A validated reverse-phase high performance liquid chromatography (RP-HPLC) method for the quantification of Gamma- tocotrienol in tocotrienol rich fractions of crude palm oil

Background: Palm oil and its constituents have wide applications in food, cosmetics and pharmaceutical industries. The tocotrienol-rich fractions of crude palm oil have drawn greater research interest in recent years due to its potent health benefits. Therefore, reliable and validated analytical methods are essential for the quantification of tocotrienols. Objective: This study aimed to develop a simple and economical RP-HPLC method for the quantification of gamma-tocotrienol in tocotrienol rich fractions of crude palm oil. Methods: An Agilent HPLC system supplied with a Diode Array detector and an auto-injector system was used for the method development, and the wavelength was set 295 nm. A reversed-phase C18 column maintained at 30 °C using a mobile phase composition of methanol: water (95:05) at a flow rate of 1 mL/min was used for the analysis. The developed method was validated according to ICH guidelines. Results and discussion: A symmetrical peak of gamma-tocotrienol was observed at 8.7 minutes with minimal peak tailing (between 0.76 and 0.78), and an acceptable resolution above 2.0. Excellent linearity was evident with R2 values 0.9996 and 0.9991 for intra-day and inter-day respectively. The method demonstrated a high precision (%RSD values ≤ 5.8%) and accuracy (%RE<9.6%). The LOD and LOQ of gamma-tocotrienol were determined as 1.4 μg/mL and 4.2 μg/mL respectively. The system suitability studies indicate that the chromatographic parameters are well within the acceptable limit. Conclusion: In conclusion, the developed RP-HPLC method is rapid, precise, stable and economical for the quantification of gamma-tocotrienol.

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.

Depletion of Gut Microbiota Suppresses the Metabolite Formation of Vitamin E Forms Including Delta-tocotrienol and Its Long-chain Metabolite 13’-carboxychromanol in Mice

Objectives We recently show that supplementation of delta-tocotrienol (δTE), a vitamin E form and its metabolite δTE-13’-carboxychromanol (δTE-13’) modulated gut microbiota and meanwhile increased metabolites in feces. Since gut microbiota has been shown to metabolize phenolic compounds, we hypothesize that gut bacteria may play a role in metabolizing δTE and δTE-13’. This hypothesis was addressed in the comparison of metabolites formation between antibiotic cocktail (ABX)-treated mice and non-ABX treated mice. Methods Male Balb/c mice were given ABX or water daily for 7 days to remove the gut microbiota. Subsequently ABX or water-treated mice were given a single gavage of δTE/gamma-tocotrienol (δTE/γTE at 8:1) or δTE-13’ at 42mg/kg bw. 24-hr later, mice were sacrificed. We collected 24-hr accumulative fecal samples, adipose, plasma, colon and liver tissues and quantified the concentrations of vitamin E forms and metabolites in these samples. Results Compared with non-ABX controls, ABX-treated mice had decreased weights of liver, spleen and colon, while had doubled the amount of 24-hr fecal output. In δTE-gavaged animals, ABX treatment decreased fecal amounts of δTE and its metabolites by 61% and 98% respectively, while increased δTE level in the adipose tissue. Similarly, in animals gavaged with δTE-13’, ABX treatment led to a 98% reduction in its downstream metabolites. Additionally, ABX treatment decreased fecal excretion of metabolites from other vitamin E forms including α, γ, δ-tocopherols and γTE. Conclusions These results demonstrate that without the gut microbiota, fecal concentrations of vitamin E metabolites declined dramatically, suggesting potential role of the gut microbiota in metabolizing vitamin E forms. Funding Sources Purdue Center for Cancer Research.

Reduced infiltration of T-regulatory cells in tumours from mice fed daily with gamma-tocotrienol supplementation

Gamma-tocotrienol (γT3) is an analogue of vitamin E with beneficial effects on the immune system, including immune-modulatory properties. This study reports the immune-modulatory effects of daily supplementation of γT3 on host T-helper (Th) and T-regulatory (Treg) populations in a syngeneic mouse model of breast cancer. Female BALB/c mice were fed with either γT3 or vehicle (soy oil) for 2-weeks via oral gavage before they were inoculated with syngeneic 4T1 mouse mammary cancer cells (4T1 cells). Supplementation continued until the mice were sacrificed. Mice (n=6) were sacrificed at specified time-points for various analysis (blood leucocyte, cytokine production, and immunohistochemistry). Tumour volume was measured once every seven days. Gene expression studies were carried out on tumour-specific T-lymphocytes isolated from splenic cultures. Supplementation with γT3 increased CD4+ (p<0.05), CD8+ (p<0.05) T-cells and natural killer cells (p<0.05) but suppressed Treg cells (p<0.05) in peripheral blood when compared to animals fed with the vehicle. Higher interferon-gamma (IFN) and lower transforming growth factor-beta (TGF-) levels were noted in the T3 fed mice. Immunohistochemistry findings revealed higher infiltration of CD4+ cells, increased expression of interleukin-12 receptor-beta-2 (IL-122R), interleukin-24 (IL-24) and reduced expression of cells that express the forkhead box P3 (FoxP3) in tumours from the T3 fed animals. Gene expression studies showed the downregulation of seven prominent genes in splenic CD4+ T-cells isolated from γT3-fed mice. Supplementation with γT3 from palm oil-induced T-cell dependent cell-mediated immune responses and suppressed Treg cells in the tumour microenvironment in a syngeneic mouse model of BC.

Gamma Tocotrienol Protects Mice From Targeted Thoracic Radiation Injury

Radiation injury will result in multiorgan dysfuntion leading to multiorgan failure. In addition to many factors such as radiation dose, dose rate, the severity of the injury will also depend on organ systems which are exposed. Here, we report the protective property of gamma tocotrienol (GT3) in total as well as partial body irradiation (PBI) model in C3H/HeN male mice. We have carried out PBI by targeting thoracic region (lung-PBI) using Small Animal Radiation Research Platform, an X-ray irradiator with capabilities of an image guided irradiation with a variable collimator with minimized exposure to non-targeted tissues and organs. Precise and accurate irradiation of lungs was carried out at either 14 or 16 Gy at an approximate dose rate of 2.6 Gy/min. Though a low throughput model, it is amenable to change the field size on the spot. No damage to other non-targeted organs was observed in histopathological evaluation. There was no significant change in peripheral blood counts of irradiated mice in comparison to naïve mice. Femoral bone marrow cells had no damage in irradiated mice. As expected, damage to the targeted tissue was observed in the histopathological evaluation and non-targeted tissue was found normal. Regeneration and increase of cellularity and megakaryocytes on GT3 treatment was compared to significant loss of cellularity in saline group. Peak alveolitis was observed on day 14 post-PBI and protection from alveolitis by GT3 was noted. In irradiated lung tissue, thirty proteins were found to be differentially expressed but modulated by GT3 to reverse the effects of irradiation. We propose that possible mode of action of GT3 could be Angiopoietin 2-Tie2 pathway leading to AKT/ERK pathways resulting in disruption in cell survival/angiogenesis.