Effects of Statin and Annatto-extracted Tocotrienol Supplementation on Glucose Homeostasis, Bone Microstructure, and Gut Microbiota Composition in Obese Mice.

BACKGROUND/AIM: This study examined the effects of tocotrienols (TT) in conjunction with statin on glucose homeostasis, bone microstructure, gut microbiome, and systemic and liver inflammatory markers in obese C57BL/6J mice. MATERIALS AND METHODS: Forty male C57BL/6J mice were fed a high-fat diet (HFD) and assigned into four groups in a 2 (no statin vs. 120 mg statin/kg diet)×2 (no TT vs. 400 mg TT/kg diet) factorial design for 14 weeks. RESULTS: Statin and TT improved glucose tolerance only when each was given alone, and only statin supplementation decreased insulin resistance. Consistently, only statin supplementation decreased serum insulin levels and HOMA-IR. Pancreatic insulin was also increased with statin treatment. Statin and TT, alone or in combination, reduced the levels of serum IL-6, but only TT attenuated the increased serum leptin levels induced by a HFD. Statin supplementation increased bone area/total area and connectivity density at LV-4, while TT supplementation increased bone area/total area and trabecular number, but decreased trabecular separation at the distal femur. Statin supplementation, but not TT, reduced hepatic inflammatory cytokine gene expression. Neither TT supplementation nor statin supplementation statistically altered microbiome species evenness or richness. However, they altered the relative abundance of certain microbiome species. Most notably, both TT and statin supplementation increased the relative abundance of Lachnospiraceae UCG-006. CONCLUSION: TT and statin collectively benefit bone microstructure, glucose homeostasis, and microbial ecology in obese mice. Such changes may be, in part, associated with suppression of inflammation in the host.

Tocotrienol-Rich Vitamin E (Tocovid) Improved Nerve Conduction Velocity in Type 2 Diabetes Mellitus Patients in a Phase II Double-Blind, Randomized Controlled Clinical Trial.

Diabetic peripheral neuropathy (DPN) is the most common microvascular complication of diabetes that affects approximately half of the diabetic population. Up to 53% of DPN patients experience neuropathic pain, which leads to a reduction in the quality of life and work productivity. Tocotrienols have been shown to possess antioxidant, anti-inflammatory, and neuroprotective properties in preclinical and clinical studies. This study aimed to investigate the effects of tocotrienol-rich vitamin E (Tocovid SuprabioTM) on nerve conduction parameters and serum biomarkers among patients with type 2 diabetes mellitus (T2DM). A total of 88 patients were randomized to receive 200 mg of Tocovid twice daily, or a matching placebo for 12 months. Fasting blood samples were collected for measurements of HbA1c, renal profile, lipid profile, and biomarkers. A nerve conduction study (NCS) was performed on all patients at baseline and subsequently at 2, 6, 12 months. Patients were reassessed after 6 months of washout. After 12 months of supplementation, patients in the Tocovid group exhibited highly significant improvements in conduction velocity (CV) of both median and sural sensory nerves as compared to those in the placebo group. The between-intervention-group differences (treatment effects) in CV were 1.60 m/s (95% CI: 0.70, 2.40) for the median nerve and 2.10 m/s (95% CI: 1.50, 2.90) for the sural nerve. A significant difference in peak velocity (PV) was also observed in the sural nerve (2.10 m/s; 95% CI: 1.00, 3.20) after 12 months. Significant improvements in CV were only observed up to 6 months in the tibial motor nerve, 1.30 m/s (95% CI: 0.60, 2.20). There were no significant changes in serum biomarkers, transforming growth factor beta-1 (TGFß-1), or vascular endothelial growth factor A (VEGF-A). After 6 months of washout, there were no significant differences from baseline between groups in nerve conduction parameters of all three nerves. Tocovid at 400 mg/day significantly improve tibial motor nerve CV up to 6 months, but median and sural sensory nerve CV in up to 12 months of supplementation. All improvements diminished after 6 months of washout.

Effects of tocotrienols supplementation on markers of inflammation and oxidative stress: A systematic review and meta-analysis of randomized controlled trials.

Studies investigating the effects of tocotrienols on inflammation and oxidative stress have yielded inconsistent results. This systematic review and meta-analysis aimed to evaluate the effects of tocotrienols supplementation on inflammatory and oxidative stress biomarkers. We searched PubMed, Scopus, and Cochrane Central Register of Controlled Trials from inception until 13 July 2020 to identify randomized controlled trials supplementing tocotrienols and reporting circulating inflammatory or oxidative stress outcomes. Weighted mean difference (WMD) and corresponding 95% confidence interval (CI) were determined by pooling eligible studies. Nineteen studies were included for qualitative analysis, and 13 studies were included for the meta-analyses. A significant reduction in C-reactive protein levels (WMD: -0.52 mg/L, 95% CI: -0.73, -0.32, p < 0.001) following tocotrienols supplementation was observed, but this finding was attributed to a single study using δ-tocotrienols, not mixed tocotrienols. There were no effects on interleukin-6 (WMD: 0.03 pg/mL, 95% CI: -1.51, 1.58, p = 0.966), tumor necrosis factor-alpha (WMD: -0.28 pg/mL, 95% CI: -1.24, 0.68, p = 0.571), and malondialdehyde (WMD: -0.42 µmol/L, 95% CI: -1.05, 0.21, p = 0.189). A subgroup analysis suggested that tocotrienols at 400 mg/day might reduce malondialdehyde levels (WMD: -0.90 µmol/L, 95% CI: -1.20, -0.59, p < 0.001). Future well-designed studies are warranted to confirm the effects of tocotrienols on inflammatory and oxidative stress biomarkers, particularly on different types and dosages of supplementation. PROSPERO registration number: CRD42020198241.

Dietary Annatto-Extracted Tocotrienol Reduces Inflammation and Oxidative Stress, and Improves Macronutrient Metabolism in Obese Mice: A Metabolic Profiling Study.

Obesity and its related complications are a world-wide health problem. Dietary tocotrienols (TT) have been shown to improve obesity-associated metabolic disorders, such as hypercholesterolemia, hyperglycemia, and gut dysbiosis. This study examined the hypothesis that the antioxidant capacity of TT alters metabolites of oxidative stress and improves systemic metabolism. C57BL/6J mice were fed either a high-fat diet (HFD control) or HFD supplemented with 800 mg annatto-extracted TT/kg (HFD+TT800) for 14 weeks. Sera from obese mice were examined by non-targeted metabolite analysis using UHPLC/MS. Compared to the HFD group, the HFD+TT800 group had higher levels of serum metabolites, essential amino acids (lysine and methionine), sphingomyelins, phosphatidylcholine, lysophospholipids, and vitamins (pantothenate, pyridoxamine, pyridoxal, and retinol). TT-treated mice had lowered levels of serum metabolites, dicarboxylic fatty acids, and inflammatory/oxidative stress markers (trimethylamine N-oxide, kynurenate, 12,13-DiHOME, and 13-HODE + 9-HODE) compared to the control. The results suggest that TT supplementation lowered inflammation and oxidative stress (oxidized glutathione and GSH/GSSH) and improved macronutrient metabolism (carbohydrates) in obese mice. Thus, TT actions on metabolites were beneficial in reducing obesity-associated hypercholesterolemia/hyperglycemia. The effects of a non-toxic dose of TT in mice support the potential for clinical applications in obesity and metabolic disease.

The Combination of Berberine, Tocotrienols and Coffee Extracts Improves Metabolic Profile and Liver Steatosis by the Modulation of Gut Microbiota and Hepatic miR-122 and miR-34a Expression in Mice.

Non-alcoholic-fatty liver disease (NAFLD) is spreading worldwide. Specific drugs for NAFLD are not yet available, even if some plant extracts show beneficial properties. We evaluated the effects of a combination, composed by Berberis Aristata, Elaeis Guineensis and Coffea Canephora, on the development of obesity, hepatic steatosis, insulin-resistance and on the modulation of hepatic microRNAs (miRNA) levels and microbiota composition in a mouse model of liver damage. C57BL/6 mice were fed with standard diet (SD, n = 8), high fat diet (HFD, n = 8) or HFD plus plant extracts (HFD+E, n = 8) for 24 weeks. Liver expression of miR-122 and miR-34a was evaluated by quantitativePCR. Microbiome analysis was performed on cecal content by 16S rRNA sequencing. HFD+E-mice showed lower body weight (p < 0.01), amelioration of insulin-sensitivity (p = 0.021), total cholesterol (p = 0.014), low-density-lipoprotein-cholesterol (p < 0.001), alanine-aminotransferase (p = 0.038) and hepatic steatosis compared to HFD-mice. While a decrease of hepatic miR-122 and increase of miR-34a were observed in HFD-mice compared to SD-mice, both these miRNAs had similar levels to SD-mice in HFD+E-mice. Moreover, a different microbial composition was found between SD- and HFD-mice, with a partial rescue of dysbiosis in HFD+E-mice. This combination of plant extracts had a beneficial effect on HFD-induced NAFLD by the modulation of miR-122, miR-34a and gut microbiome.

Therapeutic potential of annatto tocotrienol with self-emulsifying drug delivery system in a rat model of postmenopausal bone loss.

Tocotrienol has been shown to prevent bone loss in animal models of postmenopausal osteoporosis, but the low oral bioavailability might limit its use. A self-emulsifying drug delivery system (SEDDS) could increase the bioavailability of tocotrienol. However, evidence of this system in improving the skeletal effects of tocotrienol is scanty. This study aims to evaluate the therapeutic efficacy of annatto tocotrienol with SEDDS in a rat model of postmenopausal bone loss. Ten-month-old female Sprague Dawley rats were randomized into six groups. The baseline group was euthanatized at the onset of the study. Four other groups underwent ovariectomy to induce estrogen deficiency. The sham underwent similar surgery procedure, but their ovaries were retained. Eight weeks after surgery, the ovariectomized rats received one of the four different regimens orally daily: (a) SEDDS, (b) annatto tocotrienol [60 mg/kg body weight (b.w.)] without SEDDS, (c) annatto-tocotrienol (60 mg/kg b.w.) with SEDDS, (d) raloxifene (1 mg/kg b.w.). After eight weeks of treatment, blood was collected for the measurement of delta-tocotrienol level and oxidative stress markers. The rats were euthanized and their bones were harvested for the evaluation of the bone microstructure, calcium content and strength. Circulating delta-tocotrienol level was significantly higher in rats receiving annatto tocotrienol with SEDDS compared to the group receiving unformulated annatto-tocotrienol (p < 0.05). Treatment with unformulated or SEDDS-formulated annatto tocotrienol improved cortical bone thickness, preserved bone calcium content, increased bone biomechanical strength and increased antioxidant enzyme activities compared with the ovariectomized group (p < 0.05). Only SEDDS-formulated annatto tocotrienol improved trabecular microstructure, bone stiffness and lowered malondialdehyde level (p < 0.05 vs the ovariectomized group). The improvement caused by annatto tocotrienol was comparable to raloxifene. In conclusion, SEDDS improves the bioavailability and skeletal therapeutic effects of annatto tocotrienol in a rat model of postmenopausal bone loss. This formulation should be tested in a human clinical trial to validate its efficacy.

A Phase IIb Randomized Controlled Trial Investigating the Effects of Tocotrienol-Rich Vitamin E on Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is a debilitating complication of diabetes, which develops in 40% of the diabetic population and is responsible for up to 50% of end-stage renal disease (ESRD). Tocotrienols have shown to be a potent antioxidant, anti-inflammatory, and antifibrotic agent in animal and clinical studies. This study evaluated the effects of 400 mg tocotrienol-rich vitamin E supplementation daily on 59 DKD patients over a 12-month period. Patients with stage 3 chronic kidney disease (CKD) or positive urine microalbuminuria (urine to albumin creatinine ratio; UACR > 20-200 mg/mmol) were recruited into a randomized, double-blind, placebo-controlled trial. Patients were randomized into either intervention group (n = 31) which received tocotrienol-rich vitamin E (Tocovid SupraBioTM; Hovid Berhad, Ipoh, Malaysia) 400 mg daily or a placebo group which received placebo capsules (n = 28) for 12 months. HbA1c, renal parameters (i.e., serum creatinine, eGFR, and UACR), and serum biomarkers were collected at intervals of two months. Tocovid supplementation significantly reduced serum creatinine levels (MD: -4.28 ± 14.92 vs. 9.18 ± 24.96), p = 0.029, and significantly improved eGFR (MD: 1.90 ± 5.76 vs. -3.29 ± 9.24), p = 0.011 after eight months. Subgroup analysis of 37 patients with stage 3 CKD demonstrated persistent renoprotective effects over 12 months; Tocovid improved eGFR (MD: 4.83 ± 6.78 vs. -1.45 ± 9.18), p = 0.022 and serum creatinine (MD: -7.85(20.75) vs. 0.84(26.03), p = 0.042) but not UACR. After six months post washout, there was no improvement in serum creatinine and eGFR. There were no significant changes in the serum biomarkers, TGF-ß1 and VEGF-A. Our findings verified the results from the pilot phase study where tocotrienol-rich vitamin E supplementation at two and three months improved kidney function as assessed by serum creatinine and eGFR but not UACR.

Application of Partial Hydrogenation for the Generation of Minor Tocochromanol Homologs and Functional Evaluation of Hydrogenated Tocotrienol-rich Vitamin E Oil in Diabetic Obese Mice.

Recent research has identified minor homologs of vitamin E with one or two double bonds in the side-chain, namely tocomonoenol (T1) and tocodienol (T2), in natural products. We first explored the effectiveness of partial hydrogenation for generating minor tocochromanols from tocotrienol (T3). During hydrogenation with pure α-T3 as a substrate, the side-chain was partially saturated in a time-dependent manner, and a large amount of α-T1 and α-T2 was obtained. To investigate the beneficial effects of the hydrogenated product, we fed diabetic obese KK-A y mice with a hydrogenated T3 mixture (HT3). Feeding HT3 revealed tissue-specific accumulation of tocochromanols, ameliorated hyperglycemia and improved ratio of high-density lipoprotein cholesterol to total cholesterol in serum, with invariant body weight and fat mass. Hence, we propose that hydrogenation is a useful method for generating T1 and T2 homologs, which can be applied to explore the structure-related function of tocochromanols.

An evaluation of tocotrienol ethosomes for transdermal delivery using Strat-M® membrane and excised human skin.

Tocotrienol (TRF) ethosomes were developed and evaluated in vitro for potential transdermal delivery against melanoma. The optimised TRF ethosomal size ranged between 64.9 ± 2.2 nm to 79.6 ± 3.9 nm and zeta potential (ZP) between -53.3 mV to -62.0 ± 2.6 mV. Characterisation of the ethosomes by ATR-FTIR indicated the successful formation of TRF-ethosomes. Scanning electron microscopy (SEM) images demonstrated the spherical shape of ethosomes, and the entrapment efficiencies of all the formulations were above 66%. In vitro permeation studies using full-thickness human skin showed that the permeation of gamma-T3 from the TRF ethosomal formulations was significantly higher (p < 0.05) than from the control. The cumulative amount of gamma-T3 permeated from TRF ethosome after 48 hours was 1.03 ± 0.24 µg cm-2 with a flux of 0.03 ± 0.01 µg cm-2 h-1. Furthermore, the flux of gamma-T3 across the Strat-M ® and the epidermal membrane was significantly higher than that across full-thickness human skin (p < 0.05). In vitro cytotoxicity studies on HaCat cells showed significantly higher cell viability than the pure drug solution (p < 0.05). The enhanced skin permeation and high cell viability associated with this formulation suggest a promising carrier for transdermal delivery.

Effects of Calcium and Annatto Tocotrienol Supplementation on Bone Loss Induced by Pantoprazole in Male Rats.

PURPOSE: Prolonged use of proton pump inhibitors may cause bone loss, and limited therapeutic agents are available to prevent this skeletal side effect. The combination of annatto tocotrienol, a bone anabolic agent, with calcium presents a novel strategy to prevent bone loss caused by proton pump inhibitors. This study aims to compare the effects of calcium alone and in combination with annatto tocotrienol or vitamin D3 (Caltrate Plus) in preventing bone loss caused by pantoprazole. METHODS: Three-month-old Sprague Dawley male rats (n=30) were randomised into five groups (n=6/group). Bone loss was induced by pantoprazole (3 mg/kg p.o.) in four groups, and they were treated concurrently with either calcium carbonate (77 mg p.o.), calcium carbonate (77 mg p.o.) plus annatto tocotrienol (60 mg/kg p.o.) or Caltrate Plus (31 mg p.o.) for 60 days. The rats were euthanised at the end of the experiment, and their femurs were harvested for X-ray micro-computed tomography, bone cellular histomorphometry and bone mechanical strength analysis. RESULTS: Pantoprazole caused significant deterioration of trabecular bone microstructures but did not affect other skeletal indices. Calcium supplementation with or without annatto tocotrienol prevented the deterioration of trabecular microstructures at the femur but did not improve other skeletal indices. Annatto tocotrienol did not enhance the skeletal actions of calcium, whereas Caltrate Plus did not affect the bone health indices in these rats. CONCLUSION: Calcium supplementation per se can prevent the deterioration of bone trabecular microstructures in rats receiving long-term treatment of pantoprazole.

Platelet function in stroke/transient ischemic attack patients treated with tocotrienol.

The purpose of this study was to characterize the effects of tocotrienol form of vitamin E (TCT) on platelet function in patients with stroke or transient ischemic attack (TIA). A double blind, randomized, single center phase II clinical trial was conducted comparing placebo (PBO) and 400 and 800 mg TCT daily for a year in 150 patients with a sentinel ischemic stroke or TIA event in the prior 6 months. Platelet function was measured at baseline and then, at 3 month intervals for a year, using light transmission aggregometry. The incidence of aspirin resistance in aspirin-treated patients or platelet inhibition in patients on clopidogrel alone was compared between the three treatment groups. Results showed that in patients taking aspirin and clopidogrel, the incidence of aspirin resistance was significantly decreased from 40% in PBO-treated patients to 9% in the 400 mg TCT group and 25% in the TCT 800 mg group (P = .03). In conclusion, patients on aspirin and clopidogrel had a higher incidence of aspirin resistance than all patients treated with aspirin alone and TCT decreased the frequency of aspirin resistance in this group.

Metabolic benefits of annatto-extracted tocotrienol on glucose homeostasis, inflammation, and gut microbiome.

Emerging evidence suggests that the gut microbiome plays an important role in the pathophysiology of both obesity and type 2 diabetes mellitus. We previously reported that dietary annatto-extracted tocotrienol exerts beneficial effects by modulating inflammatory responses in mice fed a high-fat diet (HFD). The purpose of this study was to test the hypothesis that tocotrienol supplementation when combined with an HFD would result in an altered gut microbiota composition. For 14 weeks, forty-eight male C57BL/6J mice were assigned to 4 groups-low-fat diet, HFD, HFD supplemented with annatto-extracted tocotrienol at 800 mg/kg diet (AT), and HFD supplemented with metformin at 200 mg/kg diet. Glucose homeostasis was assessed by glucose and insulin tolerance tests, serum and pancreas insulin levels, and histological assessments of insulin and glucagon in pancreatic tissue. The concentrations of adipokines were measured in white adipose tissues. For the gut microbiome analysis, cecal content was collected, DNA was extracted, and 16S rRNA gene sequencing was performed. AT supplementation improved glucose homeostasis and lowered resistin, leptin, and interleukin-6 levels in white adipose tissue. Relative to the HFD group, AT-supplemented mice showed a decrease in the Firmicutes to Bacteroidetes ratio and had a lower abundance of Ruminococcus lactaris, Dorea longicatena, and Lachnospiraceae family. The relative abundance of Akkermansia muciniphila was increased in the AT group compared to the low-fat diet group. The association between the metabolic improvements and the identified bacterial taxa suggests a potential metabolic modulation caused by AT supplementation through the gut microbiota composition in mice fed an HFD.

Antioxidant status following postprandial challenge of two different doses of tocopherols and tocotrienols.

OBJECTIVE: Tocotrienols (T3s) have been hypothesized to have greater antioxidant capacity than tocopherols (Ts) due to differences in biokinetics that affect their absorption and function. The present trial compares the antioxidant effectiveness following postprandial challenge of two different doses of α-T or palm T3-rich fraction (TRF) treatments and evaluates their dose-response effects on antioxidant status. METHODS: Ten healthy volunteers were given four different doses of vitamin E formulations (268 mg α-T, 537 mg α-T, 263 mg TRF or 526 mg TRF) in a cross-over postprandial trial. Blood was sampled at 0, 2, 4, 5, 6 and 8 hours after meal consumption and plasma antioxidant status including total glutathione, superoxide dismutase, malondialdehyde (MDA), ferric reducing antioxidant potential and trolox-equivalent antioxidant capacity, was analyzed. RESULTS: Supplementation with the different doses of either α-T or TRF did not significantly improve overall antioxidant status. There was no significant difference in overall antioxidant status among treatments at the different doses compared. However, a significant dose-response effect was observed for plasma MDA throughout the 8-hour postprandial period. MDA was significantly lower after the 537 mg α-T treatment, compared to the 268 mg α-T treatment; it was also lower after the 526 mg TRF treatment compared to the 263 mg TRF treatment (P < 0.05). CONCLUSION: T3 and α-T demonstrated similar antioxidant capacity, despite markedly lower levels of T3 in blood and lipoproteins, compared to α-T.

Investigation of the curative effects of palm vitamin E tocotrienols on autoimmune arthritis disease in vivo.

The tocotrienol-rich fraction (TRF) from palm oil contains vitamin E, which possesses potent antioxidant and anti-inflammatory activities. Rheumatoid arthritis (RA) is a chronic joint inflammatory disease characterised by severe joint pain, cartilage destruction, and bone erosion owing to the effects of various pro-inflammatory mediators and cytokines. Here, we investigated the therapeutic effects of TRF in a rat model of collagen-induced arthritis (CIA). Arthritis was induced by a single intradermal injection of collagen type II in Dark Agouti (DA) rats. Rats were then treated with or without TRF by oral gavage from day 28 after the first collagen injection. Arthritic rats supplemented with TRF showed decreased articular index scores, ankle circumferences, paw volumes, and radiographic scores when compared with untreated rats. The untreated arthritic rats showed higher plasma C-reactive protein levels (p < 0.05) and production of pro-inflammatory cytokines than arthritic rats fed TRF. Moreover, there was a marked reduction in the severity of histopathological changes observed in arthritic rats treated with TRF compared with that in untreated arthritic rats. Overall, the results show that TRF had beneficial effects in this rat model of RA.

Oral Supplementation of Tocotrienol-Rich Fraction Alleviates Severity of Ulcerative Colitis in Mice.

Ulcerative colitis (UC) is characterized by damaged colonic mucosa and submucosa layers that are caused by excessive inflammatory reactions and oxidative stress. This study aimed to examine the use of tocotrienol-rich fraction (TRF) in mitigating damages caused by UC on the colon epithelium. Dextran sulfate sodium (DSS)-induced UC mice were treated with vehicle control, TRF, alpha-tocopherol (αTP) and 5-aminosalicylic acid (5-ASA). Observable clinical signs, quality of stool, histopathological scoring, inflammatory and oxidative markers were assessed. Vitamin E levels of colons and plasma were quantified. Oral supplementation of TRF significantly reduced the severity of DSS-induced UC by lowering the disease activity index (DAI) and histopathological inflammatory scoring. TRF also attenuated the DSS-induced enlargement of spleen and shortening of the colon. TRF has demonstrated marked anti-inflammatory and antioxidative properties indicated by the attenuation of DSS-induced upregulation of inflammation and oxidative stress markers including interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), nitric oxide (NO), malondialdehyde (MDA) and pNF-κB. These improvements were similar to that of 5-aminosalicylic acid (5-ASA) treatment. In contrast, αTP did not demonstrate evident clinical and histopathological improvements. The superior protective effect of TRF may be ascribed to the preferential absorption of TRF by the gut mucosa. TRF alleviated the signs and symptoms of acute UC in murine model via the reduction of local inflammatory reactions and oxidative stress. These effects suggested that TRF could serve as a gut health supplement for preventive measures for UC condition in patients.

Tocotrienol Rich Fraction Supplementation Modulate Brain Hippocampal Gene Expression in APPswe/PS1dE9 Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and other cognitive abilities. AD is associated with aggregation of amyloid-ß (Aß) deposited in the hippocampal brain region. Our previous work has shown that tocotrienol rich fraction (TRF) supplementation was able to attenuate the blood oxidative status, improve behavior, and reduce fibrillary-type Aß deposition in the hippocampus of an AD mouse model. In the present study, we investigate the effect of 6 months of TRF supplementation on transcriptome profile in the hippocampus of APPswe/PS1dE9 double transgenic mice. TRF supplementation can alleviate AD conditions by modulating several important genes in AD. Moreover, TRF supplementation attenuated the affected biological process and pathways that were upregulated in the AD mouse model. Our findings indicate that TRF supplementation can modulate hippocampal gene expression as well as biological processes that can potentially delay the progression of AD.

Tocotrienol-rich fraction supplementation prevents foetal loss in females mated with corticosterone-treated male Sprague-Dawley rats.

This study examined whether tocotrienol supplementation to corticosterone-treated male rats could prevent foetal loss in females upon their mating. Epididymides of adult male Sprague-Dawley (SD) rats with proven fertility were surgically separated at the testis-caput junction. Twenty-four hours post-surgery, these animals received for 7 days either: tocopherol-stripped corn oil (Control), corticosterone 25 mg/kg s.c. (CORT), CORT 25 mg/kg s.c. and tocotrienol-rich fraction (TRF) 100 mg/kg orally (CORT + TRF) or TRF 100 mg/kg orally (TRF). On day 8, males were cohabited with proestrus females. A spermatozoa-positive vaginal smear indicated pregnancy. Males were euthanised for analysis of testosterone and antioxidant activities. Reproductive organs were weighed. On day 8 of pregnancy, females were laparotomised to count the number of implantation sites. Pregnancy was continued until term. Number of pups delivered and their weights were determined. Data were analysed using ANOVA. Malondialdehyde levels were significantly lower in CORT + TRF group compared with CORT group. Enzymatic antioxidant activities, testosterone level and reproductive organ weights were significantly higher in CORT + TRF group compared with CORT group. Number of implantation sites and live pups delivered, and their birth weights from females mated with CORT + TRF males were significantly higher compared to CORT group. Therefore, TRF prevents foetal loss in females mated with CORT + TRF-treated males.

Exploring the potential of tocotrienol from Bixa orellana as a single agent targeting metabolic syndrome and bone loss.

Metabolic syndrome (MetS) is associated with osteoporosis due to the underlying inflammatory and hormonal changes. Annatto tocotrienol has been shown to improve medical complications associated with MetS or bone loss in animal studies. This study aimed to investigate the effects of annatto tocotrienol as a single treatment for MetS and osteoporosis in high-carbohydrate high-fat (HCHF) diet-induced MetS animals. Three-month-old male Wistar rats were randomly divided into five groups. The baseline group was euthanized at the onset of the study. The normal group received standard rat chow and tap water. The remaining groups received HCHF diet and treated with three different regimens orally daily: (a) tocopherol-stripped corn oil (the vehicle of tocotrienol), (b) 60 mg/kg annatto tocotrienol, and (c) 100 mg/kg annatto tocotrienol. At the end of the study, measurements of MetS parameters, body compositions, and bone mineral density were performed in animals before sacrifice. Upon euthanasia, blood and femur of the rats were harvested for the evaluations of bone microstructure, biomechanical strength, remodelling activities, hormonal changes, and inflammatory response. Treatment with annatto tocotrienol improved all MetS parameters (except abdominal obesity), trabecular bone microstructure, bone strength, increased osteoclast number, normalized hormonal changes and inflammatory response in the HCHF animals. In conclusion, annatto tocotrienol is a potential agent for managing MetS and osteoporosis concurrently. The beneficial effects of annatto tocotrienol may be attributed to its ability to prevent the hormonal changes and pro-inflammatory state in animals with MetS.

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.

Efficacy of Oral Mixed Tocotrienols in Diabetic Peripheral Neuropathy: A Randomized Clinical Trial.

Importance: Management of painful diabetic peripheral neuropathy remains challenging. Most therapies provide symptomatic relief with varying degrees of efficacy. Tocotrienols have modulatory effects on the neuropathy pathway and may reduce neuropathic symptoms with their antioxidative and anti-inflammatory activities. Objective: To evaluate the efficacy of oral mixed tocotrienols for patients with diabetic peripheral neuropathy. Design, Setting, and Participants: The Vitamin E in Neuroprotection Study (VENUS) was a parallel, double-blind, placebo-controlled trial that recruited participants from January 30, 2011, to December 7, 2014, with 12 months of follow-up. This trial screened 14 289 patients with diabetes from 6 health clinics and ambulatory care units from 5 public hospitals in Malaysia. A total of 391 patients who reported neuropathic symptoms were further assessed with Total Symptom Score (TSS) and Neuropathy Impairment Score (NIS). Patients 20 years or older with a TSS of 3 or higher and an NIS of 2 or higher were recruited. Interventions: Patients were randomized to receive 200 mg of mixed tocotrienols twice daily or matching placebo for 12 months. Patients with hyperhomocysteinemia (homocysteine level ≥2.03 mg/L) received oral folic acid, 5 mg once daily, and methylcobalamin, 500 µg thrice daily, in both groups. Main Outcomes and Measures: The primary outcome was patient-reported neuropathy TSS (lancinating pain, burning pain, paresthesia, and asleep numbness) changes at 12 months. The secondary outcomes were NIS and sensory nerve conduction test result. Results: Of 391 eligible patients, 300 were recruited (130 [43.3%] male; mean [SD] age, 57.6 [8.9] years; mean [SD] duration of diabetes, 11.4 [7.8] years) and 229 (76.3%) completed the trial. The TSS changes between the tocotrienols and placebo groups at 12 months (-0.30; 95% CI, -1.16 to 0.56; P = .49) were similar. No significant differences in NIS (0.60; 95% CI, -1.37 to 2.65; P = .53) and sensory nerve conduction test assessments were found between both groups. In post hoc subgroup analyses, tocotrienols reduced lancinating pain among patients with hemoglobin A1C levels greater than 8% (P = .03) and normohomocysteinemia (homocysteine level <2.03 mg/L; P = .008) at 1 year. Serious adverse events in both groups were similar, except more infections were observed in the tocotrienols group (6.7% vs 0.7%, P = .04). Results reported were of modified intention-to-treat analyses. Conclusions and Relevance: Supplementation of oral mixed tocotrienols, 400 mg/d for 1 year, did not improve overall neuropathic symptoms. The preliminary observations on lancinating pain among subsets of patients require further exploration. Trial Registration: National Medical Research Registry Identifier: NMRR-10-948-7327 and clinicaltrials.gov Identifier: NCT01973400.