Formation of Previously Undescribed Δ7-Phytosterol Oxidation Products and Tocopherylquinone Adducts in Pumpkin Seed Oil during Roasting, Screw-Pressing, and Simulated Culinary Processing at Elevated Temperatures.

The influence of pumpkin seed roasting conditions (110-140 °C) and screw-pressing on the formation of previously undescribed Δ7-phytosterol oxidation products and tocopherylquinone adducts with nucleophilic phosphatidylethanolamine species was investigated. The roasting process of pumpkin seed paste at a temperature above 120 °C for 30 min considerably enhanced the formation of Δ7-oxysterols. Targeted analysis [electron impact mass spectrometry (MS), 1D-nuclear magnetic resonance] led to the identification of five novel markers of pumpkin paste roasting, among which (3ß,5α,22E,24S)-stigmasta-7,22-dien-6-one-3-ol (6-oxo-α-spinasterol), stereoisomers of (3ß,5α,22E)-7,8-epoxystigmast-22-en-3-ol (7,8-epoxy-α-spinasterol), and (3ß,5α)-22,23-epoxystigmast-7-en-3-ol (7,8-epoxy-α-spinasterol) were reported in edible oils for the first time. Simulated culinary processing provided novel stereoisomers of (3ß,5α,22E)-stigmasta-7,22-dien-3,6-diol, unusual (3ß,5α,22E)-stigmasta-7,22-dien-6,15-dione-3-ol, and (5α,22E)-stigmasta-7,22-dien-3-one accompanied by minor stereoisomers of (3ß,5α)-7,8;22,23-diepoxystigmastan-3-ol. Moreover, a clear relationship between the pumpkin seed oil stability index and synergistic effect of glycerophospholipids with present tocochromanols was found. High-resolution atmospheric pressure chemical ionization-MS experiments clearly demonstrated the formation of various γ-tocopherylquinone adducts with primary amines, namely, octylamine. The mitigation strategy of potentially detrimental oxysterols from pumpkin seed oil included optimization of processing parameters while maintaining the formation of desirable sensory-active compounds.

Comparison of delta-tocotrienol and alpha-tocopherol effects on hepatic steatosis and inflammatory biomarkers in patients with non-alcoholic fatty liver disease: A randomized double-blind active-controlled trial.

OBJECTIVE: We aimed to compare the efficacy of δ-tocotrienol with α-tocopherol in the treatment of patients with non-alcoholic fatty liver disease (NAFLD). DESIGN AND INTERVENTIONS: This study was a double-blinded, active-controlled trial. The patients with NAFLD were randomly assigned to receive either δ-tocotrienol 300 mg or α-tocopherol 268 mg twice daily for 48 weeks. ENDPOINTS: The primary endpoints were change from baseline in fatty liver index (FLI), liver-to-spleen attenuation ratio (L/S ratio), and homeostatic model assessment for insulin resistance (HOMA-IR) at 48 weeks. Key secondary endpoints were change in markers of inflammation, oxidative stress, and hepatocyte apoptosis. Clinical assessment, biochemical analysis, and computed tomography scan of the liver were conducted at baseline, 24 and 48 weeks. RESULTS: A total of 100 patients (δ-tocotrienol = 50, α-tocopherol = 50) were randomized and included in the intention to treat analysis. Compared with baseline, there was a significant improvement (p < .001) in FLI, L/S ratio, HOMA-IR, and serum malondialdehyde in both groups at 48 weeks that was not significant between the two groups. However, there was a significantly greater decrease in body weight, serum interleukin-6, tumor necrosis factor-alpha, leptin, cytokeratin-18, and increase in adiponectin in the δ-tocotrienol group compared to the α-tocopherol group at 48 weeks (p < .05). No adverse events were reported. CONCLUSION: δ-tocotrienol and α-tocopherol exerted equally beneficial effects in terms of improvement in hepatic steatosis, oxidative stress, and insulin resistance in patients with NAFLD. However, δ-tocotrienol was more potent than α-tocopherol in reducing body weight, inflammation, and apoptosis associated with NAFLD. TRIAL REGISTRATION: Sri Lankan Clinical Trials Registry (https://slctr.lk/SLCTR/2019/038).

Effects of delta-tocotrienol supplementation on glycaemic control in individuals with prediabetes: A randomized controlled study.

OBJECTIVE: To study the effects of delta-tocotrienol on glycaemic control parameters in individuals with pre-diabetes. METHODS: The randomised control trial was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from July 15 to November 15, 2019, and comprised individuals aged 18-60 years having fasting plasma glucose of 5.6 to 6.9 mmol/L or glycosylated haemoglobin of 5.7 to 6.4%. They were randomised into group A receiving 300mg delta-tocotrienol and group B receiving a placebo once daily for 12 weeks. Weight, height, waist circumference, fasting plasma glucose, insulin and glycosylated haemoglobin were measured at the beginning and end of the trial to assess any change. Body mass index and homeostatic model assessment-insulin resistance were also calculated. Data was analysed using SPSS 21. RESULTS: Of the 77participants, 40(52%) were in group A and 37(48%) in group B. Group A showed significantly greater reduction in terms of fasting plasma glucose, glycosylated haemoglobin, insulin and homeostatic model assessment-insulin resistance index (p≤0.001) post-intervention. CONCLUSIONS: Delta-tocotrienol supplementation was found to have a significant effect in improving glycaemic control parameters in persons with pre-diabetes. Futures larger scale clinical trials are needed to confirm these findings. CLINICAL TRIAL NUMBER: SLCTR/2019/024.

Delta-tocotrienol enhances the anti-tumor effects of interferon alpha through reactive oxygen species and Erk/MAPK signaling pathways in hepatocellular carcinoma cells.

The complexity of hepatocellular carcinoma (HCC) signaling and the failure of pharmacological therapeutics reveal the significance of establishing new anti-cancer strategies. Interferon alpha (IFN-α) has been used as adjuvant therapy for reducing HCC recurrence and improving survival. Delta-tocotrienol (δ-tocotrienol), a natural unsaturated isoform of vitamin E, is a promising candidate for cancer treatment. In this study, we evaluated whether the combination of δ-tocotrienol with IFN-α displays significant advantages in the treatment of HCC cells. Results showed that the combination significantly decreased cell viability, migration and invasion of HCC cells compared with single therapies. Combining δ-tocotrienol and IFN-α enhanced the decrease in proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase (MMP) 7 and MMP-9. The combination also produced an enhancement of apoptosis together with increased Bax/Bcl-xL ratio and reactive oxygen species (ROS) generation. δ-tocotrienol induced Notch1 activation and changes in Erk and p38 MAPK signaling status. Blocking experiments confirmed that ROS and Erk are involved, at least in part, in the anti-cancer effects of the combined treatment. In conclusion, the combination of δ-tocotrienol with IFN-α therapy showed promising results for HCC cell treatment, which makes the combination of cytokine-based immunotherapy with natural products a potential strategy against liver cancer.

Evaluating Anticancer and Immunomodulatory Effects of Spirulina (Arthrospira) platensis and Gamma-Tocotrienol Supplementation in a Syngeneic Mouse Model of Breast Cancer.

Nutrition can modulate host immune responses as well as promote anticancer effects. In this study, two nutritional supplements, namely gamma-tocotrienol (γT3) and Spirulina, were evaluated for their immune-enhancing and anticancer effects in a syngeneic mouse model of breast cancer (BC). Five-week-old female BALB/c mice were fed Spirulina, γT3, or a combination of Spirulina and γT3 (Spirulina + γT3) for 56 days. The mice were inoculated with 4T1 cells into their mammary fat pad on day 28 to induce BC. The animals were culled on day 56 for various analyses. A significant reduction (p < 0.05) in tumor volume was only observed on day 37 and 49 in animals fed with the combination of γT3 + Spirulina. There was a marked increase (p < 0.05) of CD4/CD127+ T-cells and decrease (p < 0.05) of T-regulatory cells in peripheral blood from mice fed with either γT3 or Spirulina. The breast tissue of the combined group showed abundant areas of necrosis, but did not prevent metastasis to the liver. Although there was a significant increase (p < 0.05) of MIG-6 and Cadherin 13 expression in tumors from γT3-fed animals, there were no significant (p > 0.05) differences in the expression of MIG-6, Cadherin 13, BIRC5, and Serpine1 upon combined feeding. This showed that combined γT3 + Spirulina treatment did not show any synergistic anticancer effects in this study model.

Effects of delta-tocotrienol supplementation on Glycemic Control, oxidative stress, inflammatory biomarkers and miRNA expression in type 2 diabetes mellitus: A randomized control trial.

The study aimed to ascertain the effects of delta-tocotrienol (δT3) supplementation on glycemic control, oxidative stress, inflammation and related micro-ribonucleic acid (miRNA) expression in patients with type 2 diabetes mellitus (T2DM). Total 110 patients of T2DM on oral hypoglycemic agents, were randomly divided into tocotrienol and placebo groups and given 250 mg δT3 or cellulose soft gel capsule once daily respectively for 24 weeks. Glycemic control, oxidative stress, inflammatory biomarkers, and miRNAs expression were measured in serum at baseline and end of the intervention by using standard laboratory methods. Compared to the placebo, δT3 supplementation resulted in a significant (p ≤ .05) reduction [mean difference (95% confidence interval)] in plasma glucose [-0.48 (-0.65, -0.30)], insulin [-1.19 (-1.51, -0.87)], homeostatic model assessment of insulin resistance [-0.67 (-0.86, -0.49)], glycosylated hemoglobin [-0.53 (-0.79, -0.28)], malondialdehyde [-0.34 (-0.45, -0.22)], high sensitive-C-reactive protein[-0.35 (-0.54, -0.16)], tumor necrosis factor-alpha [-1.22 (-1.62, -0.83)], and interleukin-6[-2.30 (-2.91, -1.68)]. More than twofold downregulation in miRNA-375, miRNA-34a, miRNA-21, and upregulation in miRNA-126, miRNA-132 expression was observed in the δT3 group compared to the placebo. The study demonstrated that δT3 supplementation in addition to oral hypoglycemic agents, improved glycemic control, inflammation, oxidative stress, and miRNA expression in T2DM without any adverse effect. Thus, δT3 might be considered as an effective dietary supplement to prevent long-term diabetic complications.

γ-Tocotrienol-Loaded Liposomes for Radioprotection from Hematopoietic Side Effects Caused by Radiotherapeutic Drugs.

With the successful development and increased use of targeted radionuclide therapy for treating cancer comes the increased risk of radiation injury to bone marrow-both direct suppression and stochastic effects, leading to neoplasia. Herein, we report a novel radioprotector drug, a liposomal formulation of γ-tocotrienol (GT3), or GT3-Nano for short, to mitigate bone marrow radiation damage during targeted radionuclide therapy. Methods: GT3 was loaded into liposomes using passive loading. 64Cu-GT3-Nano and 3H-GT3-Nano were synthesized to study the in vivo biodistribution profile of the liposome and GT3 individually. The radioprotection efficacy of GT3-Nano was assessed after acute 137Cs whole-body irradiation at a sublethal (4 Gy), a lethal (9 Gy), or a single high-dose administration of 153Sm-ethylenediamine-N,N,N',N'-tetrakis(methylene phosphonic acid) (EDTMP). Flow cytometry and fluorescence microscopy were used to analyze hematopoietic cell population dynamics and the cellular site of GT3-Nano localization in the spleen and bone marrow, respectively. Results: Bone marrow uptake and retention (percentage injected dose per gram of tissue) at 24 h was 6.98 ± 2.34 for 64Cu-GT3-Nano and 7.44 ± 2.52 for 3H-GT3-Nano. GT3-Nano administered 24 h before or after 4 Gy of total-body irradiation (TBI) promoted rapid and complete hematopoietic recovery, whereas recovery of controls stalled at 60%. GT3-Nano demonstrated dose-dependent radioprotection, achieving 90% survival at 50 mg/kg against lethal 9-Gy TBI. Flow cytometry of the bone marrow indicated that progenitor bone marrow cells MPP2 and CMP were upregulated in GT3-Nano-treated mice. Immunohistochemistry showed that GT3-Nano accumulates in CD105-positive sinusoid epithelial cells. Conclusion: GT3-Nano is highly effective in mitigating the marrow-suppressive effects of sublethal and lethal TBI in mice. GT3-Nano can facilitate rapid recovery of hematopoietic components in mice treated with the endoradiotherapeutic agent 153Sm-EDTMP.

Achiote (Bixa orellana) Lipophilic Extract, Bixin, and δ-tocotrienol Effects on Lifespan and Stress Resistance in Caenorhabditis elegans.

The onset of many degenerative diseases related to aging has been associated with a decrease in the activity of antistress systems, and pharmacological interventions increasing stress resistance could be effective to prevent the development of such diseases. Achiote is a valuable source of carotenoid and tocotrienols, which have antioxidant activity. In this work, we explore the capacity of an achiote seed extract and its main compounds to modulate the lifespan and antistress responses on Caenorhabditis elegans, as well as the mechanisms involved in these effects. Achiote lipophilic extract, bixin, and δ-tocotrienol were applied on nematodes to carry out lifespan, stress resistance, and fertility assays. The achiote seed extract increased the median and maximum lifespan up to 35% and 27% and increased resistance against oxidative and thermal stresses without adverse effects on fertility. The beneficial effects were mimicked by a bixin+δ-tocotrienol mixture. All the effects on lifespan and stress resistance were independent of caloric restriction but dependent on the insulin/insulin growth factor-1 pathway. This study could provide insights for further research on a new beneficial use of this important crop in health and nutraceutical applications beyond its use as a source of natural pigments.

Delta-tocotrienol supplementation improves biochemical markers of hepatocellular injury and steatosis in patients with nonalcoholic fatty liver disease: A randomized, placebo-controlled trial.

OBJECTIVE: The aim of this study was to examine the effects of delta-tocotrienol (δ-tocotrienol) supplementation on biochemical markers of hepatocellular injury and steatosis in patients with nonalcoholic fatty liver disease (NAFLD). DESIGN: The study design was a two-group, randomized, double-blind, placebo-controlled trial. The patients with NAFLD were randomly assigned to receive δ-tocotrienol 300 mg twice daily or placebo for 24 weeks. ENDPOINTS: The primary endpoints were change from baseline in fatty liver index (FLI) and homeostasis model of insulin resistance (HOMA-IR) after 24 weeks. Secondary endpoints included change from baseline in high sensitivity C-reactive protein (hs-CRP), malondialdehyde (MDA), alanine transaminase (ALT), aspartate transaminase (AST) and grading of hepatic steatosis on ultrasound. Between-group differences were tested for significance using ANCOVA. Mean differences (MD) with 95 % CIs are reported. RESULTS: A total of 71 patients (tocotrienol=35, placebo=36) were randomized and included in the intention to treat analysis. Compared with placebo, δ-tocotrienol significantly reduced (MD [95 % CI]) FLI (-8.52 [-10.7, -6.3]; p < 0.001); HOMA-IR (-0.37 [-0.53, -0.21]; p < 0.001), hs-CRP (-0.61[-0.81, -0.42]; p < 0.001), MDA (-0.91 [-1.20, -0.63]; p < 0.001), ALT (-8.86 [-11.5, -6.2]; p < 0.001) and AST (-6.6 [-10.0, -3.08]; p < 0.001). Hepatic steatosis was also reduced by a significantly greater extent with tocotrienol than with placebo (p =0.047). No adverse events were reported. CONCLUSION: δ-tocotrienol effectively improved biochemical markers of hepatocellular injury and steatosis in patients with NAFLD. δ-tocotrienol supplementation might be considered as a therapeutic option in the management of patients with NAFLD. TRIAL REGISTRATION: Sri Lankan Clinical Trials Registry (SLCTR/2015/023, 2015-10-03).

Cellular uptake and anti-inflammatory effects of palm oil-derived delta (δ)-tocotrienol in microglia.

Tocopherols long dominated studies on vitamin E, although interest has shifted to tocotrienols. It was previously shown that δ-tocotrienol derived from palm oil reduced nitric oxide released by BV2 microglia as early as 18 h after lipopolysaccharide stimulation. The current study measured δ-tocotrienol uptake by BV2 over a 24 h incubation period and its anti-inflammatory effects on primary microglia. Uptake of 17.5 µg/mL δ-tocotrienol by BV2 microglia began as early as 5 min and rose steeply to 21 ± 3% of the amount administered at 24 h. The amount of δ-tocotrienol retained in the lipopolysaccharide-stimulated microglia at 24 h was 14 ± 2%, with no substantial difference seen in unstimulated microglia. The same δ-tocotrienol regimen reduced nitric oxide levels by 82% at 24 h after lipopolysaccharide stimulation (p < 0.05). This was accompanied by decreased inducible nitric oxide synthase protein expression by 67 ± 5% compared to untreated controls (p < 0.05). In primary microglia, δ-tocotrienol downregulated IL-1ß production, but TNF-α and IL-6 were not affected. δ-Tocotrienol also reduced prostaglandin E2 production by ~78%% and decreased transcription of COX-2 and 5-LOX, but not COX-1. This study showed the anti-inflammatory effects of δ-tocotrienol derived from palm oil and opens up interest for tocotrienol supplementation to reduce the effects of inflammatory conditions.

Food for Bone: Evidence for a Role for Delta-Tocotrienol in the Physiological Control of Osteoblast Migration.

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 µg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/ß-catenin signalling pathway activation. In fact, δ-TT increased ß-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/ß-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of ß-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

Structure-based design, semi-synthesis and anti-inflammatory activity of tocotrienolic amides as 5-lipoxygenase inhibitors.

Inflammation contributes to the development of various pathologies, e.g. asthma, cardiovascular diseases, some types of cancer, and metabolic disorders. Leukotrienes (LT), biosynthesized from arachidonic acid by 5-lipoxygenase (5-LO), constitute a potent family of pro-inflammatory lipid mediators. δ-Garcinoic acid (δ-GA) (1), a natural vitamin E analogue, was chosen for further structural optimization as it selectively inhibited 5-LO activity in cell-free and cell-based assays without impairing the production of specialized pro-resolving mediators by 15-LO. A model of semi-quantitative prediction of 5-LO inhibitory potential developed during the current study allowed the design of 24 garcinamides that were semi-synthesized. In accordance with the prediction model, biological evaluations showed that eight compounds potently inhibited human recombinant 5-LO (IC50 < 100 nM). Interestingly, four compounds were substantially more potent than 1 in activated primary human neutrophils assays. Structure - activity relationships shed light on a supplementary hydrophobic pocket in the allosteric binding site that could be fitted with an aromatic ring.

A computational study of effects on membrane recruitment of the polar linkers in Vitamin E derivatives.

BACKGROUND: Previous studies found that Vitamin E (VE) could recruit protein kinase B (Akt1) to the membrane by targeting its unconventional lipid-binding site, which led to the dephosphorylation of Akt1 at Ser473, eventually deactivating the enzyme. METHODS: A series of VE-like compounds with varying types and lengths of the linker groups are designed to study the VE-driven membrane recruitment of Akt1 using a combined molecular docking and molecular dynamics (MD) simulation approach. RESULTS: We find that the linker groups with only one methylene linker and multiple hydrogen bond donors are optimal for achieving a balance between binding to the protein and partitioning into the membrane to form a stable protein-ligand-membrane ternary complex. These polar linkers are found to form stable hydrogen bonds with the lipid head groups during the MD simulations, which turns out critical for ensuring that the chromanol ring of the VE-like compounds resides above the membrane surface to fully engage in the protein. CONCLUSIONS: Our results reveal the molecular determinants of the linker groups for VE derivatives' ability to anchor Akt1 to the membrane. GENERAL SIGNIFICANCE: These findings will facilitate the design of membrane interfacial compounds to recruit specific proteins to the membrane to modulate the protein function.

Identification of two α-tocodienol isomers in palm oil after countercurrent chromatographic enrichment.

Countercurrent chromatography (CCC) was used for the enrichment of α-tocodienol (α-T2), a rare vitamin E-related minor compound previously tentatively detected in palm oil. Hitherto, only one isomer has been mentioned to occur at traces in palm oil. However, CCC fractionation followed by GC/MS measurements of all fractions resulted in the detection of two α-T2 isomers in five different palm oil vitamin E dietary supplement capsules. Five repetitive CCC separations of ~ 1 g sample and additional purification steps by column chromatography provided ~ 2 mg of two equally abundant α-T2 isomers with a purity of ~ 85%. The positions of the double bonds in the alkyl side chain could be assigned by means of two characteristic chemical shifts in the 1H NMR spectrum. Accordingly, the structures of the α-T2 isomers were 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridec-3,11-dienyl)chroman-6-ol (double bonds in 3',11'-position) and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridec-7,11-dienyl)chroman-6-ol (double bonds in 7',11'-position). Natural occurrence of both isomers was proven by GC/MS screening of crude palm oil after saponification and CCC separation. Moreover, GC/MS analysis allowed the tentative assignment of γ-tocomonoenol (γ-T1) and ß-tocomonoenol (ß-T1) as trace compounds in palm oil.

Suppression of colorectal cancer cell growth by combined treatment of 6-gingerol and γ-tocotrienol via alteration of multiple signalling pathways.

Our previous study reported that combined treatment of γ-tocotrienol with 6-gingerol showed promising anticancer effects by synergistically inhibiting proliferation of human colorectal cancer cell lines. This study aimed to identify and elucidate molecular mechanisms involved in the suppression of SW837 colorectal cancer cells modulated by combined treatment of γ-tocotrienol and 6-gingerol. Total RNA from both untreated and treated cells was prepared for transcriptome analysis using RNA sequencing techniques. We performed high-throughput sequencing at approximately 30-60 million coverage on both untreated and 6G + γT3-treated cells. The results showed that cancer-specific differential gene expression occurred and functional enrichment pathway analysis suggested that more than one pathway was modulated in 6G + γT3-treated cells. Combined treatment with 6G + γT3 augmented its chemotherapeutic effect by interfering with the cell cycle process, downregulating the Wnt signalling pathway and inducing apoptosis mainly through caspase-independent programmed cell death through mitochondrial dysfunction, activation of ER-UPR, disruption of DNA repair mechanisms and inactivation of the cell cycle process through the downregulation of main genes in proliferation such as FOXM1 and its downstream genes. The combined treatment exerted its cytotoxic effect through upregulation of genes in stress response activation and cytostatic effects demonstrated by downregulation of main regulator genes in the cell cycle. Selected genes involved in particular pathways including ATF6, DDIT3, GADD34, FOXM1, CDK1 and p21 displayed concordant patterns of gene expression between RNA sequencing and RT-qPCR. This study provides new insights into combined treatment with bioactive compounds not only in terms of its pleiotropic effects that enhance multiple pathways but also specific target genes that could be exploited for therapeutic purposes, especially in suppressing cancer cell growth.

Omega 3-DHA and Delta-Tocotrienol Modulate Lipid Droplet Biogenesis and Lipophagy in Breast Cancer Cells: the Impact in Cancer Aggressiveness.

Omega 3-docosahexaenoic acid (DHA) and vitamin E Delta-tocotrienol (Delta-T3) are extensively studied as protective nutrients against cancer development. Little is known about the biological mechanisms targeted by these bioactive molecules on lipid droplet (LD) biogenesis, an important breast cancer aggressiveness marker, and the occurrence of lipophagy in breast cancer cells. The aim of this study was to investigate the effect of DHA, Delta-T3 and DHA plus Delta-T3 co-treatment in LD biogenesis and lipophagy process in triple negative breast cancer cell line MDA-MB-231. Cells were treated with 50 µM DHA and/or 5 µM Delta-T3. Our results demonstrated that DHA can trigger an increase in LD biogenesis and co-treatment with Delta-T3 was able to reduce this LD biogenesis. In addition, we showed that a higher cytoplasmic LD content is associated with a higher breast cancer cells malignance and proliferation. Reduction of cytoplasmic LD content by silencing ADRP (adipose differentiation-related protein), a structural LD protein, also decreased cell proliferation in MDA-MB-231 cells. Treatment with DHA and Delta-T3 alone or co-treatment did not reduce cell viability. Moreover, we showed here that DHA can trigger lipophagy in MDA-MB-231 cells and DHA plus Delta-T3 co-treatment was able to enhance this lipophagy process. Our findings demonstrated that co-treatment with DHA plus Delta-T3 in MDA-MB-231 cells could reduce LD biogenesis and potentiate lipophagy in these cells, possibly having a positive impact to inhibit breast cancer malignancy. Therefore, suitable doses of DHA and Delta-T3 vitamin E isoform supplementation can be a prominent tool in therapeutic treatments against breast cancer.

The vitamin E derivative garcinoic acid from Garcinia kola nut seeds attenuates the inflammatory response.

The plant Garcinia kola is used in African ethno-medicine to treat various oxidation- and inflammation-related diseases but its bioactive compounds are not well characterized. Garcinoic acid (GA) is one of the few phytochemicals that have been isolated from Garcinia kola. We investigated the anti-inflammatory potential of the methanol extract of Garcinia kola seeds (NE) and purified GA, as a major phytochemical in these seeds, in lipopolysaccharide (LPS)-activated mouse RAW264.7 macrophages and its anti-atherosclerotic potential in high fat diet fed ApoE-/- mice. This study outlines an optimized procedure for the extraction and purification of GA from Garcinia kola seeds with an increased yield and a purity of >99%. We found that LPS-induced upregulation of iNos and Cox2 expression, and the formation of the respective signaling molecules nitric oxide and prostanoids, were significantly diminished by both the NE and GA. In addition, GA treatment in mice decreased intra-plaque inflammation by attenuating nitrotyrosinylation. Further, modulation of lymphocyte sub-populations in blood and spleen have been detected, showing immune regulative properties of GA. Our study provides molecular insights into the anti-inflammatory activities of Garcinia kola and reveals GA as promising natural lead for the development of multi-target drugs to treat inflammation-driven diseases.

Gamma-Tocotrienol Induces Apoptosis in Prostate Cancer Cells by Targeting the Ang-1/Tie-2 Signalling Pathway.

Emerging evidence suggests that gamma-tocotrienol (γ-T3), a vitamin E isomer, has potent anti-cancer properties against a wide-range of cancers. γ-T3 not only inhibited the growth and survival of cancer cells in vitro, but also suppressed angiogenesis and tumour metastasis under in vivo conditions. Recently, γ-T3 was found to target cancer stem cells (CSCs), leading to suppression of tumour formation and chemosensitisation. Despite its promising anti-cancer potential, the exact mechanisms responsible for the effects of γ-T3 are still largely unknown. Here, we report the identification of Ang-1 (Angiopoietin-1)/Tie-2 as a novel γ-T3 downstream target. In prostate cancer cells, γ-T3 treatment leads to the suppression of Ang-1 at both the mRNA transcript and protein levels. Supplementing the cells with Ang-1 was found to protect them against the anti-CSC effect of γ-T3. Intriguingly, inactivation of Tie-2, a member receptor that mediates the effect of Ang-1, was found to significantly enhance the cytotoxic effect of γ-T3 through activation of AMP-activated protein kinase (AMPK) and subsequent interruption of autophagy. Our results highlighted the therapeutic potential of using γ-T3 in combination with a Tie-2 inhibitor to treat advanced prostate cancer.

Annatto (Bixa orellana) δ-TCT Supplementation Protection against Embryonic Malformations through Alterations in PI3K/Akt-Cyclin D1 Pathway.

Protective action by annatto-derived delta-tocotrienol (δ-TCT) and soy-derived alpha-tocopherol (α-TOC) through the regulation of the PI3K/Akt-cyclin D1 pathway against nicotine-induced DNA damage is the focus of the present study. Nicotine, which has been widely reported to have numerous adverse effects on the reproductive system, was used as a reproductive toxicant. 48 female balb/c mice (6⁻8 weeks) (23⁻25 g) were randomly divided into eight groups (Grp.1⁻Grp.8; n = 6) and treated with either nicotine or/and annatto δ-TCT/soy α-TOC for seven consecutive days. On Day 8, the females were superovulated and mated before euthanization for embryo collection (46 h post-coitum). Fifty 2-cell embryos from each group were used in gene expression analysis using Affymetrix QuantiGene Plex2.0 assay. Findings indicated that nicotine (Grp.2) significantly decreased (p < 0.05) the number of produced 2-cell embryos compared to the control (Grp.1). Intervention with mixed annatto δ-TCT (Grp.3) and pure annatto δ-TCT (Grp.4) significantly increased the number of produced 2-cell embryos by 127% and 79%, respectively compared to Grp.2, but these were lower than Grp.1. Concurrent treatment with soy α-TOC (Grp.5) decreased embryo production by 7%. Supplementations with δ-TCT and α-TOC alone (Grp.6-Grp.8) significantly increased (p < 0.05) the number of produced 2-cell embryos by 50%, 36%, and 41%, respectively, compared to control (Grp.1). These results were found to be associated with alterations in the PI3K/Akt-Cyclin D1 genes expressions, indicating the inhibitory effects of annatto δ-TCT and soy α-TOC against nicotinic embryonic damage. To our knowledge, this is the first attempt in studying the benefits of annatto δ-TCT on murine preimplantation 2-cell embryos.

Increased plasma levels of the lipoperoxyl radical-derived vitamin E metabolite α-tocopheryl quinone are an early indicator of lipotoxicity in fatty liver subjects.

Lipid peroxidation is one of the earliest pathogenic events of non-alcoholic fatty liver disease (NAFLD). In this context, an increased oxidation of the lipoperoxyl radical scavenger α-tocopherol (α-TOH) should occur already in the subclinical phases of the disease to compensate for the increase oxidation of the lipid excess of liver and possibly of other tissues. However, this assumption remains unsupported by direct analytical evidence. In this study, GC-MS/MS and LC-MS/MS procedures have been developed and applied for the first time to measure the vitamin E oxidation metabolite α-tocopheryl quinone (α-TQ) in plasma of fatty liver (FL) subjects that were compared in a pilot cross-sectional study with healthy controls. The protein adducts of 4-hydroxynonenal (4-HNE) and the free form of polyunsaturated free fatty acids (PUFA) were measured as surrogate indicators of lipid peroxidation. α-TQ formation was also investigated in human liver cells after supplementation with α-TOH and/or fatty acids (to induce steatosis). Compared with controls, FL subjects showed increased (absolute and α-TOH-corrected) levels of plasma α-TQ and 4-HNE, and decreased concentrations of PUFA. α-TQ levels positively correlated with indices of liver damage and metabolic dysfunction, such as alanine aminotransferase, bilirubin and triglycerides, and negatively correlated with HDL cholesterol. Fatty acid supplementation in human hepatocytes stimulated the generation of cellular oxidants and α-TOH uptake leading to increased α-TQ formation and secretion in the extracellular medium - both were markedly stimulated by α-TOH supplementation. In conclusion, plasma α-TQ represents an early biomarker of the lipoperoxyl radical-induced oxidation of vitamin E and lipotoxicity of the fatty liver.