based on immunohistochemistry. Both TT and GTP individually increased soleus muscle weight of mice; while only GTP increased gastrocnemius muscle weight of mice. The TT+GTP group had the greatest gastrocnemius muscle cross sectional area than other groups. GTP, not TT, induced cytochrome c oxidase activity and reduced thiobarbituric acid reactive substances levels in soleus muscle. Our results suggest that TT and GTP, individually or synergistically have the potential to improve skeletal muscle metabolism in obese mice by improving glucose homeostasis, reducing lipid peroxidation, and increasing rate limiting enzymes of oxidative phosphorylation.

Annatto-extracted tocotrienols improve glucose homeostasis and bone properties in high-fat diet-induced type 2 diabetic mice by decreasing the inflammatory response.

Diabetes is a risk factor for osteoporosis. Annatto-extracted tocotrienols (TT) have proven benefits in preserving bone matrix. Here, we evaluated the effects of dietary TT on glucose homeostasis, bone properties, and liver pro-inflammatory mRNA expression in high-fat diet (HFD)-induced type 2 diabetic (T2DM) mice. 58 male C57BL/6 J mice were divided into 5 groups: low-fat diet (LFD), HFD, HFD + 400 mgTT/kg diet (T400), HFD + 1600 mgTT/kg diet (T1600), and HFD + 200 mg metformin/kg (Met) for 14 weeks. Relative to the HFD group, both TT-supplemented groups (1) improved glucose homeostasis by lowering the area under the curve for both glucose tolerance and insulin tolerance tests, (2) increased serum procollagen I intact N-terminal propeptide (bone formation) level, trabecular bone volume/total volume, trabecular number, connectivity density, and cortical thickness, (3) decreased collagen type 1 cross-linked C-telopeptide (bone resorption) levels, trabecular separation, and structure model index, and (4) suppressed liver mRNA levels of inflammation markers including IL-2, IL-23, IFN-γ, MCP-1, TNF-α, ITGAX and F4/80. There were no differences in glucose homeostasis and liver mRNA expression among T400, T1600, and Met. The order of osteo-protective effects was LFD ≥T1600 ≥T400 = Met >HFD. Collectively, these data suggest that TT exerts osteo-protective effects in T2DM mice by regulating glucose homeostasis and suppressing inflammation.

Effects of delta-tocotrienol on obesity-related adipocyte hypertrophy, inflammation and hepatic steatosis in high-fat-fed mice.

Inflammation is a major underlying cause for obesity-associated metabolic diseases. Hence, anti-inflammatory dietary components may improve obesity-related disorders. We hypothesized that delta-tocotrienol (δT3), a member of the vitamin E family, reduces adiposity, insulin resistance and hepatic triglycerides through its anti-inflammatory properties. To test this hypothesis, C57BL/6J male mice were fed a high-fat diet (HF) with or without supplementation of δT3 (HF+δT3) at 400 mg/kg and 1600 mg/kg for 14 weeks, and they were compared to mice fed a low-fat diet (LF) or HF supplemented with metformin as an antidiabetic control. Glucose tolerance tests were administered 2 weeks prior to the end of treatments. Histology, quantitative polymerase chain reaction and protein analyses were performed to assess inflammation and fatty acid metabolism in adipose and liver tissues. Significant improvements in glucose tolerance, and reduced hepatic steatosis and serum triglycerides were observed in δT3-supplemented groups compared to the HF group. Body and fat pad weights were not significantly reduced in HF+δT3 groups; however, we observed smaller fat cell size and reduced macrophage infiltration in their adipose tissues compared to other groups. These changes were at least in part mechanistically explained by a reduction of mRNA and protein expression of proinflammatory adipokines and increased expression of anti-inflammatory adipokines in HF+δT3 mice. Moreover, δT3 dose-dependently increased markers of fatty acid oxidation and reduced markers of fatty acid synthesis in adipose tissue and liver. In conclusion, our studies suggest that δT3 may promote metabolically healthy obesity by reducing fat cell hypertrophy and decreasing inflammation in both liver and adipose tissue.

Safety Evaluation of Green Tea Polyphenols Consumption in Middle-aged Ovariectomized Rat Model.

This work evaluates chronic safety in middle-aged ovariectomized rats supplemented with different dosages of green tea polyphenols (GTP) in drinking water. The experiment used 6-mo-old sham (n = 39) and ovariectomized (OVX, n = 143) female rats. All sham (n = 39) and 39 of the OVX animals received no GTP treatment and their samples were collected for outcome measures at baseline, 3 mo, and 6 mo (n = 13 per group for each). The remaining OVX animals were randomized into 4 groups receiving 0.15%, 0.5%, 1%, and 1.5% (n = 26 for each) of GTP (wt/vol), respectively, in drinking water for 3 and 6 mo. No mortality or abnormal treatment-related findings in clinical observations or ophthalmologic examinations were noted. No treatment-related macroscopic or microscopic findings were noted for animals administered 1.5% GTP supplementation. Throughout the study, there was no difference in the body weight among all OVX groups. In all OVX groups, feed intake and water consumption significantly decreased with GTP dose throughout the study period. At 6 mo, GTP intake did not affect hematology, clinical chemistry, and urinalysis, except for phosphorus and blood urea nitrogen (increased), total cholesterol, lactate dehydrogenase, and urine pH (decreased). This study reveals that the no-observed-adverse-effect level (NOAEL) of GTP is 1.5% (wt/vol) in drinking water, the highest dose used in this study.

Safety and efficacy of tocotrienol supplementation for bone health in postmenopausal women: protocol for a dose-response double-blinded placebo-controlled randomised trial.

INTRODUCTION: Osteoporosis is a major health concern in postmenopausal women, and oxidative stress contributes to the development of bone loss. Cellular studies and ovariectomised rat model mimicking bone loss in postmenopausal women show the bone-protective effect of tocotrienols (TTs) with antioxidant capability. We aim to access the safety and efficacy of TT consumption for bone health in postmenopausal women. METHODS AND ANALYSIS: In this 12-week randomised double-blinded placebo-controlled trial for the effects of dietary TT supplementation in postmenopausal women, postmenopausal women aged 45 years and older with at least 1 year after menopause and bone mineral density T-score at the spine and/or hip 2.5 or more below the reference values will be randomly assigned to 3 daily supplements: (1) placebo group receiving 860 mg olive oil, (2) low TT group receiving 430 mg of 70% pure TTs (containing 300 mg TT) and (3) high TT group receiving 860 mg of 70% pure TTs (600 mg TT). The primary outcome measure will be urinary N-terminal telopeptide. The secondary outcome measures will be serum bone-specific alkaline phosphatase, receptor activator of nuclear factor-κB ligand, osteoprotegerin, urinary 8-hydroxy-2'-deoxyguanosine and quality of life. At 0, 6 and 12 weeks, the following will be assessed: (1) primary and secondary outcome measures; (2) serum TT and tocopherol concentrations; (3) physical activity and food frequency questionnaires. Liver function will be monitored every 6 weeks for safety. 'Intent-to-treat' principle will be employed for data analysis. A model of repeated measurements with random effect error terms will be applied. Analysis of covariance, χ2 analysis and regression will be used for comparisons. ETHICS AND DISSEMINATION: This study was approved by the Bioethics Committee of the Texas Tech University Health Sciences Center. The findings of this trial will be submitted to a peer-reviewed journal in the areas of bone or nutrition and international conferences. TRIAL REGISTRATION NUMBER: NCT02058420; results.